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Proteasome inhibitors induce auditory hair cell death through peroxisome dysfunction.
Lee, Joon No; Kim, Seul-Gi; Lim, Jae-Young; Kim, Se-Jin; Choe, Seong-Kyu; Park, Raekil
Even though bortezomib, a proteasome inhibitor, is a powerful chemotherapeutic agent used to treat multiple myeloma (MM) and other lymphoma cells, recent clinical reports suggest that the proteasome inhibitor therapy may be associated with severe bilateral hearing loss. We herein investigated the adverse effect of proteasome inhibitor on auditory hair cells. Treatment of a proteasome inhibitor destroys stereocilia bundles of hair cells resulting in the disarray of stereocilia in the organ of Corti explants. Since proteasome activity may be potentially important for biogenesis and function of the peroxisome, we tested whether proteasome activity is necessary for maintaining functional peroxisomes. Our results showed that treatment of a proteasome inhibitor significantly decreases both the number of peroxisomes and expression of peroxisomal proteins such as PMP70 and Catalase. In addition, we also found that proteasome inhibitor impairs the import pathway of PTS1-peroxisome matrix proteins. Taken together, our findings support recent clinical reports of hearing loss associated with proteasome inhibition. Mechanistically, peroxisome dysfunction may contribute to hair cell damage and hearing loss in response to the treatment of a proteasome inhibitor. PMID:25446082
Proteasome inhibitors induce peroxisome proliferator-activated receptor transactivation through RXR accumulation and a protein kinase C-dependent pathway
Tsao, W.-C. [Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Wu, H.-M. [Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Chi, K.-H. [Cancer Center, Veterans General Hospital, Taipei, Taiwan (China); Chang, Y.-H. [Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Lin, W.-W. [Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan (China)]. E-mail: email@example.com
Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of nuclear hormone receptors, forms a heterodimeric DNA binding complex with retinoid X receptor (RXR) and serves as a transcriptional regulator of gene expression. In this study, using luciferase assay of a reporter gene containing PPAR response element (PPRE), we found PPRE transactivity was additively induced by PPAR{gamma} activator (15dPGJ{sub 2}) and RXR activator (9-cis retinoic acid, 9-cis RA). Proteasome inhibitors MG132 and MG262 also stimulate PPRE transactivity in a concentration-dependent manner, and this effect is synergistic to 15dPGJ{sub 2} and 9-cis RA. PKC activation by 12-myristate 13-acetate (PMA) and ingenol 3,20-dibenzoate (IDB) also led to an increased PPRE activation, and this action was additive to PPAR{gamma} activators and 9-cis RA, but not to proteasome inhibitors. Results indicate that the PPAR{gamma} enhancing effect of proteasome inhibitors was attributed to redox-sensitive PKC activation. Western blot analysis showed that the protein level of RXR{alpha}, but not PPAR{gamma}, RXR{beta}, or PKC isoforms, was accumulated in the presence of proteasome inhibitors. Taken together, we conclude that proteasome inhibitors can upregulate PPRE activity through RXR{alpha} accumulation and a PKC-dependent pathway. The former is due to inhibition of RXR{alpha} degradation through ubiquitin-dependent proteasome system, while the latter is mediated by reactive oxygen species (ROS) production.
Hydrogen peroxide generation in peroxisome proliferator-induced oncogenesis
Anjana V Yeldandi; M. Sambasiva Rao; Janardan K Reddy
Peroxisome proliferators are a structurally diverse group of non-genotoxic chemicals that induce predictable pleiotropic responses including the development of liver tumors in rats and mice. These chemicals interact variably with peroxisome proliferator-activated receptors (PPARs), which are members of the nuclear receptor superfamily. Evidence derived from mice with PPAR? gene disruption indicates that of the three PPAR isoforms (?, ?\\/? and
Dysfunction of peroxisomes in twitcher mice brain: A possible mechanism of psychosine-induced disease
Haq, Ehtishamul [Department of Pediatrics and The Children's Research Institute, Medical University of South Carolina, Charleston, SC 29425 (United States); Contreras, Miguel A. [Department of Pediatrics and The Children's Research Institute, Medical University of South Carolina, Charleston, SC 29425 (United States); Giri, Shailendra [Department of Pediatrics and The Children's Research Institute, Medical University of South Carolina, Charleston, SC 29425 (United States); Singh, Inderjit [Department of Pediatrics and The Children's Research Institute, Medical University of South Carolina, Charleston, SC 29425 (United States); Singh, Avtar K. [Department of Pathology and Laboratory Medicine, Medical University of South Carolina and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29425 (United States)]. E-mail: firstname.lastname@example.com
Psychosine (galactosylsphingosine) accumulates in Brain of Krabbe disease (KD) patients as well as twitcher mice, a murine model of KD, resulting in loss of oligodendrocytes and myelin. This study documents progressive loss of peroxisomal proteins/functions and induction of expression of inflammatory cytokine TNF-{alpha} in twitcher brain. The observed decrease in peroxisomal proteins was accompanied by decreased level of peroxisome proliferator-activated receptor-alpha (PPAR-{alpha}), one of the transcription factors required for expression of peroxisomal protein genes. The role of psychosine in down-regulation of PPAR-{alpha} activity was further supported by decreased PPAR-{alpha} mediated PPRE transcriptional activity in cells transfected with PPAR-{alpha} and PPRE reporters. The psychosine-induced down-regulation of PPAR activity and cell death was attenuated by sPLA{sub 2} inhibitor. Therefore, this study provides First evidence of peroxisomal abnormality in a lysosomal disorder, suggesting that such dysfunction of peroxisomes may play a role in the pathogenesis of Krabbe disease.
PEX11? induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development
Mark A Fox; Logan A Walsh; Michelle Nieuwesteeg; Sashko Damjanovski
BackgroundĀĀPeroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much\\u000a attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in\\u000a part regulated by proteins such as Pex11, which can facilitate the division
Expression level of methanol-inducible peroxisomal proteins and peroxisome morphology are affected by oxygen conditions and mitochondrial respiratory pathway function in the methylotrophic yeast Candida boidinii.
Fujimura, Shuki; Yurimoto, Hiroya; Kurimoto, Shota; Matsufuji, Yoshimi; Ito, Takashi; Hayakawa, Takashi; Tomizuka, Noboru; Sakai, Yasuyoshi; Nakagawa, Tomoyuki
In the methylotrophic yeast, Candida boidinii, methanol-inducible peroxisomal proteins, for example alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and peroxisomal glutathione peroxidase (Pmp20), were induced only under aerobic conditions, while expression of PMP47 encoding peroxisomal integral membrane protein Pmp47 was independent of oxygen conditions. Expression of the methanol-inducible peroxisomal enzymes was repressed by inhibition of the mitochondrial respiratory chain. In the respiratory-deficient (?0) mutant strain, their induction was at very low levels despite the presence of oxygen, whereas the expression of PMP47 was unaffected. Taken together, these facts indicate that C. boidinii can sense oxygen conditions, and that mitochondrial respiratory function may have a profound effect on induction of methanol-inducible gene expression of peroxisomal proteins. Peroxisome morphology was also affected by oxygen conditions and respiratory function. Under hypoxic conditions or respiration-inhibited conditions, cells induced by methanol contained small peroxisomes, indicating that peroxisome biogenesis and the protein import machinery were not affected by oxygen conditions but that peroxisome morphology was dependent on induction of peroxisomal matrix proteins. PMID:23448597
Endothelial Peroxisomal Dysfunction and Impaired Pexophagy Promotes Oxidative Damage in Lipopolysaccharide-Induced Acute Kidney Injury
Ratliff, Brian B.; Bohr, Stefan; Nadel, Ellen; Chen, Jun; Xavier, Sandhya; Chander, Praveen; Goligorsky, Michael S.
Abstract Aims: We examined that (a) how the endotoxic stress affects peroxisomal function and autophagic degradation of peroxisomesŚpexophagy, (b) how a superimposed dysfunction of lysosomes and pexophagy modifies responses to lipopolysaccharide (LPS), and (c) the mechanisms of peroxisomal contribution to renal injury. To accomplish this, we used lysosome-defective Lyst-mice in vivo and primary endothelial cells in vitro, and compared the responses with wild-type (WT) littermates. Results: LPS induced pexophagic degradation, followed by proliferation of peroxisomes in WT mice, which was abolished in Lyst-mice. Lyst-mice exhibited impaired activation of catalase, which together with preserved hydrogen peroxide-generating ?-oxidation resulted in redox disequilibrium. LPS treatment induced a heightened inflammatory response, increased oxidative damage, and aggravated renal injury in Lyst-mice. Similarly, as in vivo, LPS-activated lysosomal (LYS) pexophagy and transiently repressed peroxisomes in vitro, supported by reduced peroxisomal density in the vicinity of lysosomes. Peroxisomal dynamics was also abolished in lysosome-defective cells, which accumulated peroxisomes with compromised functions and intraorganellar redox imbalance. Innovation: We demonstrated that pexophagy is a default response to endotoxic injury. However, when LYS dysfunction (a frequent companion of chronic diseases) is superimposed, recycling and functioning of peroxisomes are impaired, and an imbalance between hydrogen peroxide-generating ?-oxidation and hydrogen peroxide-detoxifying catalase ensues, which ultimately results in peroxisomal burnout. Conclusion: Our data strongly suggest that pexophagy, a cellular mechanism per se, is essential in functional maintenance of peroxisomes during LPS exposure. Inhibition of pexophagy results in accumulation of impaired peroxisomes, redox disequilibrium, and aggravated renal damage. Antioxid. Redox Signal. 19, 211¢230. PMID:23088293
Peroxisome proliferator-activated receptor alpha-retinoid X receptor agonists induce beta-cell protection against palmitate toxicity.
Hellemans, Karine; Kerckhofs, Karen; Hannaert, Jean-Claude; Martens, Geert; Van Veldhoven, Paul; Pipeleers, Daniel
Fatty acids can stimulate the secretory activity of insulin-producing beta-cells. At elevated concentrations, they can also be toxic to isolated beta-cells. This toxicity varies inversely with the cellular ability to accumulate neutral lipids in the cytoplasm. To further examine whether cytoprotection can be achieved by decreasing cytoplasmic levels of free acyl moieties, we investigated whether palmitate toxicity is also lowered by stimulating its beta-oxidation. Lower rates of palmitate-induced beta-cell death were measured in the presence of L-carnitine as well as after addition of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, conditions leading to increased palmitate oxidation. In contrast, inhibition of mitochondrial beta-oxidation by etomoxir increased palmitate toxicity. A combination of PPARalpha and retinoid X receptor (RXR) agonists acted synergistically and led to complete protection; this was associated with enhanced expression levels of genes involved in mitochondrial and peroxisomal beta-oxidation, lipid metabolism, and peroxisome proliferation. PPARalpha-RXR protection was abolished by the carnitine palmitoyl transferase 1 inhibitor etomoxir. These observations indicate that PPARalpha and RXR regulate beta-cell susceptibility to long-chain fatty acid toxicity by increasing the rates of beta-oxidation and by involving peroxisomes in fatty acid metabolism. PMID:17970749
PPAR? activation induces N(?)-Lys-acetylation of rat liver peroxisomal multifunctional enzyme type 1.
Contreras, Miguel A; Alzate, Oscar; Singh, Avtar K; Singh, Inderjit
Peroxisomes are ubiquitous subcellular organelles that participate in metabolic and disease processes, with few of its proteins undergoing posttranslational modifications. As the role of lysine-acetylation has expanded into the cellular intermediary metabolism, we used a combination of differential centrifugation, organelle isolation by linear density gradient centrifugation, western blot analysis, and peptide fingerprinting and amino acid sequencing by mass spectrometry to investigate protein acetylation in control and ciprofibrate-treated rat liver peroxisomes. Organelle protein samples isolated by density gradient centrifugation from PPAR?-agonist treated rat liver screened with an anti-N(?)-acetyl lysine antibody revealed a single protein band of 75 kDa. Immunoprecipitation with this antibody resulted in the precipitation of a protein from the protein pool of ciprofibrate-induced peroxisomes, but not from the protein pool of non-induced peroxisomes. Peptide mass fingerprinting analysis identified the protein as the peroxisomal multifunctional enzyme type 1. In addition, mass spectrometry-based amino acid sequencing resulted in the identification of unique peptides containing 4 acetylated-Lys residues (K╣??, K╣?│, K╣??, and K??│). This is the first report that demonstrates posttranslational acetylation of a peroxisomal enzyme in PPAR?-dependent proliferation of peroxisomes in rat liver. PMID:24092543
Effects of peroxisome proliferator-activated receptor agonists on LPS-induced neuronal death in mixed cortical neurons: associated with iNOS and COX2
Eun Joo Kim; Kyoung Ja Kwon; Jee-Young Park; Soo Hwan Lee; Chang-Hyun Moon; Eun Joo Baik
In neurodegenerative disease, the use of non-steroidal anti-inflammatory drugs (NSAIDs) has been regarded as beneficial. The NSAID, an inhibitor of cyclooxygenase (COX), has been also suggested as a ligand of the peroxisome proliferator-activated receptor (PPAR). In cortical neuron¢glial co-cultures, we examined the effect of PPAR agonists on lipopolysaccharide(LPS)-induced neuronal death, which has been known to be NO-dependent. LPS induced iNOS
Hepatocyte proliferation induced by a single dose of a peroxisome proliferator.
Ohmura, T.; Ledda-Columbano, G. M.; Piga, R.; Columbano, A.; Glemba, J.; Katyal, S. L.; Locker, J.; Shinozuka, H.
In compensatory hyperplasia after partial hepatectomy or liver cell injury, hepatocyte proliferation is triggered by coordinated actions of growth factor such as hepatocyte growth factor and transforming growth factor-alpha and -beta. Initiation of hepatocyte DNA synthesis is preceded by the activation of the set of early growth response genes mediated by enhanced nuclear factor-kappa B binding to DNA. Using an experimental model to induce hepatocyte DNA synthesis in vivo by a single dose of a peroxisome proliferator, which does not induce liver cell necrosis (direct hyperplasia), we investigated whether peroxisome proliferator-induced hepatocyte proliferation involved an induction of known growth factors, an activation of early growth response genes, and nuclear factor-kappa B. A single intragastric administration of 250 mg/kg BR931 (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide) to male wistar rats induced a wave of hepatocyte DNA synthesis starting after 12 hours and peaking at approximately 24 to 36 hours. The response was dose dependent. The treatment also induced the expression of the mRNA for the peroxisomal bifunctional enzyme, one of the peroxisome-related fatty acid beta-oxidation enzymes. Pretreatment of rats with dexamethasone (2 mg/kg) inhibited both hepatocyte DNA synthesis and the induction of the peroxisomal bifunctional enzyme gene. Northern blot analyses of liver RNA during a period preceding the onset of DNA synthesis revealed no induction of hepatocyte growth factor, transforming growth factor-alpha, or tumor necrosis factor-alpha mRNAs. No induction of early growth response genes, liver regeneration factor-1, or c-myc was detected. Furthermore, gel mobility shift assays showed no enhanced nuclear factor-kappa B binding to its DNA consensus sequence after BR931 treatment, whereas control studies demonstrated a distinct increase in binding after partial hepatectomy or lead nitrate treatment. The results suggest that peroxisome-proliferator-induced hepatocyte proliferation may be triggered by signal transduction pathways different from those after partial hepatectomy and that the binding of peroxisome proliferators to their nuclear receptors may play a role in stimulation of DNA synthesis and peroxisome proliferation. Images Figure 2 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:8774136
Visfatin is induced by peroxisome proliferator-activated receptor gamma in human macrophages
Mayi, ThķrĶse HĶrvķe; Duhem, Christian; Copin, Corinne; Bouhlel, Mohamed Amine; Rigamonti, Elena; Pattou, Franńois; Staels, Bart; Chinetti-Gbaguidi, Giulia
Obesity is a low grade chronic inflammatory disease associated with an increased number of macrophages (ATM) in adipose tissue. Within the adipose tissue, ATM are the major source of visfatin/PBEF/NAMPT. The nuclear receptor Peroxisome Proliferator-Activated Receptor (PPAR)? exerts anti-inflammatory effects in macrophages by inhibiting cytokine production and enhancing alternative differentiation. In this study, we investigated whether PPAR? modulates visfatin expression in murine (BMDM) and human (RM, M1, M2, ATM) macrophage models and preadipocyte-derived adipocytes. We show that synthetic PPAR? ligands increased visfatin gene expression in a PPAR?-dependent manner in primary human macrophages (RM) and ATM, but not in adipocytes. The increase of visfatin mRNA (3-fold) was paralleled by an increase of protein expression (30%) and secretion (30%). Electrophoretic Mobility Shift Assay (EMSA) experiments and transient transfection assays indicated that PPAR? induces visfatin promoter activity in human macrophages by binding to a DR1-PPAR? response element. Finally, we show that PPAR? ligands increase NAD+ production in primary human macrophages and this regulation is dampened in the presence of visfatin siRNA or by the visfatin-specific inhibitor FK866. Taken together, our results suggest that PPAR? regulates the expression of visfatin in macrophages leading to increased NAD+ levels. PMID:20608974
Activation of Peroxisome Proliferator-Activated Receptor Ā by Substituted Urea-Derived Soluble Epoxide Hydrolase Inhibitors
Xiang Fang; Shanming Hu; Takaho Watanabe; Neal L. Weintraub; Gary D. Snyder; Jianrong Yao; Yi Liu; John Y.-J. Shyy; Bruce D. Hammock; Arthur A. Spector
Soluble epoxide hydrolase (sEH) plays a major role in regulating vascular epoxyeicosatrienoic acid metabolism and function, and substituted urea derivatives that inhibit sEH activity reduce blood pressure in hypertensive rats. We found that substituted urea derivatives containing a dodecanoic acid group, besides effectively inhibiting sEH, increased peroxisome proliferator- activated receptor (PPAR) activity. In PPAR transfected COS-7 cells, treatment with 10
Oleuropein as an inhibitor of peroxisome proliferator-activated receptor gamma.
Svobodova, Michaela; Andreadou, Ioanna; Skaltsounis, Alexios-Leandros; Kopecky, Jan; Flachs, Pavel
Oleuropein, the major phenolic compound found in olive leaves and oil, exerts antioxidant, anti-inflammatory and anti-atherogenic effects and suppresses the adipocyte differentiation in vitro. Herein, we characterized molecular mechanisms underlying the anti-adipogenic effects of oleuropein on 3T3-L1 cells and adipocytes derived from stromal-vascular fraction of dorsolumbar and gonadal fat dissected from mice. We found that oleuropein (>100Ā?M) decreased viability of preadipocytes proliferating in vitro and did not exerted any cytotoxic effects in post-confluent cells after induction of differentiation. Oleuropein (>100Ā?M) inhibited adipocyte differentiation, suppressed gene expression of peroxisome proliferator-activated receptor ? (PPAR?), CCAAT-/enhancer-binding protein ?, sterol regulatory element-binding transcription factor 1c and fatty acid synthase. Furthermore, we tested ability of oleuropein to regulate of PPAR?-, PPAR?- or PPAR?-/PPAR?-mediated ?-lactamase expression in appropriate reporter gene assays. Oleuropein between 10 and 400Ā?M concentrations did not affect activity of PPAR? or PPAR?/?. Contrary, PPAR? activity, either basal or rosiglitazone activated, was inhibited by oleuropein. Our data suggest that oleuropein exerts anti-adipogenic effect through direct inhibition of PPAR? transcriptional activity. PMID:24323842
Peroxisome Proliferator-Activated Receptor activation induces 11-Hydroxysteroid Dehydrogenase type 1 activity in human alternative macrophages
Boyer, Edmond
1 Peroxisome Proliferator-Activated Receptor activation induces 11-Hydroxysteroid Dehydrogenase type 1 activity in human alternative macrophages Giulia Chinetti-Gbaguidi 1,2,3,4* , Mohamed Amine;32(3):677-85" DOI : 10.1161/ATVBAHA.111.241364 #12;2 Abstract Objectives - 11-hydroxysteroid dehydrogenase type 1
Modulation of VEGF-Induced Retinal Vascular Permeability by Peroxisome Proliferator-Activated Receptor-?/?
Suarez, Sandra; McCollum, Gary W.; Bretz, Colin A.; Yang, Rong; Capozzi, Megan E.; Penn, John S.
Purpose. Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision-threatening condition. Peroxisome proliferator-activated receptor ?/? (PPAR?/?) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPAR?/? in VEGF-induced retinal hyperpermeability. Methods. Transendothelial electrical resistance (TEER) measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/Claudin-5 localization in HRMEC monolayers was determined by immunocytochemistry. Extracellular signal-regulated protein kinases 1 and 2 (Erk 1/2) phosphorylation, VEGF receptor 1 (VEGFR1) and R2 were assayed by Western blot analysis. Expression of VEGFR1 and R2 was measured by quantitative RT-PCR. Last, retinal vascular permeability was assayed in vivo by Evans blue extravasation. Results. Human retinal microvascular endothelial cell monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10 ?M) and PPAR?/?-directed siRNA (20 ?M). In HRMEC treated with VEGF, GSK0660 stabilized tight-junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. Peroxisome proliferator-activated receptor ?/? siRNA had a similar effect on VEGFR expression and Claudin-1, supporting the specificity of GSK0660 in our experiments. Last, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1and R2 levels in C57BL/6 mice. Conclusions. These data suggest a protective effect for PPAR?/? antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPAR?/? siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation. PMID:25406289
Identification of transcriptional networks involved in peroxisome proliferator chemical-induced hepatocyte proliferation
Peroxisome proliferator chemical (PPC) exposure leads to increases in rodent liver tumors through a non-genotoxic mode of action (MOA). The PPC MOA includes increased oxidative stress, hepatocyte proliferation and decreased apoptosis. We investigated the putative genetic regulato...
Peroxisome proliferator-activated receptor ? attenuates serotonin-induced pulmonary artery smooth muscle cell proliferation and apoptosis inhibition involving ERK1/2 pathway.
Han, Xinyuan; Chen, Chunyan; Cheng, Gong; Liang, Lei; Yao, Xiaowei; Yang, Guang; You, Penghua; Shou, Xiling
Serotonin (5-HT) has been shown to be involved in pulmonary vascular remodeling in pulmonary arterial hypertension (PAH) by inducing pulmonary artery smooth muscle cells (PASMCs) proliferation and inhibiting PASMC apoptosis. Peroxisome proliferator-activated receptor ? (PPAR?) plays a crucial role in regulating proliferation and apoptosis of many cell types. Moreover, recently, loss of PPAR? has also been reported to be associated with the development of PAH. The present study is aimed to assess whether PPAR? is involved in 5-HT induced PASMC proliferation and apoptosis inhibition and the possible mechanism. We found that 5-HT could induce PASMC proliferation and inhibit PASMC apoptosis in a dose-dependent manner. Furthermore, we found that 5-HT negatively regulated PPAR? expression and gene promoter activity in PASMCs and 5-HT induced PASMC proliferation and apoptosis resistance could be abolished by PPAR? agonists and enhanced by PPAR? inhibitor. In addition, we found that extracellular signal-regulated kinase (ERK) signaling pathway mediated the 5-HT-induced inhibition of PPAR? expression. Our results might provide novel insights into the mechanisms for the pro-remodeling action of 5-HT in pulmonary vasculature. PMID:25937083
Fatty aldehyde dehydrogenase is up-regulated by polyunsaturated fatty acid via peroxisome proliferator-activated receptor alpha and suppresses polyunsaturated fatty acid-induced endoplasmic reticulum stress.
Ashibe, Bunichiro; Motojima, Kiyoto
Fatty aldehyde dehydrogenase (FALDH; also known as ALDH3A2 or ALDH10) oxidizes medium- or long-chain aliphatic aldehydes. FALDH deficiency in humans is known to be the cause of Sj÷gren-Larsson syndrome, in which individuals display neurological symptoms and cutaneous abnormality. FALDH-V, a splice isoform of FALDH, is localized in the peroxisome and contributes to the oxidization of pristanal, an intermediate of the alpha-oxidation pathway. FALDH-N, another splice isoform of FALDH, is induced by peroxisomal proliferator-activated receptor alpha ligands, although its activation mechanism has not been clarified. In the present study, we show that transcriptional activation of FALDH is directly regulated by peroxisomal proliferator-activated receptor alpha through a direct repeat-1 site located in the FALDH promoter. In addition, FALDH is efficiently induced by linoleic acid in rat hepatoma Fao cells through transcriptional activation by peroxisomal proliferator-activated receptor alpha. Furthermore, ectopic expression of endoplasmic reticulum-localizing FALDH-N, but not peroxisome-localizing FALDH-V, suppresses endoplasmic reticulum stress caused by linoleic acid in HEK293 cells. These results suggest the autocatalytic nature of the FALDH-N system against endoplasmic reticulum stress that is induced by polyunsaturated fatty acid; polyunsaturated fatty acid binds to peroxisomal proliferator-activated receptor alpha to activate the expression of FALDH-N, which then detoxifies polyunsaturated fatty acid-derived fatty aldehydes and protects cells from endoplasmic reticulum stress. PMID:19860831
Peroxisome Proliferator-Activated Receptor ? Activation Induces Hepatic Steatosis, Suggesting an Adverse Effect
Yan, Fang; Wang, Qi; Xu, Chao; Cao, Mingfeng; Zhou, Xiaoming; Wang, Tingting; Yu, Chunxiao; Jing, Fei; Chen, Wenbin; Gao, Ling; Zhao, Jiajun
Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic triglyceride accumulation, ranging from steatosis to steatohepatitis and cirrhosis. NAFLD is a risk factor for cardiovascular diseases and is associated with metabolic syndrome. Antihyperlipidemic drugs are recommended as part of the treatment for NAFLD patients. Although fibrates activate peroxisome proliferator-activated receptor ? (PPAR?), leading to the reduction of serum triglyceride levels, the effects of these drugs on NAFLD remain controversial. Clinical studies have reported that PPAR? activation does not improve hepatic steatosis. In the present study, we focused on exploring the effect and mechanism of PPAR? activation on hepatic triglyceride accumulation and hepatic steatosis. Male C57BL/6J mice, Ppar?-null mice and HepG2 cells were treated with fenofibrate, one of the most commonly used fibrate drugs. Both low and high doses of fenofibrate were administered. Hepatic steatosis was detected through oil red O staining and electron microscopy. Notably, in fenofibrate-treated mice, the serum triglyceride levels were reduced and the hepatic triglyceride content was increased in a dose-dependent manner. Oil red O staining of liver sections demonstrated that fenofibrate-fed mice accumulated abundant neutral lipids. Fenofibrate also increased the intracellular triglyceride content in HepG2 cells. The expression of sterol regulatory element-binding protein 1c (SREBP-1c) and the key genes associated with lipogenesis were increased in fenofibrate-treated mouse livers and HepG2 cells in a dose-dependent manner. However, the effect was strongly impaired in Ppar?-null mice treated with fenofibrate. Fenofibrate treatment induced mature SREBP-1c expression via the direct binding of PPAR? to the DR1 motif of the SREBP-1c gene. Taken together, these findings indicate the molecular mechanism by which PPAR? activation increases liver triglyceride accumulation and suggest an adverse effect of fibrates on the pathogenesis of hepatic steatosis. PMID:24926685
Cucurbitane Triterpenoid from Momordica charantia Induces Apoptosis and Autophagy in Breast Cancer Cells, in Part, through Peroxisome Proliferator-Activated Receptor ? Activation
Weng, Jing-Ru; Bai, Li-Yuan; Chiu, Chang-Fang; Hu, Jing-Lan; Chiu, Shih-Jiuan; Wu, Chia-Yung
Although the antitumor activity of the crude extract of wild bitter gourd (Momordica charantia L.) has been reported, its bioactive constituents and the underlying mechanism remain undefined. Here, we report that 3?,7?-dihydroxy-25-methoxycucurbita-5,23-diene-19-al (DMC), a cucurbitane-type triterpene isolated from wild bitter gourd, induced apoptotic death in breast cancer cells through peroxisome proliferator-activated receptor (PPAR) ? activation. Luciferase reporter assays indicated the ability of DMC to activate PPAR?, and pharmacological inhibition of PPAR? protected cells from DMC's antiproliferative effect. Western blot analysis indicated that DMC suppressed the expression of many PPAR?-targeted signaling effectors, including cyclin D1, CDK6, Bcl-2, XIAP, cyclooxygenase-2, NF-?B, and estrogen receptor ?, and induced endoplasmic reticulum stress, as manifested by the induction of GADD153 and GRP78 expression. Moreover, DMC inhibited mTOR-p70S6K signaling through Akt downregulation and AMPK activation. The ability of DMC to activate AMPK in liver kinase (LK) B1-deficient MDA-MB-231 cells suggests that this activation was independent of LKB1-regulated cellular metabolic status. However, DMC induced a cytoprotective autophagy presumably through mTOR inhibition, which could be overcome by the cotreatment with the autophagy inhibitor chloroquine. Together, the ability of DMC to modulate multiple PPAR?-targeted signaling pathways provides a mechanistic basis to account for the antitumor activity of wild bitter gourd. PMID:23843889
A peroxisomal ABC transporter promotes seed germination by inducing pectin degradation under the control of ABI5.
Kanai, Masatake; Nishimura, Mikio; Hayashi, Makoto
Seed dormancy is essential for most plants to control the timing of germination. In Arabidopsis thaliana, PED3 is a single-copy gene encoding an ATP-binding cassette transporter that is required for peroxisomal fatty acid beta-oxidation. PED3 is involved in the import of several biologically important molecules into the peroxisome, including very-long-chain fatty acids associated with the breakdown of seed-reserve lipids, and precursors of auxin and jasmonic acid. The germination of ped3 mutants is significantly impaired, suggesting that PED3 regulates dormancy and germination. A transcriptome analysis revealed that many genes containing the core motif of the ABA responsive element (ABRE) in their promoter regions, and the ABA insensitive 5 (ABI5) transcription factor that binds to ABRE, are abnormally up-regulated in imbibed ped3 seeds. Expression of polygalacturonase inhibiting proteins (PGIPs) is also up-regulated specifically in ped3 after imbibition. By contrast, the ped3 abi5 double mutant does not show any of these expression patterns. The results indicate that the abi5 mutation normalizes PGIP expression and rescues the impaired germination phenotype of the ped3 mutant. PGIPs are known to act as inhibitors of polygalacturonases that degrade pectin. The amount of PGIP1 transcript regulates the timing of radicle protrusion. The impaired germination of ped3 could also be rescued by removal of pectin from the seed coat using exogenous polygalacturonase or acidic conditions. Overall, our results suggest that PED3, a peroxisomal ABC transporter, promotes seed germination by suppressing PGIPs under the control of ABI5. PMID:20345608
Peroxisome proliferator-activated receptor gamma induces apoptosis and inhibits autophagy of human monocyte-derived macrophages via induction of cathepsin L: potential role in atherosclerosis.
Mahmood, Dler Faieeq Darweesh; Jguirim-Souissi, Imene; Khadija, El-Hadri; Blondeau, Nicolas; Diderot, Vimala; Amrani, Souliman; Slimane, Mohamed-Naceur; Syrovets, Tatiana; Simmet, Thomas; Rouis, Mustapha
Macrophages play a pivotal role in the pathophysiology of atherosclerosis. These cells express cathepsin L (CatL), a cysteine protease that has been implicated in atherogenesis and the associated arterial remodeling. In addition, macrophages highly express peroxisome proliferator-activated receptor (PPAR) ?, a transcription factor that regulates numerous genes important for lipid and lipoprotein metabolism, for glucose homeostasis, and inflammation. Hence, PPAR? might affect macrophage function in the context of chronic inflammation such as atherogenesis. In the present study, we examined the effect of PPAR? activation on the expression of CatL in human monocyte-derived macrophages (HMDM). Activation of PPAR? by the specific agonist GW929 concentration-dependently increased the levels of CatL mRNA and protein in HMDM. By promoter analysis, we identified a functional PPAR response element-like sequence that positively regulates CatL expression. In addition, we found that PPAR?-induced CatL promotes the degradation of Bcl2 without affecting Bax protein levels. Consistently, degradation of Bcl2 could be prevented by a specific CatL inhibitor, confirming the causative role of CatL. PPAR?-induced CatL was found to decrease autophagy through reduction of beclin 1 and LC3 protein levels. The reduction of these proteins involved in autophagic cell death was antagonized either by the CatL inhibitor or by CatL knockdown. In conclusion, our data show that PPAR? can specifically induce CatL, a proatherogenic protease, in HMDM. In turn, CatL inhibits autophagy and induces apoptosis. Thus, the proatherogenic effect of CatL could be neutralized by apoptosis, a beneficial phenomenon, at least in the early stages of atherosclerosis. PMID:21700710
Peroxisome Proliferator-activated Receptor ? Induces Apoptosis and Inhibits Autophagy of Human Monocyte-derived Macrophages via Induction of Cathepsin L
Mahmood, Dler Faieeq Darweesh; Jguirim-Souissi, Imene; Khadija, El-Hadri; Blondeau, Nicolas; Diderot, Vimala; Amrani, Souliman; Slimane, Mohamed-Naceur; Syrovets, Tatiana; Simmet, Thomas; Rouis, Mustapha
Macrophages play a pivotal role in the pathophysiology of atherosclerosis. These cells express cathepsin L (CatL), a cysteine protease that has been implicated in atherogenesis and the associated arterial remodeling. In addition, macrophages highly express peroxisome proliferator-activated receptor (PPAR) ?, a transcription factor that regulates numerous genes important for lipid and lipoprotein metabolism, for glucose homeostasis, and inflammation. Hence, PPAR? might affect macrophage function in the context of chronic inflammation such as atherogenesis. In the present study, we examined the effect of PPAR? activation on the expression of CatL in human monocyte-derived macrophages (HMDM). Activation of PPAR? by the specific agonist GW929 concentration-dependently increased the levels of CatL mRNA and protein in HMDM. By promoter analysis, we identified a functional PPAR response element-like sequence that positively regulates CatL expression. In addition, we found that PPAR?-induced CatL promotes the degradation of Bcl2 without affecting Bax protein levels. Consistently, degradation of Bcl2 could be prevented by a specific CatL inhibitor, confirming the causative role of CatL. PPAR?-induced CatL was found to decrease autophagy through reduction of beclin 1 and LC3 protein levels. The reduction of these proteins involved in autophagic cell death was antagonized either by the CatL inhibitor or by CatL knockdown. In conclusion, our data show that PPAR? can specifically induce CatL, a proatherogenic protease, in HMDM. In turn, CatL inhibits autophagy and induces apoptosis. Thus, the proatherogenic effect of CatL could be neutralized by apoptosis, a beneficial phenomenon, at least in the early stages of atherosclerosis. PMID:21700710
Liver fatty acid-binding protein: specific mediator of the mitogenesis induced by two classes of carcinogenic peroxisome proliferators.
Khan, S H; Sorof, S
Peroxisome proliferators (PP) are a diverse group of chemicals that induce dramatic increases in peroxisomes in rodent hepatocytes, followed by hypertrophy, hepatomegaly, alterations in lipid metabolism, mitogenesis, and finally hepatocarcinomas. Termed nongenotoxic carcinogens, they do not interact with DNA, are not mutagenic in bacterial assays, and fail to elicit many of the phenotypes associated with classic genotoxic carcinogens. We report here that the mitogenesis induced by the major PP class, the amphipathic carboxylates, and by the tetrazole-substituted acetophenones specifically requires liver fatty acid-binding protein (L-FABP) in cultured rat hepatoma cells transfected with the sense cDNA of L-FABP, in contrast to L-FABP-nonexpressing cells transfected with its antisense cDNA. The mitogenic actions of L-FABP were protein-specific, inasmuch as no other protein in the nonexpressing cells could act like L-FABP. L-FABP was previously shown not only (i) to interact covalently with metabolites of the two genotoxic carcinogens 2-acetylaminofluorene and aminoazo dyes during liver carcinogenesis, but also (ii) to bind noncovalently the two classes of PP in vitro with avidities that correlate with their abilities to elicit peroxisomal enzymatic responses, and (iii) together with unsaturated fatty acids, especially linoleic acid, to promote multiplication of the transfected hepatoma cells in culture. The convergence of the two types of genotoxic carcinogens with the two classes of PP nongenotoxic carcinogens, and also with unsaturated fatty acids, at L-FABP actions in inducing mitogenesis allows the following hypothesis. During tumor promotion of carcinogenesis in vivo, these groups of genotoxic and nongenotoxic carcinogens act on the normal process by which L-FABP, functioning as a specific receptor of unsaturated fatty acids or their metabolites, promotes hepatocyte proliferation. Images PMID:8302856
Peroxisomes in cardiomyocytes and the peroxisome / peroxisome proliferator-activated receptor-loop.
Colasante, Claudia; Chen, Jiangping; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline
It is well established that the heart is strongly dependent on fatty acid metabolism. In cardiomyocytes there are two distinct sites for the ?-oxidisation of fatty acids: the mitochondrion and the peroxisome. Although the metabolism of these two organelles is believed to be tightly coupled, the nature of this relationship has not been fully investigated. Recent research has established the significant contribution of mitochondrial function to cardiac ATP production under normal and pathological conditions. In contrast, limited information is available on peroxisomal function in the heart. This is despite these organelles harbouring metabolic pathways that are potentially cardio-protective, and findings that patients with peroxisomal diseases, such as adult Refsum┤s disease, can develop heart failure. In this article, we provide a comprehensive overview on the current knowledge of peroxisomes and the regulation of lipid metabolism by PPARs in cardiomyocytes. We also present new experimental evidence on the differential expression of peroxisome-related genes in the heart chambers and demonstrate that even a mild peroxisomal biogenesis defect (Pex11?-/-) can induce profound alterations in the cardiomyocyte┤s peroxisomal compartment and related gene expression, including the concomitant deregulation of specific PPARs. The possible impact of peroxisomal dysfunction in the heart is discussed and a model for the modulation of myocardial metabolism via a peroxisome/PPAR-loop is proposed. PMID:25608554
Failure of the peroxisome proliferator WY-14,643 to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment.
Cattley, R C; Smith-Oliver, T; Butterworth, B E; Popp, J A
Peroxisome proliferator hepatocarcinogens lack genotoxic activity in numerous in vitro assays using non-target cells which do not respond with peroxisome proliferation. Therefore, the effect of in vivo treatment with WY-14,643 on DNA repair was quantitated in rat hepatocytes, the target cell for carcinogenesis. Palmitoyl CoA oxidase and carnitine acetyltransferase activities in isolated hepatocytes were elevated by WY-14,643 (50 mg/kg/day by gavage for up to 5 consecutive days) and by WY-14,643 (0.1%) or di(2-ethyl-hexyl)phthalate (DEHP) (1.2%) feeding (for up to 28 days), indicating peroxisome proliferation had occurred. DNA repair as unscheduled DNA synthesis (UDS) was measured autoradiographically as net nuclear grains following thymidine incorporation in primary hepatocyte cultures. Treatment of rats with WY-14,643 (gavage or feeding) or DEHP (feeding) did not induce UDS. Addition of 2-acetylaminofluorene to replicate cultures demonstrated that WY-14,643 or DEHP treatment did not prevent repair response. Additional cultures were treated with H2O2 (0.8 mM H2O2 3x at 1-h intervals) to evaluate the ability of UDS to detect any repair which may be induced by peroxisomal metabolism. H2O2 did not induce UDS at this concentration, nor did it prevent 2-acetylaminofluorene-induced repair. UDS was, however, observed in a separate experiment using a higher concentration of H2O2. In summary, a highly carcinogenic peroxisome proliferator did not induce UDS in the target cell for carcinogenesis in spite of peroxisome proliferation following in vivo treatment. PMID:3383337
Peroxisomal Localization of Hypoxia-Inducible Factors and Hypoxia-Inducible Factor Regulatory Hydroxylases in Primary Rat Hepatocytes Exposed to Hypoxia-Reoxygenation
Khan, Zahida; Michalopoulos, George K.; Stolz, Donna Beer
Many signals involved in pathophysiology are controlled by hypoxia-inducible factors (HIFs), transcription factors that induce expression of hypoxia-responsive genes. HIFs are post-translationally regulated by a family of O2-dependent HIF hydroxylases: four prolyl 4-hydroxylases and an asparaginyl hydroxylase. Most of these enzymes are abundant in resting liver, which is itself unique because of its physiological O2 gradient, and they can exist in both nuclear and cytoplasmic pools. In this study, we analyzed the cellular localization of endogenous HIFs and their regulatory hydroxylases in primary rat hepatocytes cultured under hypoxia-reoxygenation conditions. In hepatocytes, hypoxia targeted HIF-1? to the peroxisome, rather than the nucleus, where it co-localized with von Hippel-Lindau tumor suppressor protein and the HIF hydroxylases. Confocal immunofluorescence microscopy demonstrated that the HIF hydroxylases translocated from the nucleus to the cytoplasm in response to hypoxia, with increased accumulation in peroxisomes on reoxygenation. These results were confirmed via immunotransmission electron microscopy and Western blotting. Surprisingly, in resting liver tissue, perivenous localization of the HIF hydroxylases was observed, consistent with areas of low pO2. In conclusion, these studies establish the peroxisome as a highly relevant site of subcellular localization and function for the endogenous HIF pathway in hepatocytes. PMID:17003483
Combination therapy of an intestine-specific inhibitor of microsomal triglyceride transfer protein and peroxisome proliferator-activated receptor ? agonist in diabetic rat.
Sakata, Shohei; Mera, Yasuko; Kuroki, Yukiharu; Nashida, Reiko; Kakutani, Makoto; Ohta, Takeshi
We investigated effects on glucose and lipid metabolism in combination of JTT-130, a novel intestine-specific microsomal triglyceride transfer protein (MTP) inhibitor, and pioglitazone, peroxisome proliferator-activated receptor (PPAR) ? agonist. Male Zucker diabetic fatty rats were divided into 4 groups: control group, JTT-130 treatment group, pioglitazone treatment group, and combination group. The Zucker diabetic fatty rats were fed a regular powdered diet with JTT-130 and/or pioglitazone as a food admixture for 6 weeks. Effects on glucose and lipid metabolism were compared mainly between JTT-130 treatment group and combination group. JTT-130 treatment showed good glycemic control, while the plasma glucose and glycated hemoglobin levels in combination group were significantly decreased as compared with those JTT-130 treatment group. The reduction in the plasma triglyceride and free fatty acid levels in combination group was higher than that in JTT-130 treatment group, and glucose utilization was significantly elevated in adipose tissues. In Zucker diabetic fatty rats, combination treatment of JTT-130 and pioglitazone showed better glycemic control and a strong hypolipidemic action with an enhancement of insulin sensitivity. Combination therapy of MTP inhibitor and PPAR ? agonist might be more useful in the treatment of type 2 diabetes accompanied with obesity and insulin resistance. PMID:24772450
Redox regulated peroxisome homeostasis.
Wang, Xiaofeng; Li, Shuo; Liu, Yu; Ma, Changle
Peroxisomes are ubiquitous organelles present in nearly all eukaryotic cells. Conserved functions of peroxisomes encompass beta-oxidation of fatty acids and scavenging of reactive oxygen species generated from diverse peroxisomal metabolic pathways. Peroxisome content, number, and size can change quickly in response to environmental and/or developmental cues. To achieve efficient peroxisome homeostasis, peroxisome biogenesis and degradation must be orchestrated. We review the current knowledge on redox regulated peroxisome biogenesis and degradation with an emphasis on yeasts and plants. PMID:25545794
Neurotoxicity induced by antineoplastic proteasome inhibitors.
Alķ, Albert; Bruna, Jordi; Navarro, Xavier; Udina, Esther
In the last ten years, the proteasome has become one of the most attractive targets for the treatment of several cancer malignancies. Like other types of antineoplastic agents, proteasome inhibitors cause toxic peripheral neuropathy, which indeed is one of the limiting side effects of these treatments, and which thus curtails its potential effectiveness. Bortezomib was the first proteasome inhibitor approved for clinical use and is currently the first line treatment for multiple myeloma. The incidence of neuropathy induced by bortezomib is around 30-60%. Although the neurotoxic mechanisms are not completely understood, experimental studies suggest that aggresome formation, endoplasmic reticulum stress, mitotoxicity, inflammatory response, and DNA damage could contribute to this neurotoxicity. Additionally, the second generation of proteasome inhibitors, headed by carfilzomib, is currently being developed in order to reduce the toxic profile, with promising results. However, more extensive clinical experience and further experimental research are needed in order to determine the potential benefits of the second generation over bortezomib. The present review summarizes the main clinical features and mechanistic events related to the neuropathy induced by proteasome-inhibitors. PMID:24525285
Fenofibrate, a peroxisome proliferator-activated receptor ?-agonist, blocks lipopolysaccharide-induced inflammatory pathways in mouse liver
Backgrounds/Aims During the acute phase response, cytokines induce marked alterations in lipid metabolism including an increase in serum triglyceride levels and a decrease in hepatic fatty acid oxidation, in bile acid synthesis, and in high-density lipoprotein levels. Methods Peroxisome proliferator-activated receptors (PPARs: PPAR?, ?/?, and ?) regulate fatty acid metabolism, glucose homeostasis, cell proliferation, differentiation and inflammation. Proinflammatory profiles including tumor necrosis factor ? (TNF-?), interleukin-1? (IL-1?), and interleukin-6 (IL-6) are the important pathological factors in inflammatory responses during the pathological progression of the acute phase response. Lipopolysaccarides (LPS) induced the expression of TNF-?, IL-1?, and IL-6. LPS-induced inflammation decrease the expression of peroxisome proliferator-activated receptor ? (PPAR?), PPAR?/?, PPAR?, and coactivators PPAR? co-activator 1 ? (PGC-1?), PGC-1? messenger RNA (mRNA) in the liver of Balb/c mouse. In addition, LPS-induced inflammation diminishes the protein level of PPAR?, PPAR?/?, and PPAR?. Proinflammatory cytokines including TNF?, IL-1?, and IL-6 are the principal reducer of PPARs. However, the knockout mouse model against TNF? and IL-6 does not block decrease of PPARs in serum and liver. The mice were pretreated with fenofibrate at 100 mg/kg for 2 days. Results These treatment protocols increased the amount of PPARs mRNA in the liver. Fenofibrate inhibited LPS-induced TNF-?, IL-1?, and IL-6 production in the serum and liver. Similar results were obtained when human hepatoma HepG2 cells exposed to LPS were co-incubated with fenofibrate. LPS-treated HepG2 cells decreased expression of I?B. Moreover, activation of PPARs abrogated LPS-induced degradation of I?B, thus suppressing LPS-induced NF-?B activities. Conclusions Therefore, fenofibrate decreases the expression and secretion of TNF-?, IL-1?, and IL-6 via the NF-?B signaling pathway, thus serving as therapeutic targets to attenuate inflammation that is involved in hepatic pathological progression.
GSK-3 inhibitors induce chromosome instability
Tighe, Anthony; Ray-Sinha, Arpita; Staples, Oliver D; Taylor, Stephen S
Background Several mechanisms operate during mitosis to ensure accurate chromosome segregation. However, during tumour evolution these mechanisms go awry resulting in chromosome instability. While several lines of evidence suggest that mutations in adenomatous polyposis coli (APC) may promote chromosome instability, at least in colon cancer, the underlying mechanisms remain unclear. Here, we turn our attention to GSK-3 ¢ a protein kinase, which in concert with APC, targets ?-catenin for proteolysis ¢ and ask whether GSK-3 is required for accurate chromosome segregation. Results To probe the role of GSK-3 in mitosis, we inhibited GSK-3 kinase activity in cells using a panel of small molecule inhibitors, including SB-415286, AR-A014418, 1-Azakenpaullone and CHIR99021. Analysis of synchronised HeLa cells shows that GSK-3 inhibitors do not prevent G1/S progression or cell division. They do, however, significantly delay mitotic exit, largely because inhibitor-treated cells have difficulty aligning all their chromosomes. Although bipolar spindles form and the majority of chromosomes biorient, one or more chromosomes often remain mono-oriented near the spindle poles. Despite a prolonged mitotic delay, anaphase frequently initiates without the last chromosome aligning, resulting in chromosome non-disjunction. To rule out the possibility of "off-target" effects, we also used RNA interference to selectively repress GSK-3?. Cells deficient for GSK-3? exhibit a similar chromosome alignment defect, with chromosomes clustered near the spindle poles. GSK-3? repression also results in cells accumulating micronuclei, a hallmark of chromosome missegregation. Conclusion Thus, not only do our observations indicate a role for GSK-3 in accurate chromosome segregation, but they also raise the possibility that, if used as therapeutic agents, GSK-3 inhibitors may induce unwanted side effects by inducing chromosome instability. PMID:17697341
Subchronic exposure to high-dose ACE-inhibitor moexipril induces catalase activity in rat liver.
Adeghate, E; Hasan, M Y; Ponery, A S; Nurulain, S M; Petroianu, G A
The long-term clinical effects of ACE-inhibitors have similarities with those of both fibrates and glitazones, activators of peroxisome proliferator activator receptor (PPAR) alpha and gamma, respectively. The antioxidant enzyme catalase, a heme protein that degrades hydrogen peroxide, is found at high concentrations in peroxisomes. Catalase activity is one of the recognized surrogate markers indicative of PPAR activation in the rat liver. The purpose of the study was to establish the effect of moexipril on catalase activity and to compare it with the effect of both saline controls and that of the known PPAR agonist clofibrate (positive control). Three groups of seven rats were used. All substances were applied i.p. daily for 5 days, followed by a 2-day break. The cycle was repeated eight times. After the final cycle (day 56) the animals were sacrificed and liver tissue collected. The number of catalase positive cells in both moexipril group (95% CI 57-61) and clofibrate group (95% CI 72-80) is higher than in controls (95% CI 3-16) (p < or = 0.01). The number of catalase positive cells in the clofibrate group is higher than in the moexipril group (p < or = 0.01). High-dose subchronic exposure to the ACE-inhibitor moexipril induces catalase activity in the rat liver to an extent comparable to fibrates. We suggest that some of the long-term advantages of ACE inhibitor use - beyond mere BP lowering - might be due to a PPAR mediated effect. PMID:16311918
Hepatic sirtuin 1 is dispensable for fibrate-induced peroxisome proliferator-activated receptor-? function in vivo
Bonzo, Jessica A.; Brocker, Chad; Jiang, Changtao; Wang, Rui-Hong; Deng, Chu-Xia
Peroxisome proliferator-activated receptor-? (PPAR?) mediates metabolic remodeling, resulting in enhanced mitochondrial and peroxisomal ?-oxidation of fatty acids. In addition to the physiological stimuli of fasting and high-fat diet, PPAR? is activated by the fibrate class of drugs for the treatment of dyslipidemia. Sirtuin 1 (SIRT1), an important regulator of energy homeostasis, was downregulated in fibrate-treated wild-type mice, suggesting PPAR? regulation of Sirt1 gene expression. The impact of SIRT1 loss on PPAR? functionality in vivo was assessed in hepatocyte-specific knockout mice that lack the deacetylase domain of SIRT1 (Sirt1?Liv). Knockout mice were treated with fibrates or fasted for 24 h to activate PPAR?. Basal expression of the PPAR? target genes Cyp4a10 and Cyp4a14 was reduced in Sirt1?Liv mice compared with wild-type mice. However, no difference was observed between wild-type and Sirt1?Liv mice in either fasting- or fibrate-mediated induction of PPAR? target genes. Similar to the initial results, there was no difference in fibrate-activated PPAR? gene induction. To assess the relationship between SIRT1 and PPAR? in a pathophysiological setting, Sirt1?Liv mice were maintained on a high-fat diet for 14 wk, followed by fibrate treatment. Sirt1?Liv mice exhibited increased body mass compared with control mice. In the context of a high-fat diet, Sirt1?Liv mice did not respond to the cholesterol-lowering effects of the fibrate treatment. However, there were no significant differences in PPAR? target gene expression. These results suggest that, in vivo, SIRT1 deacetylase activity does not significantly impact induced PPAR? activity. PMID:24496310
Antioxidant cytoprotection by peroxisomal peroxiredoxin-5.
Walbrecq, Geoffroy; Wang, Bo; Becker, Sarah; Hannotiau, Amandine; Fransen, Marc; Knoops, Bernard
Peroxiredoxin-5 (PRDX5) is a thioredoxin peroxidase that reduces hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This enzyme is present in the cytosol, mitochondria, peroxisomes, and nucleus in human cells. Antioxidant cytoprotective functions have been previously documented for cytosolic, mitochondrial, and nuclear mammalian PRDX5. However, the exact function of PRDX5 in peroxisomes is still not clear. The aim of this work was to determine the function of peroxisomal PRDX5 in mammalian cells and, more specifically, in glial cells. To study the role of PRDX5 in peroxisomes, the endogenous expression of PRDX5 in murine oligodendrocyte 158N cells was silenced by RNA interference. In addition, human PRDX5 was also overexpressed in peroxisomes using a vector coding for human PRDX5, whose unconventional peroxisomal targeting sequence 1 (PTS1; SQL) was replaced by the prototypical PTS1 SKL. Stable 158N clones were obtained. The antioxidant cytoprotective function of peroxisomal PRDX5 against peroxisomal and mitochondrial KillerRed-mediated reactive oxygen species production as well as H2O2 was examined using MTT viability assays, roGFP2, and C11-BOBIPY probes. Altogether our results show that peroxisomal PRDX5 protects 158N oligodendrocytes against peroxisomal and mitochondrial KillerRed- and H2O2-induced oxidative stress. PMID:25772011
4-Hydroxydocosahexaenoic acid, a potent peroxisome proliferator-activated receptor {gamma} agonist alleviates the symptoms of DSS-induced colitis
Yamamoto, Keiko [Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543 (Japan); Ninomiya, Yuichi; Iseki, Mioko [Division of Translational Research, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241 (Japan); Nakachi, Yutaka; Kanesaki-Yatsuka, Yukiko [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241 (Japan); Yamanoue, Yu; Itoh, Toshimasa [Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 (Japan); Nishii, Yasuho [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241 (Japan); Petrovsky, Nikolai [Diabetes and Endocrinology, Flinders Medical Centre, Bedford Park, SA 5042 (Australia); Okazaki, Yasushi [Division of Translational Research, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241 (Japan); Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241 (Japan)], E-mail: email@example.com
(5E,7Z,10Z,13Z,16Z,19Z)-4-Hydroxy-5,7,10,13,16,19-docosahexaenoic acid (4-OHDHA) is a potential agonist of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and antidiabetic agent as has been previously reported. As PPAR{gamma} agonists may also have anti-inflammatory functions, in this study, we investigated whether 4-OHDHA has an inhibitory effect on expression of inflammatory genes in vitro and whether 4-OHDHA could relieve the symptoms of dextran sodium sulfate (DSS)-induced colitis in a murine model of inflammatory bowel disease. 4-OHDHA inhibited production of nitric oxide and expression of a subset of inflammatory genes including inducible nitric oxide synthase (Nos2/iNOS) and interleukin 6 (Il6) by lipopolysaccharide (LPS)-activated macrophages. In addition, 4-OHDHA-treated mice when compared to control mice not receiving treatment recovered better from the weight loss caused by DSS-induced colitis. Changes in disease activity index (DAI) of 4-OHDHA-treated mice were also more favorable than for control mice and were comparable with mice treated with a typical anti-inflammatory-drug, 5-aminosalichylic acid (5-ASA). These results suggest that 4-OHDHA has potentially clinically useful anti-inflammatory effects mediated by suppression of inflammatory gene expression.
Peroxisome Proliferator Activator Receptor (PPAR)-? Ligand, but Not PPAR-?, Ameliorates Cyclophosphamide-Induced Oxidative Stress and Inflammation in Rat Liver
El-Sheikh, Azza A. K.; Rifaai, Rehab A.
Hepatoprotective potential of peroxisome proliferator activator receptor (PPAR)-? and -? agonists, fenofibrate (FEN), and pioglitazone (PIO), respectively, against cyclophosphamide (CP)-induced toxicity has been investigated in rat. FEN and PIO (150 and 10?mg/kg/day, resp.) were given orally for 4 weeks. In separate groups, CP (150?mg/kg, i.p.) was injected as a single dose 5 days before the end of experiment, with or without either PPAR agonist. CP induced hepatotoxicity, as it caused histopathological alterations, with increased serum alanine and aspartate transaminases, total bilirubin, albumin, alkaline phosphatase and lactate dehydrogenase. CP caused hepatic oxidative stress, indicated by decrease in tissue reduced glutathione, with increase in malondialdehyde and nitric oxide levels. CP also caused decrease in hepatic antioxidant enzyme levels, including catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferase. Furthermore, CP increased serum and hepatic levels of the inflammatory marker tumor necrosis factor (TNF)-?, evaluated using ELISA. Preadministration of PIO, but not FEN, prior to CP challenge improved hepatic function and histology, and significantly reversed oxidative and inflammatory parameters. In conclusion, activation of PPAR-?, but not PPAR-?, conferred protection against CP-induced hepatotoxicity, via activation of antioxidant and anti-inflammatory mechanisms, and may serve as supplement during CP chemotherapy. PMID:24803924
The Peroxisomal Proliferator-Activated Receptor (PPAR) ? Agonist, Fenofibrate, Prevents Fractionated Whole-Brain Irradiation-Induced Cognitive Impairment
Greene-Schloesser, Dana; Payne, Valerie; Peiffer, Ann M.; Hsu, Fang-Chi; Riddle, David R.; Zhao, Weiling; Chan, Michael D.; Metheny-Barlow, Linda; Robbins, Mike E.
We hypothesized that dietary administration of the peroxisomal proliferator-activated receptor ? agonist, fenofibrate, to young adult male rats would prevent the fractionated whole-brain irradiation (fWBI)-induced reduction in cognitive function and neurogenesis and prevent the fWBI-induced increase in the total number of activated microglia. Eighty 12¢14-week-old young adult male Fischer 344 ū Brown Norway rats received either: (1) sham irradiation, (2) 40 Gy of fWBI delivered as two 5 Gy fractions/week for 4 weeks, (3) sham irradiation + dietary fenofibrate (0.2% w/w) starting 7 days prior to irradiation, or (4) fWBI + fenofibrate. Cognitive function was measured 26¢29 weeks after irradiation using: (1) the perirhinal cortex (PRh)-dependent novel object recognition task; (2) the hippocampal-dependent standard Morris water maze (MWM) task; (3) the hippocampal-dependent delayed match-to-place version of the MWM task; and (4) a cue strategy preference version of the MWM to distinguish hippocampal from striatal task performance. Neurogenesis was assessed 29 weeks after fWBI in the granular cell layer and subgranular zone of the dentate gyrus using a doublecortin antibody. Microglial activation was assessed using an ED1 antibody in the dentate gyrus and hilus of the hippocampus. A significant impairment in perirhinal cortex-dependent cognitive function was measured after fWBI. In contrast, fWBI failed to alter hippocampal-dependent cognitive function, despite a significant reduction in hippocampal neurogenesis. Continuous administration of fenofibrate prevented the fWBI-induced reduction in perirhinal cortex-dependent cognitive function, but did not prevent the radiation-induced reduction in neurogenesis or the radiation-induced increase in activated microglia. These data suggest that fenofibrate may be a promising therapeutic for the prevention of some modalities of radiation-induced cognitive impairment in brain cancer patients. PMID:24397438
Peroxisomes and Hepatotoxicity
N. Latruffe; C. Pacot; P. Passilly; M. Petit; O. Bardot; F. Caira; M. Cherkaoui Malki; B. Jannin; M. C. Clemencet; P. Deslex
Peroxisomes are ubiquitous organelles of eukaryotic cells and are present in significant amounts in hepatic liver cells. Peroxisomal enzymes contribute to several metabolic pathways including fatty acid, purine and amino acid catabolism or bile acid synthesis. The peroxisomal oxidative reactions produce hydrogen peroxide, mostly degraded by catalase which prevents oxidative stress. Moreover, peroxisomes are involved in arylderivative drug detoxification through
Activation of cerebral peroxisome proliferator-activated receptors gamma exerts neuroprotection by inhibiting oxidative stress following pilocarpine-induced status epilepticus
Xin Yu; Xiao-Guang Shao; Hong Sun; Yong-Nan Li; Jun Yang; Yan-Chun Deng; Yuan-Gui Huang
Status epilepticus (SE) can cause severe neuronal loss and oxidative damage. As peroxisome proliferator-activated receptor gamma (PPAR?) agonists possess antioxidative activity, we hypothesize that rosiglitazone, a PPAR? agonist, might protect the central nervous system (CNS) from oxidative damage in epileptic rats. Using a lithium-pilocarpine-induced SE model, we found that rosiglitazone significantly reduced hippocampal neuronal loss 1Āweek after SE, potently suppressed
Peroxisome Proliferator-activated Receptor ?/? Induces Myogenesis by Modulating Myostatin Activity*
Bonala, Sabeera; Lokireddy, Sudarsanareddy; Arigela, Harikumar; Teng, Serena; Wahli, Walter; Sharma, Mridula; McFarlane, Craig; Kambadur, Ravi
Classically, peroxisome proliferator-activated receptor ?/? (PPAR?/?) function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPAR?/? during skeletal muscle growth and regeneration. Although PPAR?/? has been implicated in regulating myogenesis, little is presently known about the role and, for that matter, the mechanism(s) of action of PPAR?/? in regulating postnatal myogenesis. Here we report for the first time, using a PPAR?/?-specific ligand (L165041) and the PPAR?/?-null mouse model, that PPAR?/? enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (growth and differentiation factor-associated serum protein-1) as a novel downstream target of PPAR?/? in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPAR?/?-null mice muscle tissue. We further report that a functional PPAR-responsive element within the 1.5-kb proximal Gasp-1 promoter region is critical for PPAR?/? regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of myostatin; consistent with this, we found that enhanced secretion of Gasp-1, increased Gasp-1 myostatin interaction and significantly reduced myostatin activity upon L165041-mediated activation of PPAR?/?. Moreover, we analyzed the ability of hGASP-1 to regulate myogenesis independently of PPAR?/? activation. The results revealed that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data revealed that PPAR?/? is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating myostatin activity via a mechanism involving Gasp-1. PMID:22362769
Identification of small molecule inhibitors of neurite loss induced by A? peptide using high content screening.
Ofengeim, Dimitry; Shi, Peng; Miao, Benchun; Fan, Jing; Xia, Xiaofeng; Fan, Yubo; Lipinski, Marta M; Hashimoto, Tadafumi; Polydoro, Manuela; Yuan, Junying; Wong, Stephen T C; Degterev, Alexei
Multiple lines of evidence indicate a strong relationship between ?? peptide-induced neurite degeneration and the progressive loss of cognitive functions in Alzheimer disease (AD) patients and in AD animal models. This prompted us to develop a high content screening assay (HCS) and Neurite Image Quantitator (NeuriteIQ) software to quantify the loss of neuronal projections induced by A? peptide neurons and enable us to identify new classes of neurite-protective small molecules, which may represent new leads for AD drug discovery. We identified thirty-six inhibitors of A?-induced neurite loss in the 1,040-compound National Institute of Neurological Disorders and Stroke (NINDS) custom collection of known bioactives and FDA approved drugs. Activity clustering showed that non-steroidal anti-inflammatory drugs (NSAIDs) were significantly enriched among the hits. Notably, NSAIDs have previously attracted significant attention as potential drugs for AD; however their mechanism of action remains controversial. Our data revealed that cyclooxygenase-2 (COX-2) expression was increased following A? treatment. Furthermore, multiple distinct classes of COX inhibitors efficiently blocked neurite loss in primary neurons, suggesting that increased COX activity contributes to A? peptide-induced neurite loss. Finally, we discovered that the detrimental effect of COX activity on neurite integrity may be mediated through the inhibition of peroxisome proliferator-activated receptor ? (PPAR?) activity. Overall, our work establishes the feasibility of identifying small molecule inhibitors of A?-induced neurite loss using the NeuriteIQ pipeline and provides novel insights into the mechanisms of neuroprotection by NSAIDs. PMID:22277654
Activators of Peroxisome Proliferator-Activated Receptors Protect Human Skin from Ultraviolet-B-Light-Induced Inflammation
Stefan Kippenberger; Stefan Marcel Loitsch; Marcella Grundmann-Kollmann; Stephanie Simon; Tu-Anh Dang; Katja Hardt-Weinelt; Roland Kaufmann; August Bernd
Peroxisome proliferator-activated receptors (PPAR) are members of a nuclear receptor superfamily, which were initially described in the context of fatty acid degradation and adipocyte differentiation. In this study we tested the hypothesis that peroxisome proliferator-activated receptor activation also controls inflammation. In an in vitro model with human keratinocytes inflammation was mimicked by irradiation with ultraviolet B light (150 mJ per
Proton pump inhibitor-induced hypomagnesemic hypoparathyroidism
Swaminathan, Krishnan
Proton pump inhibitors are the one of the most widely used drugs in the world. Hypomagnesemic hypoparathyroidism has been reported with different proton pump inhibitors with prolonged oral use. We report the first reported case of possible such effect with intravenous preparation of proton pump inhibitor. This case report raises awareness among physicians worldwide of this often unknown association, as life-threatening cardiac and neuromuscular complications can arise with unrecognized hypocalcemia and hypomagnesemia with proton pump inhibitors. PMID:26069375
Peroxisome proliferator-activated receptor (PPAR) alpha activation and its consequences in humans
Rachel Hertz; Jacob Bar-Tana
Amphipathic carboxylates collectively defined as peroxisome proliferators (PP) induce in rodents a pleiotropic effect, mediated by the peroxisome proliferator-activated receptor ? (PPAR?). Treatment with PP results in rodents in hypolipidemia, peroxisome proliferation and liver hypertrophy and hyperplasia leading to non-genotoxic hepatocarcinogenesis. In contrast to rodents, the hypolipidemic effect exerted by PP in humans is not accompanied by peroxisome proliferation nor
Statins enhance peroxisome proliferator-activated receptor ? coactivator-1? activity to regulate energy metabolism
Wenxian Wang; Chi-Wai Wong
Peroxisome proliferator-activated receptor ? coactivator-1? (PGC-1?) serves as an inducible coactivator for a number of transcription\\u000a factors to control energy metabolism. Insulin signaling through Akt kinase has been demonstrated to phosphorylate PGC-1? at\\u000a serine 571 and downregulate its activity in the liver. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors\\u000a that reduce cholesterol synthesis in the liver. In this study, we
Arterioscler Thromb Vasc Biol . Author manuscript Peroxisome proliferator-activated receptor activation induces 11
Boyer, Edmond
-activated receptor activation induces 11 -hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages Giulia Chinetti-Gbaguidi 1 # , Mohamed Amine Bouhlel 1 # , Corinne Copin 1 , Christian Duhem 1 Objectives 11 -hydroxysteroid dehydrogenase type 1 (11 -HSD1) catalyses the intracellular reduction
Peroxisome Proliferator-Activated Receptor-/ Protects Against Chemically Induced Liver Toxicity in Mice
Omiecinski, Curtis
proliferator-activated receptor- / (PPAR / ) in skeletal muscle fatty acid catabolism and epithelial carcinogenesis have re- cently been described. Whereas PPAR / is expressed in liver, its function in this tissue is less clear. To determine the role of PPAR / in chemically induced liver toxicity, wild-type and PPAR
ABCD2 alters peroxisome proliferator-activated receptor ? signaling in vitro, but does not impair responses to fenofibrate therapy in a mouse model of diet-induced obesity.
Liu, Xiaoxi; Liu, Jingjing; Liang, Shuang; Schl³ter, Agatha; Fourcade, Stephane; Aslibekyan, Stella; Pujol, Aurora; Graf, Gregory A
Fenofibrate is a peroxisome proliferator-activated receptor (PPAR) ? ligand that has been widely used as a lipid-lowering agent in the treatment of hypertriglyceridemia. ABCD2 (D2) is a peroxisomal long-chain acyl-CoA transporter that is highly induced by fenofibrate in the livers of mice. To determine whether D2 is a modifier of fibrate responses, wild-type and D2-deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPAR? signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knockdown of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of diet-induced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes. PMID:25123288
Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops
Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554- 564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment. PMID:2544605
Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into isolated peroxisomes.
Thieringer, R; Shio, H; Han, Y S; Cohen, G; Lazarow, P B
To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. It was improved by (i) using cells that were shifted to oleic acid medium after growth to stationary phase in glucose precultures, (ii) shifting the pH from 7.2 to 6.0 during cell fractionation, and (iii) carrying out equilibrium density centrifugation with Nycodenz containing 0.25 M sucrose throughout the gradient. A concentrated peroxisomal fraction was used for in vitro import of catalase A. After 2 h of incubation, 62% of the catalase was associated with, and 16% was imported into, the organelle in a protease-resistant fashion. We introduced immunofluorescence microscopy for S. cerevisiae peroxisomes, using antibodies against thiolase, which allowed us to identify even the extremely small organelles in glucose-grown cells. Peroxisomes from media containing oleic acid were larger in size, were greater in number, and had a more intense fluorescence signal. The peroxisomes were located, sometimes in clusters, in the cell periphery, often immediately adjacent to the plasma membrane. Systematic immunofluorescence observations of glucose-grown S. cerevisiae demonstrated that all such cells contained at least one and usually several very small peroxisomes despite the glucose repression. This finding fits a central prediction of our model of peroxisome biogenesis: peroxisomes form by division of preexisting peroxisomes; therefore, every cell must have at least one peroxisome if additional organelles are to be induced in that cell. Images PMID:1986244
Myotonic Response Induced by Inhibitors of Cholesterol Biosynthesis
Nathaniel Winer; David M. Klachko; Robert D. Baer; Paul L. Langley; Thomas W. Burns
Steroid inhibitors of cholesterogenesis containing nitrogen-substituted side chains induced electromyographic myotonia in rats. Cholesterol reduction or desmosterol accumulation, per se, did not cause myotonia, and cholestrol feeding prevented drug-induced myotonia. Desmosterol accumulation in combination with a specific drug effect may cause the observed myotonia.
Myotonic response induced by inhibitors of cholesterol biosynthesis.
Winer, N; Klachko, D M; Baer, R D; Langley, P L; Burns, T W
Steroid inhibitors of cholesterogenesis containing nitrogen-substituted side chains induced electromyographic myotonia in rats. Cholesterol reduction or desmosterol accumulation, per se, did not cause myotonia, and cholestrol feeding prevented drug-induced myotonia. Desmosterol accumulation in combination with a specific drug effect may cause the observed myotonia. PMID:17780007
Peroxisome proliferator-activated receptor-? activator fenofibrate prevents high-fat diet-induced renal lipotoxicity in spontaneously hypertensive rats
Seok Joon Shin; Ji Hee Lim; Sungjin Chung; Dong-Ye Youn; Hyun Wha Chung; Hyung Wook Kim; Jeong-Hwa Lee; Yoon Sik Chang; Cheol Whee Park
We investigated the effects of a high-fat (HF) diet and peroxisome proliferator-activated receptor (PPAR)-? activation on the intrarenal lipotoxicity associated with the renin¢angiotensin system (RAS) and oxidative stress using spontaneously hypertensive (SHR) rats. Male SHR and Wistar¢Kyoto (WKY) rats at 8 weeks of age were fed either a normal-fat diet or an HF diet without or with fenofibrate treatment for
Peroxisomal disorders in neurology
R. J. A. Wanders; H. S. A. Heymans; R. B. H. Schutgens; P. G. Barth; H. van den Bosch; J. M. Tager
Although peroxisomes were initially believed to play only a minor role in mammalian metabolism, it is now clear that they catalyse essential reactions in a number of different metabolic pathways and thus play an indispensable role in intermediary metabolism. The metabolic pathways in which peroxisomes are involved include the biosynthesis of ether phospholipids and bile acids, the oxidation of very
Epigenetic Activity of Peroxisome Proliferator-Activated Receptor Gamma Agonists Increases the Anticancer Effect of Histone Deacetylase Inhibitors on Multiple Myeloma Cells
Aouali, Nassera; Broukou, Angeliki; Bosseler, Manon; Keunen, Olivier; Schlesser, Vincent; Janji, Bassam; Palissot, Valerie; Stordeur, Philippe; Berchem, Guy
Epigenetic modifications play a major role in the development of multiple myeloma. We have previously reported that the PPAR? agonist pioglitazone (PIO) enhances, in-vitro, the cytotoxic effect of the Histone deacetylase inhibitor (HDACi), valproic acid (VPA), on multiple myeloma cells. Here, we described the development of a new multiple myeloma mouse model using MOLP8 cells, in order to evaluate the effect of VPA/PIO combination on the progression of myeloma cells, by analyzing the proliferation of bone marrow plasma cells. We showed that VPA/PIO delays the progression of the disease and the invasion of myeloma cells in the bone marrow. Mechanistically, we demonstrated that VPA/PIO increases the cleavage of caspase 3 and PARP, and induces the acetylation of Histone 3 (H3). Furthermore, we provided evidence that PPAR? agonist is able to enhance the action of other HDACi such as Vorinostat or Mocetinostat. Using PPAR? antagonist or siPPAR?, we strongly suggest that, as described during adipogenesis, PIO behaves as an epigenetic regulator by improving the activity of HDACi. This study highlights the therapeutic benefit of PIO/VPA combination, compared to VPA treatment as a single-arm therapy on multiple myeloma and further highlights that such combination may constitute a new promising treatment strategy which should be supported by clinical trials. PMID:26091518
Corrosion inhibitors for chlorides induced corrosion in reinforced concrete structures
M. Ormellese; M. Berra; F. Bolzoni; T. Pastore
Reinforcements corrosion is the most important cause of premature failure on reinforced concrete structures. Phenomena promoting corrosion are the ingress of chlorides and the reaction of atmospheric CO2 with cement paste. Aim of this paper is the investigation on the effectiveness of three organic commercial inhibitors in preventing carbon steel chlorides induced corrosion in concrete, since there is not yet
Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) induces cell death through MAPK-dependent mechanism in osteoblastic cells
Kim, Sung Hun [Department of Orthopedic Surgery, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of); Yoo, Chong Il [Department of Orthopedic Surgery, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of); Medical Research Institute, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of); Kim, Hui Taek [Department of Orthopedic Surgery, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of); Medical Research Institute, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of); Park, Ji Yeon [Department of Physiology, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of); Kwon, Chae Hwa [Department of Physiology, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of); Keun Kim, Yong [Department of Physiology, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of) and Medical Research Institute, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of) and MRC for Ischemic Tissue Regeneration, College of Medicine, Pusan National University, Pusan, 602-739 (Korea, Republic of)]. E-mail: email@example.com
The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPAR{gamma} agonists in osteoblastic cells. Ciglitazone and troglitazone, PPAR{gamma} agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPAR{alpha} agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPAR{gamma} antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.
Peroxisome proliferator-activated receptor ?/? (PPAR?/?) protects against ceramide-induced cellular toxicity in rat brain astrocytes and neurons by activation of ceramide kinase.
Aleshin, Stepan; Reiser, Georg
Peroxisome proliferator-activated receptors (PPARs) are important members of the nuclear receptor superfamily. Ligands of these nuclear receptors (PPAR?, ?/? and ?) belong to a wide range of lipophilic substances. In spite of the proven neuroprotective efficacy of PPAR?/? in models of neurological diseases, the biology of PPAR?/? in the brain has been much less investigated than that of PPAR? and PPAR?. In the present study, we test the hypothesis that neuroprotection induced by PPAR?/? could rely on the regulation of ceramide metabolism. We found that preincubation of neural cells with the PPAR?/? agonist L-165041 exerts significant protection against ceramide-induced cell death. Most importantly, L-165041 protects against ceramide-induced cell death not only before the insult, but also after the onset of the insult. To identify the mechanism of protection, we show that L-165041 upregulates ceramide kinase (CerK) expression levels in neural cells. Consistent with that, we detected that pharmacological inhibition of CerK reduces the protective effects of L-165041. To further decipher the mechanism of protection, gene knockdown in astrocytes was studied. Knockdown of PPAR?/? and CerK in astrocytes was used to verify that the protective effects of L-165041 are CerK- and PPAR?/?-dependent. We demonstrate that in CerK- or PPAR?/?-knockdown astrocytes, addition of L-165041 has no protective effect. Thus, we conclude that PPAR?/? protects neural cells against ceramide-induced cell death via induction and activation of CerK. PMID:24513118
Jasmonic acid inducible aspartic proteinase inhibitors from potato.
Kreft, S; Ravnikar, M; Mesko, P; Pungercar, J; Umek, A; Kregar, I; Strukelj, B
A new cDNA clone coding for an aspartic proteinase inhibitor homologue was isolated from a potato tuber cDNA library. Southern blot analysis was used to study the structural diversity of the aspartic proteinase inhibitor gene family in several species of the Solanaceae. The existence of sequence-homologous genes was confirmed in the genomic DNA of different potato cultivars (Solanum tuberosum L. cv. Dķsirķe, Pentland Squire and Igor), tomato (Lycopersicon esculentum Mill.), aubergine (S. melongena L.) and a wild type of bittersweet (S. dulcamara L.). Northern blot hybridization of total RNA, isolated from leaves under non-stress conditions, of different solanaceous species and of potato tubers showed that the gene transcripts encoding aspartic proteinase inhibitors occur mainly in potato tubers. The presence of several cathepsin D inhibitor isoforms has been detected at the protein level. At least four isoforms were isolated by affinity chromatography on cathepsin D-Sepharose and characterized. Additionally, exogenous treatment of potato plantlets by jasmonic acid (JA) over a wide range of concentrations (0-100 microM) was performed in a stem node culture in vitro. We demonstrated that the expression of aspartic proteinase inhibitor mRNA was drastically induced in potato shoots at concentrations of 50-100 microM JA. PMID:9055446
The peroxisomal catalase gene in the methylotrophic yeast Pichia methanolica.
Nakagawa, Tomoyuki; Yoshida, Kyoko; Takeuchi, Akihito; Ito, Takashi; Fujimura, Shuki; Matsufuji, Yoshimi; Tomizuka, Noboru; Yurimoto, Hiroya; Sakai, Yasuyoshi; Hayakawa, Takashi
In this paper, we describe the CTA1 gene, which encodes a peroxisomal catalase in the methylotrophic yeast Pichia methanolica. The P. methanolica CTA1 gene (PmCTA1) comprises a 1,530-bp open reading frame corresponding to a protein of 510 amino acid residues, and its deduced amino acid sequence shows high similarity to those of Cta1ps from other methylotrophic yeasts (about 79%). Expression of PmCTA1 in a peroxisomal catalase-depleted (Cbcta1Delta) Candida boidinii strain restored the methylotrophic growth of the host strain, while the expression of PmCTA1-DeltaSRL, which lacks peroxisome targeting signal type 1, did not. In P. methanolica, expression of PmCTA1 was induced when cells were grown on peroxisome-inducing carbon sources, viz., methanol, oleate, and D-alanine. Taken together, these results indicate that PmCTA1 encodes a functional peroxisomal catalase in P. methanolica. PMID:20699560
Hypoxia-inducible Lipid Droplet-associated (HILPDA) Is a Novel Peroxisome Proliferator-activated Receptor (PPAR) Target Involved in Hepatic Triglyceride Secretion*
Mattijssen, Frits; Georgiadi, Anastasia; Andasarie, Tresty; Szalowska, Ewa; Zota, Annika; Krones-Herzig, Anja; Heier, Christoph; Ratman, Dariusz; De Bosscher, Karolien; Qi, Ling; Zechner, Rudolf; Herzig, Stephan; Kersten, Sander
Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a novel PPAR target gene and demonstrate its involvement in hepatic lipid metabolism. Microarray analysis revealed that Hilpda is one of the most highly induced genes by the PPAR? agonist Wy14643 in mouse precision cut liver slices. Induction of Hilpda mRNA by Wy14643 was confirmed in mouse and human hepatocytes. Oral dosing with Wy14643 similarly induced Hilpda mRNA levels in livers of wild-type mice but not Ppara?/? mice. Transactivation studies and chromatin immunoprecipitation showed that Hilpda is a direct PPAR? target gene via a conserved PPAR response element located 1200 base pairs upstream of the transcription start site. Hepatic overexpression of HILPDA in mice via adeno-associated virus led to a 4-fold increase in liver triglyceride storage, without any changes in key genes involved in de novo lipogenesis, ?-oxidation, or lipolysis. Moreover, intracellular lipase activity was not affected by HILPDA overexpression. Strikingly, HILPDA overexpression significantly impaired hepatic triglyceride secretion. Taken together, our data uncover HILPDA as a novel PPAR target that raises hepatic triglyceride storage via regulation of triglyceride secretion. PMID:24876382
Inhibition of rotavirus ECwt infection in ICR suckling mice by N-acetylcysteine, peroxisome proliferator-activated receptor gamma agonists and cyclooxygenase-2 inhibitors
Guerrero, Carlos Arturo; Pardo, Paula; Rodriguez, Victor; Guerrero, Rafael; Acosta, Orlando
Live attenuated vaccines have recently been introduced for preventing rotavirus disease in children. However, alternative strategies for prevention and treatment of rotavirus infection are needed mainly in developing countries where low vaccine coverage occurs. In the present work, N-acetylcysteine (NAC), ascorbic acid (AA), some nonsteroidal anti-inflammatory drugs (NSAIDs) and peroxisome proliferator-activated receptor gamma (PPAR?) agonists were tested for their ability to interfere with rotavirus ECwt infectivity as detected by the percentage of viral antigen-positive cells of small intestinal villi isolated from ECwt-infected ICR mice. Administration of 6 mg NAC/kg every 8 h for three days following the first diarrhoeal episode reduced viral infectivity by about 90%. Administration of AA, ibuprofen, diclofenac, pioglitazone or rosiglitazone decreased viral infectivity by about 55%, 90%, 35%, 32% and 25%, respectively. ECwt infection of mice increased expression of cyclooxygenase-2, ERp57, Hsc70, NF-?B, Hsp70, protein disulphide isomerase (PDI) and PPAR? in intestinal villus cells. NAC treatment of ECwt-infected mice reduced Hsc70 and PDI expression to levels similar to those observed in villi from uninfected control mice. The present results suggest that the drugs tested in the present work could be assayed in preventing or treating rotaviral diarrhoea in children and young animals. PMID:24037197
A myosin inhibitor impairs auxin-induced cell division
Carola Holweg; Anne Honsel; Peter Nick
Summary. The role of myosins for auxin-induced cell division was probed using the inhibitor 2,3-butanedione monoxime in the tobacco cell line VBI-0, where cell elongation and division are axially aligned under the control of auxin. A morphometric analysis revealed that cell division is blocked in a dose-dependent manner, whereas cell expansion continued. In addition, the polarity of terminal cells was
Potentially lethal ACE-inhibitor-induced angioedema in a child
Bukhari, Esraa; Safdar, Osama Y; Shalaby, Mohammed; AlSharif, Shafiqa MJ; Alsufiany, Khoulod; Kari, Jameela A
Key Clinical Message We report a case of a 9-year-old female with known end-stage kidney disease who presented with sudden onset tongue swelling. A diagnosis of angiotensin-converting enzyme inhibitor-induced angioedema related to bradykinin accumulation was made. Her symptoms resolved shortly after discontinuation of captopril. Early diagnosis can save patients from severe upper airway obstruction. PMID:26185642
Dysregulation of Claudin-5 in HIV-induced Interstitial Pneumonitis and Lung Vascular Injury. Protective Role of Peroxisome Proliferator¢activated Receptor-?
Li, Hong; Singh, Sangya; Potula, Raghava; Persidsky, Yuri
Rationale: HIV-1¢induced interstitial pneumonitis (IP) is a serious complication of HIV-1 infection, characterized by inflammation and cellular infiltration in lungs, often leading to respiratory failure and death. The barrier function of the pulmonary endothelium is caused in part by tight junction (TJ) proteins, such as claudin-5. Peroxisome proliferator¢activated receptor (PPAR)-? is expressed in lung tissues and regulates inflammation. We hypothesize that HIV-1 induces vascular lung injury, and HIV-1¢mediated damage of the pulmonary endothelium and IP is associated with dysregulation of PPAR-?. Objectives: Investigate the effects of HIV-1 infection on the pulmonary microvasculature and the modulatory effects of the PPAR-? ligands. Methods: Using human lung tissues, we demonstrated down-regulation of claudin-5 (marker of pulmonary barrier integrity), down-regulation of PPAR-? transcription, and expression in lung tissues of HIV-1¢infected humans with IP. Measurements and Main Results: Human lung microvascular endothelial cells expressed the TJ proteins claudin-5, ZO-1, and ZO-2; HIV-1 decreased TJ proteins expression and induced nuclear factor-?B promoter activity, which was reversed by PPAR-? agonist. Using two murine HIV/AIDS models, we demonstrated decreased claudin-5 expression and increased macrophage infiltration in the lungs of HIV-1¢infected animals. Activation of PPAR-? prevented HIV-1¢induced claudin-5 down-regulation and significantly reduced viremia and pulmonary macrophage infiltration. Conclusions: HIV-induced IP is associated with injury to the lung vascular endothelium, with decreased TJ and PPAR-? expression, and increased pulmonary macrophage infiltration. PPAR-? ligands abrogated these effects. Thus, regulation of PPAR-? can be a therapeutic approach against HIV-1¢induced vascular damage and IP in infected humans. Removal of Expression of Concern: Issues leading to the previous expression of concern for this article have been resolved after further revisions and editorial review. No further concerns exist. PMID:22345580
Activation of peroxisome proliferator-activated receptor ? induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders.
Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J; Sims, Katherine B; Berry-Kravis, Elizabeth; Pahan, Kalipada
Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) ?, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPAR?, but not PPAR? and PPAR?, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor ?, PPAR?, and PGC1? on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174
Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells
Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung; Kim, Hye Jin; Yang, Jin Mo; Ryu, Somi; Noh, Yoo Hun; Lee, Soo Hyun; Son, Meong Hi; Jung, Hye Lim; Yoo, Keon Hee [Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)] [Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Koo, Hong Hoe, E-mail: firstname.lastname@example.com [Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Sung, Ki Woong, E-mail: firstname.lastname@example.com [Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)
Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.
Peroxisome proliferator-activated receptor {alpha}-independent peroxisome proliferation
Zhang Xiuguo [Department of Metabolic Regulation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan); Tanaka, Naoki [Department of Metabolic Regulation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan) and Department of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan)]. E-mail: email@example.com; Nakajima, Takero [Department of Metabolic Regulation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan); Kamijo, Yuji [Department of Metabolic Regulation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan); Department of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan); Gonzalez, Frank J. [Laboratory of Metabolism, National Cancer Institute, Bethesda, MD 20892 (United States); Aoyama, Toshifumi [Department of Metabolic Regulation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan)
Hepatic peroxisome proliferation, increases in the numerical and volume density of peroxisomes, is believed to be closely related to peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) activation; however, it remains unknown whether peroxisome proliferation depends absolutely on this activation. To verify occurrence of PPAR{alpha}-independent peroxisome proliferation, fenofibrate treatment was used, which was expected to significantly enhance PPAR{alpha} dependence in the assay system. Surprisingly, a novel type of PPAR{alpha}-independent peroxisome proliferation and enlargement was uncovered in PPAR{alpha}-null mice. The increased expression of dynamin-like protein 1, but not peroxisome biogenesis factor 11{alpha}, might be associated with the PPAR{alpha}-independent peroxisome proliferation at least in part.
Mycobacterium bovis bacillus Calmette-Guķrin infection induces TLR2-dependent peroxisome proliferator-activated receptor gamma expression and activation: functions in inflammation, lipid metabolism, and pathogenesis.
Almeida, PatrĒcia E; Silva, Adriana R; Maya-Monteiro, Clarissa M; T÷r÷csik, Dßniel; D'Avila, Heloisa; Dezs÷, Balßzs; MagalhŃes, Kelly G; Castro-Faria-Neto, Hugo C; Nagy, Laszlo; Bozza, PatrĒcia T
Macrophages have important roles in both lipid metabolism and inflammation and are central to immunity to intracellular pathogens. Foam-like, lipid-laden macrophages are present during the course of mycobacterial infection and have recently been implicated in mycobacterial pathogenesis. In this study, we analyzed the molecular mechanisms underlying the formation of macrophage lipid bodies (lipid droplets) during Mycobacterium bovis bacillus Calmette-Guķrin (BCG) infection, focusing on the role of the lipid-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). We found that BCG infection induced increased expression of PPARgamma that paralleled the augmented lipid body formation and PGE(2) synthesis in mouse peritoneal macrophages. BCG-induced PPARgamma expression and lipid body formation were diminished in macrophages from TLR2-deficient mice, suggesting a key role for TLR2. The function of PPARgamma in modulating BCG infection was demonstrated by the capacity of the PPARgamma agonist BRL49653 to potentiate lipid body formation and PGE(2) production; furthermore, pretreatment with the PPARgamma antagonist GW9662 inhibited BCG-induced lipid body formation and PGE(2) production. BCG-induced MIP-1alpha, IL12p70, TNF-alpha, and IL6 production was not inhibited by GW9662 treatment. Nonpathogenic Mycobacterium smegmatis failed to induce PPARgamma expression or lipid body formation. Moreover, inhibition of PPARgamma by GW9662 enhanced the mycobacterial killing capacity of macrophages. Our findings show that PPARgamma is involved in lipid body biogenesis, unravels a cross-talk between the innate immune receptor TLR2 and the lipid-activated nuclear receptor PPARgamma that coordinates lipid metabolism and inflammation in BCG-infected macrophages, thereby potentially affecting mycobacterial pathogenesis. PMID:19561094
Perfluorooctanoic acid induced-developmental cardiotoxicity: are peroxisome proliferator activated receptor ? (PPAR?) and bone morphorgenic protein 2 (BMP2) pathways involved?
Jiang, Qixiao; Lust, Robert M; DeWitt, Jamie C
Perfluorooctanoic acid (PFOA) is an environmental contaminant known to induce developmental toxicity in animal models through activation of the peroxisome proliferator-activated receptor ? (PPAR?). Previously, it was demonstrated that in ovo exposure to PFOA induced cardiotoxicity in chicken embryos and hatchlings. To investigate potential PPAR?-mediated mechanisms, fertile chicken eggs were injected prior to incubation with WY 14,643, a PPAR? agonist. Cardiac morphology and function were evaluated in late-stage embryos and hatchlings. Histologically, unlike PFOA, WY 14,643 did not induce thinning of the right ventricular wall. Via echocardiography, however, WY 14,643 induced effects similar to those of PFOA, including increased left ventricular wall thickness and mass, elevated heart rate, ejection fraction, fractional shortening, and decreased stroke volume. Additionally, to investigate mechanisms associated with early heart development, a separate group of fertile chicken eggs was injected prior to incubation with PFOA or WY 14,643 and in early-stage embryos, gene expression and protein concentration associated with the bone morphogenic protein (BMP2) pathway were determined. Although changes were not statistically consistent among doses, expression of BMP2, Nkx2.5, and GATA4 mRNA in early embryos was altered by PFOA exposure; however, protein concentrations of these targets were not markedly altered by either PFOA or WY 14,643. Protein levels of pSMAD1/5, a transcriptional regulator stimulated by BMPs, were altered by both PFOA and WY 14,643, but in different directions; PFOA reduced cytoplasmic pSMAD1/5, whereas WY 14,643 decreased nuclear pSMAD1/5. Taken together, these data suggest that developmental cardiotoxicity induced by PFOA likely involves both PPAR? and BMP2 pathways. PMID:23941634
Colorado potato beetles ( leptinotarsa decemlineata) adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate
Caroline J. Bolter; Maarten A. Jongsma
Potato plants were treated with gaseous methyl jasmonate (MJ) to obtain leaves with high induced levels of cysteine and aspartic proteinase inhibitors. Induced papain inhibitor activity was estimated at 4% of total protein. Other conditions produced leaves with low and moderate levels of this inhibitor. Development of Colorado potato beetle larvae was similar when they were reared on leaves containing
Induction of peroxisomes by butyrate-producing probiotics.
Weng, Huachun; Endo, Kosuke; Li, Jiawei; Kito, Naoko; Iwai, Naoharu
We previously found that peroxisomal biogenesis factor 11a (Pex11a) deficiency is associated with a reduction in peroxisome abundance and impaired fatty acid metabolism in hepatocytes, and results in steatosis. In the present study, we investigated whether butyrate induces Pex11a expression and peroxisome proliferation, and studied its effect on lipid metabolism. C57BL/6 mice fed standard chow or a high-fat diet (HFD) were treated with tributyrin, 4-phelybutyrate acid (4-PBA), or the butyrate-producing probiotics (Clostridium butyricum MIYAIRI 588 [CBM]) plus inulin (dietary fiber), and the body weight, white adipose tissue, serum triglycerides, mRNA expression, and peroxisome abundance were evaluated. Tributyrin or 4-PBA treatment significantly decreased body weight and increased hepatic mRNA expression of peroxisome proliferator-activated receptor-? (PPAR?) and Pex11a. In addition, 4-PBA treatment increased peroxisome abundance and the expression of genes involved in peroxisomal fatty acid ?-oxidation (acyl-coenzyme A oxidase 1 and hydroxysteroid [17-beta] dehydrogenase 4). CBM and inulin administration reduced adipose tissue mass and serum triglycerides, induced Pex11a, acyl-coenzyme A oxidase 1, and hydroxysteroid (17-beta) dehydrogenase 4 genes, and increased peroxisome abundance in mice fed standard chow or an HFD. In conclusion, elevation of butyrate availability (directly through administration of butyrate or indirectly via administration of butyrate-producing probiotics plus fiber) induces PPAR? and Pex11a and the genes involved in peroxisomal fatty acid ?-oxidation, increases peroxisome abundance, and improves lipid metabolism. These results may provide a new therapeutic strategy against hyperlipidemia and obesity. PMID:25659146
An aqueous extract of Salacia oblonga root, a herb-derived peroxisome proliferator-activated receptor-alpha activator, by oral gavage over 28 days induces gender-dependent hepatic hypertrophy in rats
Xianglu Rong; Moon Sun Kim; Ning Su; Suping Wen; Yukimi Matsuo; Johji Yamahara; Michael Murray; Yuhao Li
Activation of peroxisome proliferator-activated receptor (PPAR)-alpha by natural and synthetic chemicals induces hepatic hypertrophy. An aqueous extract of Salacia oblonga root (SOW) is an Ayurvedic medicine with anti-diabetic and anti-obesity properties. In the present study, it was found that SOW (100, 300 and 900mg\\/kg, once daily by oral gavage over a 28 day period) elicited dose-related increases in liver weight
Onychopathy induced by temsirolimus, a mammalian target of rapamycin inhibitor.
Peuvrel, L; Quķreux, G; Brocard, A; Saint-Jean, M; Drķno, B
Temsirolimus belongs to the mammalian target of rapamycin (mTOR) inhibitors, targeted therapies for which indications are booming in oncology. While their tolerance is usually good, mucocutaneous toxicity is the most common, including stomatitis, rashes, edemas, pruritus, dry skin and nail disorders. The latter are common in clinical practice but have not yet been well characterized. We report 2 cases of patients who developed, after 6-7 months with temsirolimus, a dystrophy of the 20 nails with fragility, distal onycholysis, yellow discoloration, associated in 1 case with painful paronychia. Topical steroids improved the paronychia, without changing the nail dystrophy. To our knowledge, the occurrence of yellow nail discoloration with temsirolimus has never been reported before. We review the cutaneous and mucosal toxicities induced by temsirolimus and everolimus, two mTOR inhibitors used as anticancer agents and by their parent molecule sirolimus. PMID:22614575
Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor ? and the retinoid X?receptor
Tontonoz, Peter; Singer, Samuel; Forman, Barry?M.; Sarraf, Pasha; Fletcher, Jonathan?A.; Fletcher, Christopher?D.?M.; Brun, Regina?P.; Mueller, Elisabetta; Altiok, Soner; Oppenheim, Heather; Evans, Ronald?M.; Spiegelman, Bruce?M.
Induction of terminal differentiation represents a promising therapeutic approach to certain human malignancies. The peroxisome proliferator-activated receptor ? (PPAR?) and the retinoid X receptor ? (RXR?) form a heterodimeric complex that functions as a central regulator of adipocyte differentiation. Natural and synthetic ligands for both receptors have been identified. We demonstrate here that PPAR? is expressed at high levels in each of the major histologic types of human liposarcoma. Moreover, primary human liposarcoma cells can be induced to undergo terminal differentiation by treatment with the PPAR? ligand pioglitazone, suggesting that the differentiation block in these cells can be overcome by maximal activation of the PPAR pathway. We further demonstrate that RXR-specific ligands are also potent adipogenic agents in cells expressing the PPAR?/RXR? heterodimer, and that simultaneous treatment of liposarcoma cells with both PPAR?- and RXR-specific ligands results in an additive stimulation of differentiation. Liposarcoma cell differentiation is characterized by accumulation of intracellular lipid, induction of adipocyte-specific genes, and withdrawal from the cell cycle. These results suggest that PPAR? ligands such as thiazolidinediones and RXR-specific retinoids may be useful therapeutic agents for the treatment of liposarcoma. PMID:8990192
Over-expression of C/EBP-{alpha} induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-{gamma}
Wang Xueqing; Huang Guangcun; Mei Shuang; Qian Jin; Ji Juling [Department of Pathology, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032 (China); Zhang Jinsheng [Department of Pathology, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032 (China)], E-mail: firstname.lastname@example.com
Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-{alpha} (C/EBP-{alpha}) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-{alpha} gene (Ad-C/EBP-{alpha}) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-{alpha} resulted in the up-regulation of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) and P53, while P53 expression was regulated by PPAR-{gamma}. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-{gamma} and p53 in the process of apoptosis triggered by C/EBP-{alpha} in HSCs.
Curcumin protects neurons against oxygen-glucose deprivation/reoxygenation-induced injury through activation of peroxisome proliferator-activated receptor-? function.
Liu, Zun-Jing; Liu, Hong-Qiang; Xiao, Cheng; Fan, Hui-Zhen; Huang, Qing; Liu, Yun-Hai; Wang, Yu
The turmeric derivative curcumin protects against cerebral ischemic injury. We previously demonstrated that curcumin activates peroxisome proliferator-activated receptor-? (PPAR?), a ligand-activated transcription factor involved in both neuroprotective and anti-inflammatory signaling pathways. This study tested whether the neuroprotective effects of curcumin against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury of rat cortical neurons are mediated (at least in part) by PPAR?. Curcumin (10 ?M) potently enhanced PPAR? expression and transcriptional activity following OGD/R. In addition, curcumin markedly increased neuronal viability, as evidenced by decreased lactate dehydrogenase release and reduced nitric oxide production, caspase-3 activity, and apoptosis. These protective effects were suppressed by coadministration of the PPAR? antagonist 2-chloro-5-nitrobenzanilide (GW9662) and by prior transfection of a small-interfering RNA (siRNA) targeting PPAR?, treatments that had no toxic effects on healthy neurons. Curcumin reduced OGD/R-induced accumulation of reactive oxygen species and inhibited the mitochondrial apoptosis pathway, as indicated by reduced release of cytochrome c and apoptosis-inducing factor and maintenance of both the mitochondrial membrane potential and the Bax/Bcl-2 ratio. Again, GW9662 or PPAR? siRNA transfection mitigated the protective effects of curcumin on mitochondrial function. Curcumin suppressed I?B kinase phosphorylation and I?B degradation, thereby inhibiting nuclear factor-? B (NF-?B) nuclear translocation, effects also blocked by GW9662 or PPAR? siRNA. Immunoprecipitation experiments revealed that PPAR? interacted with NF-?B p65 and inhibited NF-?B activation. The present study provides strong evidence that at least some of the neuroprotective effects of curcumin against OGD/R are mediated by PPAR? activation. PMID:24975470
Analysis of the Bioactivity of Magnetically Immunoisolated Peroxisomes
Wang, Yaohua; Taylor, Thane H.; Arriaga, Edgar A.
Peroxisomes produce reactive oxygen species (ROS), which may participate in biotransformations of innate biomolecules and xenobiotics. Isolating functional peroxisomes with low levels of contaminants would be a useful tool to investigate biotransformations occurring in these organelles that are usually confounded with biotransformations occurring in other co-isolated organelles. Here we immunoisolate peroxisomes, demonstrate that the impurity level after isolation is low and that peroxisomes retain their biological activity. In this method, an antibody targeting a 70 kDa peroxisomal membrane protein was immobilized to silanized magnetic iron oxide beads (1¢4 ?m in diameter) coated with Protein A. Peroxisomes from L6 rat myoblast homogenates were magnetically captured, washed and then analyzed for subcellular composition using enzymatic assays. Based on the ratio of peroxisomal to lysosomal activity, the retained fraction is 70-fold enriched relative to the unretained fraction. Similarly, the ratio of peroxisomal activity to mitochondrial content suggests that the retained fraction is >30-fold enriched relative to the unretained fraction. H2O2 production from the ?-oxidation of palmitoyl-CoA demonstrated that the isolated peroxisomal fraction was biologically active. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) analysis confirmed that the immunopurified fractions were capable of transforming the anti-cancer drug doxorubicin and the fatty acid analog, BODIPY 500/510 C1C12. Besides its use to investigate peroxisome biotransformations in health and disease, the combination of magnetic immunoisolation with CE-LIF could be widely applicable to investigate subcellular specific biotransformations of xenobiotics occurring at immunoisolated subcellular compartments. PMID:22065344
High-dose treatment with telmisartan induces monocytic peroxisome proliferator-activated receptor-? target genes in patients with the metabolic syndrome.
Bõhr, Ilse-Nirmala; Tretter, Patrizia; Kr³ger, Janine; Stark, Renee G; Schimkus, Julia; Unger, Thomas; Kappert, Kai; Scholze, J³rgen; Parhofer, Klaus G; Kintscher, Ulrich
The present study aimed to explore the anti-inflammatory effects and peroxisome proliferator-activated receptor-? (PPAR?)-activating properties of the angiotensin type 1 receptor blocker telmisartan by analysis of serum interleukin 6 levels and monocytic PPAR? target gene expression in drug-na've patients with the metabolic syndrome. This was a 14-week, randomized, double-blind, placebo-controlled 2-center study with telmisartan 80 mg/d and telmisartan 160 mg/d in 54 patients with the metabolic syndrome. In addition to clinical laboratory measurements, peripheral monocytes were extracted by negative isolation using a Dynal Monocyte kit to evaluate ligand-activated PPAR? target gene expression (CD36 and CD163) at baseline and study end using quantitative real-time RT-PCR. In this low-risk patient population, telmisartan (80 and 160 mg) treatment did not significantly affect serum interleukin 6 levels. Expression of the PPAR? target gene CD36 in monocytes was markedly induced by telmisartan from baseline to study end (telmisartan 80 mg: 2.3▒1.5-fold change versus placebo [P value not significant]; telmisartan 160 mg: 3.5▒0.9-fold change versus placebo [P<0.05]). The recently reported PPAR? target gene CD163 was slightly induced by telmisartan (telmisartan 80 mg: 1.1▒0.3-fold change versus placebo [P value not significant]; telmisartan 160 mg: 1.4▒0.4-fold change versus placebo [P value not significant]), which did not reach statistical significance. This is the first clinical description of monocytic PPAR? target gene regulation with high-dose telmisartan treatment. These data implicate that the angiotensin type 1 receptor blocker telmisartan activates PPAR? in circulating monocytes of patients with the metabolic syndrome. PMID:21876071
The analysis of modified peroxisome proliferator responsive elements of the peroxisomal bifunctional enzyme in transfected HepG2 cells reveals two regulatory motifs
Olivier Bardot; Marie Claude Clemencet; Patricia Passilly; Norbert Latruffe
Peroxisome proliferators (PPs) are non-genotoxic carcinogens in rodents. They can induce the expression of numerous genes via the heterodimerization of two members of the steroid hormone receptor superfamily, called the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR). Many of the PP responsive genes possess a peroxisome proliferator response element (PPRE) formed by two TGACCT-related motifs. The
Peroxisome Proliferator-Activated Receptor-g Ligands Reduce Neuronal Inducible Nitric Oxide Synthase Expression and Cell Death In Vivo
Michael T. Heneka; Thomas Klockgether; Douglas L. Feinstein
Expression of the inducible form of nitric oxide synthase (iNOS) in brain may contribute to neurotoxicity in Alzheimer's disease (AD). Expression of iNOS can be induced in cerebellar granule cells (CGCs) in vivo as well as in vitro, allowing these cells to be used to study regulation of neuronal iNOS expression. We report here that microinjection of bacterial lipopolysaccharide and
Modulation of Receptor Phosphorylation Contributes to Activation of Peroxisome Proliferator Activated Receptor ? by Dehydroepiandrosterone and Other Peroxisome Proliferators
Tamasi, Viola; Miller, Kristy K. Michael; Ripp, Sharon L.; Vila, Ermin; Geoghagen, Thomas E.; Prough, Russell A.
Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor ? (PPAR?) in vivo but does not ligand-activate PPAR? in transient transfection experiments. We demonstrate that DHEA regulates PPAR? action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPAR? and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPAR? mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPAR? mRNA and protein levels as well as increased PPAR? transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region. PMID:18079279
Peroxisomal cholesterol biosynthesis and Smith-Lemli-Opitz syndrome
Weinhofer, Isabelle [Center for Brain Research, Medical University of Vienna, Vienna (Austria); Kunze, Markus [Center for Brain Research, Medical University of Vienna, Vienna (Austria); Stangl, Herbert [Department of Medical Chemistry, Medical University of Vienna, Vienna (Austria); Porter, Forbes D. [National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD (United States); Berger, Johannes [Center for Brain Research, Medical University of Vienna, Vienna (Austria)]. E-mail: firstname.lastname@example.com
Smith-Lemli-Opitz syndrome (SLOS), caused by 7-dehydrocholesterol-reductase (DHCR7) deficiency, shows variable severity independent of DHCR7 genotype. To test whether peroxisomes are involved in alternative cholesterol synthesis, we used [1-{sup 14}C]C24:0 for peroxisomal {beta}-oxidation to generate [1-{sup 14}C]acetyl-CoA as cholesterol precursor inside peroxisomes. The HMG-CoA reductase inhibitor lovastatin suppressed cholesterol synthesis from [2-{sup 14}C]acetate and [1-{sup 14}C]C8:0 but not from [1-{sup 14}C]C24:0, implicating a peroxisomal, lovastatin-resistant HMG-CoA reductase. In SLOS fibroblasts lacking DHCR7 activity, no cholesterol was formed from [1-{sup 14}C]C24:0-derived [1-{sup 14}C]acetyl-CoA, indicating that the alternative peroxisomal pathway also requires this enzyme. Our results implicate peroxisomes in cholesterol biosynthesis but provide no link to phenotypic variation in SLOS.
Peroxisomal localization in the developing mouse cerebellum: implications for neuronal abnormalities related to deficiencies in peroxisomes
Tomoko Nagase; Nobuyuki Shimozawa; Yasuhiko Takemoto; Yasuyuki Suzuki; Masayuki Komori; Naomi Kondo
In subjects with Zellweger syndrome, the most severe phenotype of peroxisomal biogenesis disorder, brain abnormalities include cortical dysplasia, neuronal heterotopia, and dysmyelination. To clarify the relationship between the lack of peroxisomes and neuronal abnormalities, we investigated peroxisomal localization in the mouse cerebellum, using double immunofluorescent staining for peroxisomal proteins.On immunostaining for peroxisomal matrix protein, while there are few peroxisomes in
A Novel Peroxisome Proliferator-activated Receptor (PPAR)? Agonist and PPAR? Antagonist, Z-551, Ameliorates High-fat Diet-induced Obesity and Metabolic Disorders in Mice.
Shiomi, Yoshihiro; Yamauchi, Toshimasa; Iwabu, Masato; Okada-Iwabu, Miki; Nakayama, Ryo; Orikawa, Yuki; Yoshioka, Yoshichika; Tanaka, Koichiro; Ueki, Kohjiro; Kadowaki, Takashi
A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPAR? agonistic and PPAR? antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPAR?-deficient mice and those of a synthetic PPAR? antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPAR? agonistic and PPAR? antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia. PMID:25907553
PPAR- ? impairment alters peroxisome functionality in primary astrocyte cell cultures.
Di Cesare Mannelli, Lorenzo; Zanardelli, Matteo; Micheli, Laura; Ghelardini, Carla
Peroxisomes provide glial cells with protective functions against the harmful effects of H2O2 on neurons and peroxisome impairment results in nervous lesions. Agonists of the ? -subtype of the Peroxisome-Proliferator-Activated-Receptors (PPAR) have been proposed as neuroprotective agents in neurodegenerative disorders. Nevertheless, the role of PPAR- ? alterations in pathophysiological mechanisms and the relevance of peroxisome functions in the PPAR- ? effects are not yet clear. In a primary cell culture of rat astrocytes, the irreversible PPAR- ? antagonist GW9662 concentration-dependently decreased the activity of catalase, the most important antioxidant defense enzyme in peroxisomes. Catalase functionality recovered in a few days and the PPAR- ? agonist rosiglitazone promoted reversal of enzymatic damage. The reversible antagonist G3335 reduced both the activity and expression of catalase in a rosiglitazone-prevented manner. G3335 reduced also the glutathione reductase expression, indicating that enzyme involved in glutathione regeneration was compromised. Neither the PPAR- ? target gene Acyl-Coenzyme-A-oxidase-1 nor the mitochondrial detoxifying enzyme NADH:ubiquinone-oxidoreductase (NDFUS3) was altered by PPAR- ? inhibition. In conclusion, PPAR- ? inhibition induced impairment of catalase in astrocytes. A general decrease of the antioxidant defenses of the cell suggests that a PPAR- ? hypofunction could participate in neurodegenerative mechanisms through peroxisomal damage. This series of experiments could be a useful model for studying compounds able to restore peroxisome functionality. PMID:24729976
Dual targeting of peroxisomal proteins
Ast, Julia; Stiebler, Alina C.; Freitag, Johannes; B÷lker, Michael
Cellular compartmentalization into organelles serves to separate biological processes within the environment of a single cell. While some metabolic reactions are specific to a single organelle, others occur in more than one cellular compartment. Specific targeting of proteins to compartments inside of eukaryotic cells is mediated by defined sequence motifs. To achieve multiple targeting to different compartments cells use a variety of strategies. Here, we focus on mechanisms leading to dual targeting of peroxisomal proteins. In many instances, isoforms of peroxisomal proteins with distinct intracellular localization are encoded by separate genes. But also single genes can give rise to differentially localized proteins. Different isoforms can be generated by use of alternative transcriptional start sites, by differential splicing or ribosomal read-through of stop codons. In all these cases different peptide variants are produced, of which only one carries a peroxisomal targeting signal. Alternatively, peroxisomal proteins contain additional signals that compete for intracellular targeting. Dual localization of proteins residing in both the cytoplasm and in peroxisomes may also result from use of inefficient targeting signals. The recent observation that some bona fide cytoplasmic enzymes were also found in peroxisomes indicates that dual targeting of proteins to both the cytoplasm and the peroxisome might be more widespread. Although current knowledge of proteins exhibiting only partial peroxisomal targeting is far from being complete, we speculate that the metabolic capacity of peroxisomes might be larger than previously assumed. PMID:24151469
Peroxisome proliferator-activated receptor-c agonists induce neuroprotection following transient focal ischemia in normotensive, normoglycemic as well as hypertensive and type-2 diabetic rodents
Kudret Tureyen; Ramya Kapadia; Kellie K. Bowen; Irawan Satriotomo; Jin Liang; Douglas L. Feinstein; Raghu Vemuganti
Thiazolidinediones (TZDs) are synthetic agonists of the lig- and-activated transcription factor peroxisome proliferator- activated receptor-c (PPARc). TZDs are known to curtail inflammation associated with peripheral organ ischemia. As inflammation precipitates the neuronal death after stroke, we tested the efficacy of TZDs in preventing brain dam- age following transient middle cerebral artery occlusion (MCAO) in adult rodents. As hypertension and diabetes
Terminal Differentiation of Human Liposarcoma Cells Induced by Ligands for Peroxisome Proliferator-Activated Receptor gamma and the Retinoid X Receptor
Peter Tontonoz; Samuel Singer; Barry M. Forman; Pasha Sarraf; Jonathan A. Fletcher; Christopher D. M. Fletcher; Regina P. Brun; Elisabetta Mueller; Soner Altiok; Heather Oppenheim; Ronald M. Evans; Bruce M. Spiegelman
Induction of terminal differentiation represents a promising therapeutic approach to certain human malignancies. The peroxisome proliferator-activated receptor gamma (PPARgamma ) and the retinoid X receptor alpha (RXRalpha ) form a heterodimeric complex that functions as a central regulator of adipocyte differentiation. Natural and synthetic ligands for both receptors have been identified. We demonstrate here that PPARgamma is expressed at high
Exposure to an obesity-inducing diet early affects the pattern of expression of peroxisome proliferator, retinoic acid, and triiodothyronine nuclear receptors in the rat
Anabelle Redonnet; Rachel Groubet; Alfredo Martinez; Paul Higueret
Since evidence has appeared that [alpha ] and [gamma ] isoforms of the peroxisome proliferator receptors (PPARs) are involved in the regulation of triglyceride homeostasis and in the control of the differentiation of adipocytes that is required for the development of obesity, a large number of studies have investigated the physiologic role of nuclear receptors in the control of energy
Mode of action framework analysis for receptor-mediated toxicity: the Peroxisome Proliferator-Activated Receptor alpha (PPARa) as a case study
Therapeutic hypolipidemic agents and industrial chemicals that cause peroxisome proliferation and induce liver tumors in rodents activate the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa). Research has elucidated the cellular and molecular events by w...
Estradiol induces proliferation of peroxisome-like microbodies and the production of 3-hydroxy fatty acid diesters, the female pheromones, in the uropygial glands of male and female mallards.
Bohnet, S; Rogers, L; Sasaki, G; Kolattukudy, P E
During the mating season the female mallards produce sex pheromones, diesters of 3-hydroxy fatty acids, in their uropygial glands. Subcellular fractionation by sucrose and Nycodenz density gradient centrifugations and electron microscopic examination of the fractions showed that diesters of 3-hydroxy acids and the enzymes that catalyze the formation and esterification of the 3-hydroxy fatty acids are located in the catalase-containing fractions, probably peroxisomes, whereas monoester synthesizing activities are located in the endoplasmic reticulum. Fatty acyl-CoA reductase that would provide fatty alcohol needed for the synthesis of monoester and diester waxes was found both in the peroxisomal and endoplasmic reticulum fraction. Upon daily intramuscular injection of estradiol into the females in the nonmating season, the short chain monoester waxes of the uropygial glands were replaced by long chain monoester waxes, and subsequently the monoester waxes were replaced by diester waxes. Injection of thyroxine with estradiol hastened the induction of the compositional changes including diester synthesis. Similar changes, including the synthesis of the female pheromones, were induced in the uropygial glands by the hormone treatment of males that do not normally produce diesters at any time during their life cycle. The structure and composition of the diesters induced by hormone treatment of both males and females were identical to those of the female pheromones produced during their mating season. Electron microscopic examination of diaminobenzidine-treated glands showed that peroxisomes proliferated in the gland of the females in the mating season and in the estradiol-treated males that produce the diesters. PMID:2033066
Inhibitory Effect of a Proline-to-Alanine Substitution at Codon 12 of Peroxisome Proliferator-Activated Receptor-? 2 on Thiazolidinedione-Induced Adipogenesis
Jiro Masugi; Yoshikazu Tamori; Hiroyuki Mori; Takashi Koike; Masato Kasuga
Peroxisome proliferator-activated receptor-? (PPAR?) is a member of the nuclear hormone receptor superfamily of transcription factors and appears to be a key regulator of adipogenesis. Members of the thiazolidinedione class of insulin-sensitizing agents act as high-affinity ligands for PPAR?, indicating that PPAR? is also important in systemic insulin action. To determine whether Pro12 ? Ala (P12A) mutation in PPAR? gene
Ligands for Peroxisome Proliferator-Activated Receptorgamma and Retinoic Acid Receptor Inhibit Growth and Induce Apoptosis of Human Breast Cancer Cells in vitro and in BNX Mice
Elena Elstner; Carsten Muller; Kozo Koshizuka; Elizabeth A. Williamson; Dorothy Park; Hiroya Asou; Peter Shintaku; Jonathan W. Said; David Heber; H. Phillip Koeffler
Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated receptor gamma (PPARgamma ) is expressed in breast cancer cells. Activation of PPARgamma through a synthetic ligand, troglitazone
Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor
Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.; Akita, Geoffrey Y.; Cheng, Seng H.; Gregory, Richard J. [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang Canwen [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)], E-mail: email@example.com
In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.
Peroxisome biogenesis and molecular defects in peroxisome assembly disorders
Yukio Fujiki; Kanji Okumoto; Hidenori Otera; Shigehiko Tamura
Peroxisome assembly in mammals requires more than 14 genes. So far, we have isolated seven complementation groups (CGs) of\\u000a peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24\\/ZP107, ZP92, ZP105\\/ZP139, ZP109, ZP110,\\u000a ZP114. Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible\\u000a for peroxisome
Successful Treatment of Epidermal Growth Factor Receptor Inhibitor-Induced Periungual Inflammation with Adapalene
Junichi Hachisuka; Kazuko Doi; Yoichi Moroi; Masutaka Furue
Epidermal growth factor receptor (EGFR) inhibitors are increasingly used for cancer treatment, but commonly carry dermatologic side effects. Periungual inflammation is a particularly painful condition that additionally worsens quality of life. In this paper, we report 3 cases of successful treatment of periungual inflammation induced by 3 different EGFR inhibitors (gefitinib, erlotinib, and cetuximab) with topically applied adapalene.
The peroxisome: still a mysterious organelle
Fahimi, H. Dariush
More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as ?-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed. PMID:18274771
Proteasome inhibitor bortezomib modulates TLR4-induced dendritic cell activation
Alessio Nencioni; Karin Schwarzenberg; Katharina M. Brauer; Susanne M. Schmidt; Alberto Ballestrero; Frank Grunebach; Peter Brossart
Evidence from the animal model sug- gests that proteasome inhibitors may have immunosuppressive properties; however, their effects on the human im- mune system remain poorly investigated. Here, we show that bortezomib, a protea- some inhibitor with anticancer activity, impairs several immune properties of hu- man monocyte-derived dendritic cells (DCs). Namely, exposure of DCs to bor- tezomib reduces their phagocytic capac-
Selective Serotonin Reuptake Inhibitor-Induced Sexual Dysfunction in Adolescents: A Review.
ERIC Educational Resources Information Center
Scharko, Alexander M.
Objective: To review the existing literature on selective serotonin reuptake inhibitor (SSRI)-induced sexual dysfunction in adolescents. Method: A literature review of SSRI-induced adverse effects in adolescents focusing on sexual dysfunction was done. Nonsexual SSRI-induced adverse effects were compared in adult and pediatric populations.ģ
Histone deacetylase inhibitor, butyrate, attenuates lipopolysaccharide-induced acute lung injury in mice
Yun-Feng Ni; Jian Wang; Xiao-Long Yan; Feng Tian; Jin-Bo Zhao; Yun-Jie Wang; Tao Jiang
BACKGROUND: Histone deacetylase (HDAC) inhibitors, developed as promising anti-tumor drugs, exhibit their anti-inflammatory properties due to their effects on reduction of inflammatory cytokines. OBJECTIVE: To investigate the protective effect of butyrate, a HDAC inhibitor, on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. METHODS: ALI was induced in Balb\\/c mice by intratracheally instillation of LPS (1 mg\\/kg). Before 1 hour
The cyclin-dependent kinase inhibitor p21 is required for TGF-?1¢induced podocyte apoptosis
The cyclin-dependent kinase inhibitor p21 is required for TGF-?1¢induced podocyte apoptosis.BackgroundReduced podocyte number is a critical determinant in the development of glomerulosclerosis. Transforming growth factor-?1 (TGF-?1) induces podocyte apoptosis, but the cell cycle events are not known. The cyclin-dependent kinase (CDK) inhibitor p21 increases in podocytes in diseases where TGF-? increases. Accordingly, we studied the role of p21 in podocyte
Histone Deacetylase Inhibitors: Inducers of Differentiation or Apoptosis of Transformed Cells
Paul A. Marks; Victoria M. Richon; Richard A. Rifkind
Histone deacetylase (HDAC) inhibitors have been shown to be potent inducers of growth arrest, differentiation, and\\/or apoptotic cell death of transformed cells in vitro and in vivo. One class of HDAC inhibitors, hydroxamic acid-based hy- brid polar compounds (HPCs), induce differentiation at mi- cromolar or lower concentrations. Studies (x-ray crystallo- graphic) showed that the catalytic site of HDAC has a
Novel selective Cox2 inhibitors induce apoptosis in Caco-2 colorectal carcinoma cell line
Reza Entezari Heravi; Farzin Hadizadeh; Mojtaba Sankian; Jalil Tavakol Afshari; Seyed Mohammad Taghdisi; Hanieh Jafarian; Javad Behravan
The cyclooxygenase-2 (COX-2) inhibitors including celecoxib inhibit cell growth and induce apoptosis in cancer cells. As COX-2 is over expressed in solid tumors such as colorectal cancer, it can be a suitable target for cancer treatment studies. In this study we designed and synthesized 4,5-bisaryl imidazolyl imidazoles as novel COX-2 inhibitors and evaluated their apoptosis inducing activities. The ability of
Peroxisome proliferation in liver of rats fed oxidized frying oil.
Chao, Pei-Min; Yang, Mei-Fang; Tseng, Yu-Na; Chang, Ko-Ming; Lu, Kuo-Shyan; Huang, Ching-Jang
Oxidized frying oil (OFO) activates peroxisome proliferator-activated receptor a (PPAR alpha) in vitro and in vivo. As most PPARalpha activators are also peroxisome proliferators (PP), this study was aimed at exploring whether OFO induces peroxisome proliferation in the liver of rats. Four groups of male weanling Sprague-Dawley rats were fed the following diets for 6 wk: a basal diet containing 5 g/100 g fresh soybean oil (LSB), high-fat diets containing 20 g/100 g of fresh soybean oil (HSB as a control). OFO (HO) or fish oil (HF, as a positive control). Hepatomegaly and peroxisome proliferation in the liver of the HO group of rats were higher than those of the HF group. In addition, the acyl-CoA oxidase (ACO) activity, as well as cytochrome P450 4A (CYP4A) protein content in the livers of the HO group were 6 fold those of the HSB group, but were 2.5 fold in those of the HF group. These results indicated that dietary OFO induced typical responses to PPARalpha signaling. Moreover. as a dietary source, the OFO prepared under our frying conditions appears to be a more potent peroxisome proliferator than fish oil. PMID:16392708
Dual-action inhibitors of HIF prolyl hydroxylases that induce binding of a second iron ion.
Yeoh, Kar Kheng; Chan, Mun Chiang; Thalhammer, Armin; Demetriades, Marina; Chowdhury, Rasheduzzaman; Tian, Ya-Min; Stolze, Ineke; McNeill, Luke A; Lee, Myung Kyu; Woon, Esther C Y; Mackeen, Mukram M; Kawamura, Akane; Ratcliffe, Peter J; Mecinovi?, Jasmin; Schofield, Christopher J
Inhibition of the hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2ĘironĘinhibitor stoichiometry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines. PMID:23151668
Caspase inhibitors, but not c-Jun NH2-terminal kinase inhibitor treatment, prevent cisplatin-induced hearing loss.
Wang, Jing; Ladrech, Sabine; Pujol, Remy; Brabet, Philippe; Van De Water, Thomas R; Puel, Jean-Luc
Cisplatin (CDDP) is a highly effective chemotherapeutic agent but with significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to an ototoxic level of CDDP. This study examines intracellular pathways involved in hair cell death induced by CDDP exposure in vivo to develop effective therapeutic strategies to protect the auditory receptor from CDDP-initiated hearing loss. Guinea pigs were treated with systemic administration of CDDP. Cochlear hair cells from CDDP-treated animals exhibited classic apoptotic alterations in their morphology. Several important signaling events that regulate the death of CDDP-injured cochlear hair cells were identified. CDDP treatment induced the activation and redistribution of cytosolic Bax and the release of cytochrome c from injured mitochondria. Activation of caspase-9 and caspase-3, but not caspase-8, was detected after treatment with CDDP, and the cleavage of fodrin by activated caspase-3 was observed within damaged hair cells. Intracochlear perfusions with caspase-3 inhibitor (z-DEVD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) prevent hearing loss and loss of sensory cells, but caspase-8 inhibitor (z-IETD-fmk) and cathepsin B inhibitor (z-FA-fmk) do not. Although the stress-activated protein kinase/c-Jun NH(2)-terminal kinase (JNK) signaling pathway is activated in response to CDDP toxicity, intracochlear perfusion of d-JNKI-1, a JNK inhibitor, did not protect against CDDP ototoxicity but instead potentiated the ototoxic effects of CDDP. The results of the present study show that blocking a critical step in apoptosis may be a useful strategy to prevent harmful side effects of CDDP ototoxicity in patients having to undergo chemotherapy. PMID:15604295
Dipeptidyl peptidase IV inhibitor lowers PPAR? agonist-induced body weight gain by affecting food intake, fat mass, and beige/brown fat but not fluid retention
Masuda, Takahiro; Fu, Yiling; Eguchi, Akiko; Czogalla, Jan; Rose, Michael A.; Kuczkowski, Alexander; Gerasimova, Maria; Feldstein, Ariel E.; Scadeng, Miriam
Peroxisome proliferator-activated receptor-? (PPAR?) agonists like pioglitazone (PGZ) are effective antidiabetic drugs, but they induce fluid retention and body weight (BW) gain. Dipeptidyl peptidase IV (DPP IV) inhibitors are antidiabetic drugs that enhance renal Na+ and fluid excretion. Therefore, we examined whether the DPP IV inhibitor alogliptin (ALG) ameliorates PGZ-induced BW gain. Male Sv129 mice were treated with vehicle (repelleted diet), PGZ (220 mg/kg diet), ALG (300 mg/kg diet), or a combination of PGZ and ALG (PGZ + ALG) for 14 days. PGZ + ALG prevented the increase in BW observed with PGZ but did not attenuate the increase in body fluid content determined by bioimpedance spectroscopy (BIS). BIS revealed that ALG alone had no effect on fat mass (FM) but enhanced the FM-lowering effect of PGZ; MRI analysis confirmed the latter and showed reductions in visceral and inguinal subcutaneous (sc) white adipose tissue (WAT). ALG but not PGZ decreased food intake and plasma free fatty acid concentrations. Conversely, PGZ but not ALG increased mRNA expression of thermogenesis mediator uncoupling protein 1 in epididymal WAT. Adding ALG to PGZ treatment increased the abundance of multilocular cell islets in sc WAT, and PGZ + ALG increased the expression of brown-fat-like ōbeigeö cell marker TMEM26 in sc WAT and interscapular brown adipose tissue and increased rectal temperature vs. vehicle. In summary, DPP IV inhibition did not attenuate PPAR? agonist-induced fluid retention but prevented BW gain by reducing FM. This involved ALG inhibition of food intake and was associated with food intake-independent synergistic effects of PPAR? agonism and DPP-IV inhibition on beige/brown fat cells and thermogenesis. PMID:24347054
Dipeptidyl peptidase IV inhibitor lowers PPAR? agonist-induced body weight gain by affecting food intake, fat mass, and beige/brown fat but not fluid retention.
Masuda, Takahiro; Fu, Yiling; Eguchi, Akiko; Czogalla, Jan; Rose, Michael A; Kuczkowski, Alexander; Gerasimova, Maria; Feldstein, Ariel E; Scadeng, Miriam; Vallon, Volker
Peroxisome proliferator-activated receptor-? (PPAR?) agonists like pioglitazone (PGZ) are effective antidiabetic drugs, but they induce fluid retention and body weight (BW) gain. Dipeptidyl peptidase IV (DPP IV) inhibitors are antidiabetic drugs that enhance renal Na(+) and fluid excretion. Therefore, we examined whether the DPP IV inhibitor alogliptin (ALG) ameliorates PGZ-induced BW gain. Male Sv129 mice were treated with vehicle (repelleted diet), PGZ (220 mg/kg diet), ALG (300 mg/kg diet), or a combination of PGZ and ALG (PGZ + ALG) for 14 days. PGZ + ALG prevented the increase in BW observed with PGZ but did not attenuate the increase in body fluid content determined by bioimpedance spectroscopy (BIS). BIS revealed that ALG alone had no effect on fat mass (FM) but enhanced the FM-lowering effect of PGZ; MRI analysis confirmed the latter and showed reductions in visceral and inguinal subcutaneous (sc) white adipose tissue (WAT). ALG but not PGZ decreased food intake and plasma free fatty acid concentrations. Conversely, PGZ but not ALG increased mRNA expression of thermogenesis mediator uncoupling protein 1 in epididymal WAT. Adding ALG to PGZ treatment increased the abundance of multilocular cell islets in sc WAT, and PGZ + ALG increased the expression of brown-fat-like "beige" cell marker TMEM26 in sc WAT and interscapular brown adipose tissue and increased rectal temperature vs. vehicle. In summary, DPP IV inhibition did not attenuate PPAR? agonist-induced fluid retention but prevented BW gain by reducing FM. This involved ALG inhibition of food intake and was associated with food intake-independent synergistic effects of PPAR? agonism and DPP-IV inhibition on beige/brown fat cells and thermogenesis. PMID:24347054
Lysine-specific demethylase 1 inhibitors protect cochlear spiral ganglion neurons against cisplatin-induced damage.
Li, Ao; He, Yingzi; Sun, Shan; Cai, Chengfu; Li, Huawei
Cisplatin is a widely used chemotherapeutic drug, but one of its side effects is ototoxicity. Epigenetic-related drugs, such as lysine-specific demethylase 1 (LSD1) inhibitors, have been reported to protect against cisplatin-induced hair cell loss by preventing demethylation of histone H3K4 (H3K4me2). However, the protective effect of LSD1 inhibitors in spiral ganglion neurons (SGNs) remains unclear. To investigate whether LSD1 inhibitors exert similar protective effects on SGNs, we treated mouse cochlear explant cultures with LSD1 inhibitors (2PCPA, S2101, or CBB1007) together with cisplatin. Low concentrations of cisplatin damaged SGNs much more than high concentrations, and blocking the demethylation of H3K4me2 with LSD1 inhibitors prevented the SGNs from injury. Reactive oxygen species are also involved in the injury process, and LSD1 inhibitors protected SGNs by increasing the expression level of the antioxidant gene Slc7a11 and decreasing the level of the pro-oxidant gene lactoperoxidase (Lpo). Our findings show that LSD1 inhibitors prevent cisplatin-induced SGN loss by regulating the demethylation of H3K4 and preventing increases of reactive oxygen species levels, which might provide a potential therapeutic strategy for cisplatin-induced hearing loss. PMID:26011390
BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells.
Baker, Emma K; Taylor, Scott; Gupte, Ankita; Sharp, Phillip P; Walia, Mannu; Walsh, Nicole C; Zannettino, Andrew Cw; Chalk, Alistair M; Burns, Christopher J; Walkley, Carl R
Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS. PMID:25944566
BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells
Baker, Emma K; Taylor, Scott; Gupte, Ankita; Sharp, Phillip P; Walia, Mannu; Walsh, Nicole C; Zannettino, Andrew CW; Chalk, Alistair M; Burns, Christopher J; Walkley, Carl R
Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS. PMID:25944566
Histone deacetylase inhibitor trichostatin A and proteasome inhibitor PS-341 synergistically induce apoptosis in pancreatic cancer cells
Bai Jirong [Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 0221 (United States)]. E-mail: firstname.lastname@example.com; Demirjian, Aram [Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 0221 (United States); Sui Jianhua [Dana-Farber Cancer Institute, Harvard Medical School, 330 Brookline Avenue, Boston, MA 0221 (United States); Marasco, Wayne [Dana-Farber Cancer Institute, Harvard Medical School, 330 Brookline Avenue, Boston, MA 0221 (United States); Callery, Mark P. [Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 0221 (United States)]. E-mail: firstname.lastname@example.com
Pancreatic cancer is a common and lethal malignancy. Pancreatic cancer cells overexpress multiple anti-apoptotic factors and death receptor decoys, and are strongly resistant to radiation and to 5-fluorouracil (5-FU)- or gemcitabine (Gem)-based chemotherapy regimens. We have found that low-dose proteasome inhibitor PS-341 and histone deacetylase inhibitor trichostatin A (TSA) synergistically induce cytotoxicity in a panel of eight diverse pancreatic cancer cell lines. Combining TSA with PS-341 effectively inactivated NF{kappa}B signaling, downregulated the predominant endogenous anti-apoptotic factor Bcl-XL overexpression, and disrupted MAP kinase pathway. The combined drug regimen effectively inflicted an average of 71.5% apoptotic cell death (55.2-80%) in diverse pancreatic cancer cell lines by activating the intrinsic apoptotic pathway. Conclusion: the TSA/PS-341 regimen may represent a potential novel therapeutic strategy for pancreatic cancer.
Fis1, DLP1, and Pex11p coordinately regulate peroxisome morphogenesis
Kobayashi, Shinta [Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 812-8581 (Japan); Tanaka, Atsushi [Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Fujiki, Yukio [Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 812-8581 (Japan) and Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan) and JST, CREST (Japan)]. E-mail: email@example.com
Dynamin-like protein 1 (DLP1) and Pex11p{beta} function in morphogenesis of peroxisomes. In the present work, we investigated whether Fis1 is involved in fission of peroxisomes. Endogenous Fis1 was morphologically detected in peroxisomes as well as mitochondria in wild-type CHO-K1 and DLP1-defective ZP121 cells. Subcellular fractionation studies also revealed the presence of Fis1 in peroxisomes. Peroxisomal Fis1 showed the same topology, i.e., C-tail anchored membrane protein, as the mitochondrial one. Furthermore, ectopic expression of FIS1 induced peroxisome proliferation in CHO-K1 cells, while the interference of FIS1 RNA resulted in tubulation of peroxisomes, hence reducing the number of peroxisomes. Fis1 interacted with Pex11p{beta}, by direct binding apparently involving the C-terminal region of Pex11p{beta} in the interaction. Pex11p{beta} also interacted with each other, whereas the binding of Pex11p{beta} to DLP1 was not detectable. Moreover, ternary complexes comprising Fis1, Pex11p{beta}, and DLP1 were detected by chemical cross-linking. We also showed that the highly conserved N-terminal domain of Pex11p{beta} was required for the homo-oligomerization of Pex11p{beta} and indispensable for the peroxisome-proliferating activity. Taken together, these findings indicate that Fis1 plays important roles in peroxisome division and maintenance of peroxisome morphology in mammalian cells, possibly in a concerted manner with Pex11p{beta} and DLP1.
Gout induced by L-dopa and decarboxylase inhibitors
Calne, D. B.; Fermaglich, J.
Two cases of clinical gout are reported in parkinsonian patients receiving L-dopa in combination with a decarboxylase inhibitor. Blockade of decarboxylation leads to major changes in the pattern of L-dopa metabolites. It is suggested that hyperuricaemia may result from accumulation of L-dopa itself or a transaminated product. PMID:1273018
Angiotensin-converting enzyme inhibitors-induced angioedema treated by C1 esterase inhibitor concentrate (Berinert«): about one case and review of the therapeutic arsenal
Lipski, Samuel Michael; Casimir, Georges; Vanlommel, Martine; Jeanmaire, Mathieu; Dolhen, Pierre
Key Clinical Message C1 esterase inhibitor (Berinert«) is generally used to treat severe attack of hereditary angioedema. We describe here the case of a patient who presented with a severe angioedema induced by angiotensin-converting enzyme inhibitors (ACEIs) endangering her life. It could be successfully treated with that medicine. PMID:25767713
Peroxisomal and Mitochondrial Fatty Acid -Oxidation in Mice Nullizygous for Both Peroxisome Proliferator-activated
Omiecinski, Curtis
Proliferator-activated Receptor and Peroxisomal Fatty Acyl-CoA Oxidase GENOTYPE CORRELATION WITH FATTY LIVER of specific fatty liver phenotype. In animal cells, mitochondria as well as peroxisomes oxidize fatty acidsPeroxisomal and Mitochondrial Fatty Acid -Oxidation in Mice Nullizygous for Both Peroxisome
Further evidence for the involvement of inhibition of cell proliferation and development in thymic and splenic atrophy induced by the peroxisome proliferator perfluoroctanoic acid in mice 3 3 Abbreviations: PFOA, perfluorooctanoic acid; PP, peroxisome proliferator; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; and DEHP, di(2-ethylhexyl)phthalate
Qian Yang; Yi Xie; Anna Messing Eriksson; B. Dean Nelson; Joseph W DePierre
We recently demonstrated that severe thymic and splenic atrophy occur upon dietary treatment of mice with potent peroxisome proliferators (PPs), e.g. perfluorooctanoic acid (PFOA), WY-14,643, nafenopin, and di(2-ethylhexyl)phthalate (DEHP). In the present study, we investigated this phenomenon further employing a relative inert PP, PFOA. Comparison of the dose-dependencies and time-courses indicated that the peroxisome proliferative effect occurred prior to atrophy
Hypothermia-induced hyperphosphorylation: a new model to study tau kinase inhibitors.
Bretteville, Alexis; Marcouiller, Franńois; Julien, Carl; El Khoury, Noura B; Petry, Franck R; Poitras, Isabelle; Mouginot, Didier; Lķvesque, Georges; Hķbert, Sķbastien S; Planel, Emmanuel
Tau hyperphosphorylation is one hallmark of Alzheimer's disease (AD) pathology. Pharmaceutical companies have thus developed kinase inhibitors aiming to reduce tau hyperphosphorylation. One obstacle in screening for tau kinase inhibitors is the low phosphorylation levels of AD-related phospho-epitopes in normal adult mice and cultured cells. We have shown that hypothermia induces tau hyperphosphorylation in vitro and in vivo. Here, we hypothesized that hypothermia could be used to assess tau kinase inhibitors efficacy. Hypothermia applied to models of biological gradual complexity such as neuronal-like cells, ex vivo brain slices and adult non-transgenic mice leads to tau hyperphosphorylation at multiple AD-related phospho-epitopes. We show that Glycogen Synthase Kinase-3 inhibitors LiCl and AR-A014418, as well as roscovitine, a cyclin-dependent kinase 5 inhibitor, decrease hypothermia-induced tau hyperphosphorylation, leading to different tau phosphorylation profiles. Therefore, we propose hypothermia-induced hyperphosphorylation as a reliable, fast, convenient and inexpensive tool to screen for tau kinase inhibitors. PMID:22761989
The multikinase inhibitor sorafenib induces caspase-dependent apoptosis in PC-3 prostate cancer cells
Huang, Rui; Chen, Xue-Qin; Huang, Ying; Chen, Ni; Zeng, Hao
The present study investigated the effects of the multikinase inhibitor sorafenib on androgen-independent cancer cells viability and intracellular signaling. Human androgen-independent PC-3 prostate cancer cells were treated with sorafenib. At concentration that suppresses extracellular signal-regulated kinase phosphorylation, sorafenib treatment reduced the mitochondrial transmembrane potential. Sorafenib also down-modulated the levels of myeloid cell leukemia 1, survivin and cellular inhibitor of apoptosis protein 2. Sorafenib induced caspase-3 cleavage and the mitochondrial release of cytochrome c. However, no nuclear translocation of apoptosis inducing factor was detected after treatment and the pan-caspase inhibitor Z-VAD-FMK had an obvious protective effect against the drug. In conclusion, sorafenib induces apoptosis through a caspase-dependent mechanism with down-regulated anti-apoptotic proteins in androgen-independent prostate cancer cells in vitro. PMID:20473320
The MR spectrum of peroxisomal disorders
M. S. Knaap; J. Valk
In the last decade an increasing number of peroxisomal disorders has been recognized. Almost all peroxisomal disorders affect the central nervous system. Many of them lead to demyelination, some of them lead to migrational disturbances. The MR pattern of X-linked adrenoleukodystrophy is well known, but the pattern of the other peroxisomal disorders is less well known. We evaluated the gray
A leukocyte elastase inhibitor reduces thrombin-induced pulmonary oedema in the rat: mechanisms of action.
Ahn, C M; Sandler, H; Saldeen, T
The effect of a selective leukocyte elastase inhibitor, ICI 200,355, on thrombin-induced pulmonary oedema was studied in rats. Thrombin administration produced an increase in lung weight (P < 0.05), wet weight/ dry weight ratio (P < 0.05), and relative lung water content (P < 0.05). The lung weight increase was reduced by the elastase inhibitor in doses of 2000, 200 and 20 micrograms/kg per h (P < 0.05), but not by 2 micrograms/kg per h. A dose of 20 micrograms/ kg per h seems to be optimal, since 10-fold and 100-fold increases in dose did not further improve the effect. Free elastase activity in lung tissue was higher after thrombin infusion than in controls, but was not depleted by the elastase inhibitor in vivo (P < 0.05). This elastase activity in the lung was, however, inhibited by the elastase inhibitor in vitro, indicating that the inhibitor can block extracellular, but not intracellular elastase activity. Thrombin infusion resulted in a significant decrease in plasma elastase inhibitory capacity (P < 0.05), which was depleted by the elastase inhibitor (20 micrograms/kg per h) (P < 0.05). Myeloperoxidase activity was significantly increased in lung tissue after thrombin infusion (P < 0.05). Lung myeloperoxidase activity 5 min after thrombin infusion was not affected by the elastase inhibitor, but the inhibitor induced a further increase in myeloperoxidase as seen 90 min after thrombin infusion, indicating that the effect of this inhibitor on pulmonary oedema is not due to reduction of leukocyte infiltration in the lungs, but may partly be exerted by prevention of neutrophil destruction. PMID:10101747
Hepatotoxic reactions induced by beta-lactamase inhibitors.
Berg, P; Hahn, E G
Since amoxicillin/clavulanate was first introduced in the UK in 1981, beta-lactamase inhibitors are used increasingly worldwide. Two more drugs of this class are currently available, sulbactam and tazobactam. Meanwhile, adverse drug reactions associated with amoxicillin/clavulanate occurring late after the end of therapy have been repeatedly published. In many cases, a cholestatic hepatitis was diagnosed that was most likely caused by the clavulanic acid component of the combination. Symptoms were mostly mild and reversible, whereas a number of cases showing a protracted, even fatal course of the disease have been documented. This article summarizes and analyzes all relevant studies and case reports dealing with hepatotoxicity caused by beta-lactamase inhibitors. The description of a typical case from our own patient population illustrates the clinical challenge associated with this adverse drug reaction. PMID:11772541
Peroxisome Proliferator-activated Receptor ? Coactivator 1 (PGC-1)- and Estrogen-related Receptor (ERR)-induced Regulator in Muscle 1 (PERM1) Is a Tissue-specific Regulator of Oxidative Capacity in Skeletal Muscle Cells*
Cho, Yoshitake; Hazen, Bethany C.; Russell, Aaron P.; Kralli, Anastasia
Mitochondrial oxidative metabolism and energy transduction pathways are critical for skeletal and cardiac muscle function. The expression of genes important for mitochondrial biogenesis and oxidative metabolism are under the control of members of the peroxisome proliferator-activated receptor ? coactivator 1 (PGC-1) family of transcriptional coactivators and the estrogen-related receptor (ERR) subfamily of nuclear receptors. Perturbations in PGC-1 and/or ERR activities have been associated with alterations in capacity for endurance exercise, rates of muscle atrophy, and cardiac function. The mechanism(s) by which PGC-1 and ERR proteins regulate muscle-specific transcriptional programs is not fully understood. We show here that PGC-1? and ERRs induce the expression of a so far uncharacterized muscle-specific protein, PGC-1- and ERR-induced regulator in muscle 1 (Perm1), which regulates the expression of selective PGC-1/ERR target genes. Perm1 is required for the basal as well as PGC-1?-enhanced expression of genes with roles in glucose and lipid metabolism, energy transfer, and contractile function. Silencing of Perm1 in cultured myotubes compromises respiratory capacity and diminishes PGC-1?-induced mitochondrial biogenesis. Our findings support a role for Perm1 acting downstream of PGC-1? and ERRs to regulate muscle-specific pathways important for energy metabolism and contractile function. Elucidating the function of Perm1 may enable novel approaches for the treatment of disorders with compromised skeletal muscle bioenergetics, such as mitochondrial myopathies and age-related/disease-associated muscle atrophies. PMID:23836911
SMK-17, a MEK1/2-specific inhibitor, selectively induces apoptosis in ?-catenin-mutated tumors
Kiga, Masaki; Nakayama, Ayako; Shikata, Yuki; Sasazawa, Yukiko; Murakami, Ryo; Nakanishi, Toshiyuki; Tashiro, Etsu; Imoto, Masaya
Although clinical studies have evaluated several MEK1/2 inhibitors, it is unlikely that MEK1/2 inhibitors will be studied clinically. BRAF mutations have been proposed as a responder marker of MEK1/2 inhibitors in a preclinical study. However, current clinical approaches focusing on BRAF mutations have shown only moderate sensitivity of MEK1/2 inhibitors. This has led to insufficient support for their promoted clinical adoption. Further characterization of tumors sensitive to MEK inhibitors holds great promise for optimizing drug therapy for patients with these tumors. Here, we report that ?-catenin mutations accelerate apoptosis induced by MEK1/2 inhibitor. SMK-17, a selective MEK1/2 inhibitor, induced apoptosis in tumor cell lines harboring ?-catenin mutations at its effective concentration. To confirm that ?-catenin mutations and mutant ?-catenin-mediated TCF7L2 (also known as TCF4) transcriptional activity is a predictive marker of MEK inhibitors, we evaluated the effects of dominant-negative TCF7L2 and of active, mutated ?-catenin on apoptosis induced by MEK inhibitor. Indeed, dominant-negative TCF7L2 reduced apoptosis induced by MEK inhibitor, whereas active, mutated ?-catenin accelerated it. Our findings show that ?-catenin mutations are an important responder biomarker for MEK1/2 inhibitors. PMID:25640451
Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization.
Meng, Zhen; Gan, Ye-Hua
Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. PMID:25770423
Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin.
Wolvetang, E J; Larm, J A; Moutsoulas, P; Lawen, A
Activation of the plasma membrane NADH-oxidoreductase (PMOR) system by addition of growth factors or extracellular electron acceptors stimulates cellular proliferation. We now show that the vanilloids capsaicin, dihydrocapsaicin, and resiniferatoxin are inhibitors of the NADH-oxidase activity of the PMOR system and that both these and two previously identified PMOR inhibitors (chloroquine and retinoic acid) induce apoptosis in human B-cell and mouse myeloid cell lines. At the optimal concentration, PMOR inhibitors can induce between 50 and 70% of apoptosis in mouse myeloid and human B-cell lines within 8-12 h, provided these cell lines do not express Bcl-2. The immunosuppressants cyclosporin A and fujimycin (tacrolimus) inhibit PMOR inhibitor-induced apoptosis. By using combinations of these immunosuppressants and excess amounts of their nonimmunosuppressive analogues, we demonstrate that in human B-cell lines the Bcl-2-sensitive apoptotic pathway triggered by PMOR inhibitors involves signaling through the protein phosphatase calcineurin. We suggest that the PMOR system is a redox sensor that can, depending on the ambient redox environment and the availability of growth factors, regulate plasma membrane calcium fluxes and signal for apoptosis through calcineurin. Bcl-2, a protein that is thought to inhibit apoptosis by regulating reactive oxygen species and calcium fluxes in the cell, inhibits this apoptotic pathway. PMID:8891335
Effects of inhibitors of radiation-induced potentially lethal damage repair on chemotherapy in murine tumors
Nakatsugawa, S.; Sugahara, T.
Enhancement of various antitumor drugs effects by inhibitors of radiation-induced potentially lethal damage (PLD) repair was studied in three murine tumors (EMT-6, RIF-1 and SQ-1). In EMT-6 tumors, PLD repair inhibitors, 3'-deoxyguanosine (3'dG) and 7904 (a derivative of 3'-deoxyadenosine) showed a marked enhancement of tumor growth inhibition by anticancerous drugs (FT-207 (a derivative of 5-FU), bleomycin, Ara-C, ACNU). However, the effects of mitomycin-C and vincristine were not potentiated by the inhibitors. In SQ-1 carcinomas, another repair inhibitor, ara-A (1-..beta..-D-arabinofuranosyladenine) (32 mg/kg) potentiated the effect of ACNU. In RIF-1 sarcomas, in which a low PLD repair function has been reported after ionizing radiation exposure, the potentiation was not so marked as in EMT-6 or SQ-1 tumors. Thus, as a possibility, the potentiation by inhibitors of radiation-induced PLD repair might be a result of the inhibition of chemical-induced PLD repair. The study of this field may contribute to the improvement of cancer treatment not only by radiotherapy but also by chemotherapy.
Na+/H+ exchanger inhibitor augments hyperosmolarity-induced vasoconstriction by enhancing actin polymerization.
Sasahara, Tomoya; Yayama, Katsutoshi; Tahara, Tsuyoshi; Onoe, Hirotaka; Okamoto, Hiroshi
Vascular smooth muscle cells (VSMCs) exhibit shrinkage-induced activation of Na(+)/H(+) exchanger isoform 1 (NHE-1) and Na(+), K(+), 2Cl(-) cotransporter (NKCC) under hyperosmotic conditions. To investigate the roles of these ion transporters in vascular smooth muscle force induced by hyperosmotic stress, we tested the effects of 5-(N, N-dimethyl)-amiloride (DMA; NHE inhibitor), cariporide (a selective NHE-1 inhibitor), and bumetanide (NKCC inhibitor) on the contractile response of rat aortic rings to hyperosmolar solutions. NHE inhibitors significantly augmented the maximum force response and contractile sensitivity to hyperosmolar sucrose, NaCl, and glucose in endothelium-denuded rings. Bumetanide elicited a comparatively modest increase in sensitivity. NHE inhibitors blocked the increase in intracellular pH and enhanced the cell volume decrease of cultured VSMCs after exposure to hyperosmolar sucrose. However, DMA had no effect on the increase in cytosolic free Ca(2+) concentration ([Ca(2+)]i) in rat VSMCs and on the increases in phosphorylation of myosin phosphatase target subunit 1 and myosin light chain (MLC) in aortic rings in response to hyperosmolar sucrose. Hyperosmolar sucrose-induced force was significantly attenuated by cytochalasin B in the presence or absence of DMA. Exposure to hyperosmolar sucrose increased the ratio of F- to G-actin; the ratio was further elevated by DMA. These results suggest that the potentiation of hyperosmotic shrinkage by NHE inhibition promotes actin polymerization in VSMCs and augments force production independent of changes in [Ca(2+)]i and MLC phosphorylation. PMID:23872622
Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells
Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5¢5 ?M induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 ?M, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10¢20 ?M), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM. PMID:25530098
Localization of peroxisomal matrix proteins by photobleaching
Buch, Charlotta [Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge (Sweden) [Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge (Sweden); Soedertoerns University, Life Sciences, SE-141 89 Huddinge (Sweden); Hunt, Mary C. [Department of Genetics, Microbiology and Toxicology, Stockholm University, Arrhenius Laboratory, Svante Arrhenius vaeg 16F, SE-106 91 Stockholm (Sweden)] [Department of Genetics, Microbiology and Toxicology, Stockholm University, Arrhenius Laboratory, Svante Arrhenius vaeg 16F, SE-106 91 Stockholm (Sweden); Alexson, Stefan E.H. [Karolinska Institutet, Department of Laboratory Medicine, Division of Clinical Chemistry C1-74, Karolinska University Hospital at Huddinge, SE-141 86 Stockholm (Sweden)] [Karolinska Institutet, Department of Laboratory Medicine, Division of Clinical Chemistry C1-74, Karolinska University Hospital at Huddinge, SE-141 86 Stockholm (Sweden); Hallberg, Einar, E-mail: email@example.com [Soedertoerns University, Life Sciences, SE-141 89 Huddinge (Sweden)] [Soedertoerns University, Life Sciences, SE-141 89 Huddinge (Sweden)
The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.
Disruption of Endothelial Peroxisome Proliferator-activated Receptor ? Accelerates Diet-induced Atherogenesis in Low-density Lipoprotein Receptor-null Mice
Qu, Aijuan; Shah, Yatrik M.; Manna, Soumen K.; Gonzalez, Frank J.
Objective Peroxisome proliferator-activated receptor ? (PPAR?) is widely expressed in vessel walls, and its activation by agonists showed beneficial effects in cardiovascular diseases. However, the role of endothelial cell (EC) PPAR? in atherogenesis is not fully understood. Methods and Results To assess the contribution of endothelial-specific PPAR? in atherosclerosis, EC-specific PPAR? disruption and low-density lipoprotein receptor (LDLR) double-knockout (Ppar??EC/Ldlr?/?) mice were developed. When challenged with a high-cholesterol diet for 4 weeks, Ppar??EC/Ldlr?/? mice exhibited severe atherosclerotic lesions compared to either their littermate controls or macrophage-specific PPAR? disruption and low-density lipoprotein receptor (LDLR) double knockout (Ppar??M?/Ldlr?/?) mice. Metabolic analysis showed severe dyslipidemia and significant increase in systolic blood pressure in the Ppar??M?/Ldlr?/? mice. Histological analysis and real-time quantitative PCR suggested an exacerbated inflammation in Ppar??EC/Ldlr?/? mice, as revealed by the increases of proinflammatory gene expression and macrophage infiltration in vivo and in vitro. Furthermore, in vivo endothelial permeability was also increased by endothelial PPAR? disruption. Bone-marrow transplantation studies, which reconstituted hematopoietic PPAR?, demonstrated that the accelerated atherogenesis was due to endothelial PPAR? deficiency. Conclusions Endothelial PPAR? plays an important protective role in atherogenesis. PMID:22015658
AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages
Kemmerer, Marina; Finkernagel, Florian; Cavalcante, Marcela Frota; Abdalla, Dulcineia Saes Parra; M³ller, Rolf; Br³ne, Bernhard; Namgaladze, Dmitry
AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor ? (PPAR?) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPAR? in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPK?1 catalytic subunit, followed by microarray expression analysis after treatment with the PPAR? agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPAR? increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPK?1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPAR?- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload. PMID:26098914
Beneficial Effect of Flavone Derivatives on A?-Induced Memory Deficit Is Mediated by Peroxisome Proliferator-Activated Receptor ? Coactivator 1?: A Comparative Study.
Arsalandeh, Farshad; Ahmadian, Shahin; Foolad, Forough; Khodagholi, Fariba; Farimani, Mahdi M; Shaerzadeh, Fatemeh
In the present study, the neuroprotective effect of 5-hydroxy-6,7,4'-trimethoxyflavone (flavone 1), a natural flavone, was investigated in comparison with another flavone, 5,7,4'-trihydroxyflavone (flavone 2) on the hippocampus of amyloid beta (A?)-injected rats. Rats were treated with the 2 flavones (1 mg/kg/d) for 1 week before A? injection. Seven days after A? administration, memory function of rats was assessed in a passive avoidance test (PAT). Changes in the levels of mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor ? coactivator 1 ? (PGC-1?), phospho-adenosine monophosphate (AMP)-activated protein kinase (pAMPK), AMPK, phospho-cAMP-responsive element-binding protein (CREB), CREB, and nuclear respiratory factor 1 (NRF-1) proteins were determined by Western blot analysis. Our results showed an improvement in memory in rats pretreated with flavonoids. At the molecular level, phosphorylation of CREB, known as the master modulator of memory processes, increased. On the other hand, the level of mitochondrial biogenesis factors, PGC-1? and its downstream molecules NRF-1 and TFAM significantly increased by dietary administration of 2 flavones. In addition, flavone 1 and flavone 2 prevented mitochondrial swelling and mitochondrial membrane potential reduction. Our results provided evidence that flavone 1 is more effective than flavone 2 presumably due to its O-methylated groups. In conclusion, it seems that in addition to classical antioxidant effect, flavones exert part of their protective effects through mitochondrial biogenesis. PMID:25972379
BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma.
Bhadury, Joydeep; Nilsson, Lisa M; Muralidharan, Somsundar Veppil; Green, Lydia C; Li, Zhoulei; Gesner, Emily M; Hansen, Henrik C; Keller, Ulrich B; McLure, Kevin G; Nilsson, Jonas A
The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi. PMID:24979794
Short Communication The nitric oxide synthase inhibitor l-NAME suppresses androgen-induced
Crews, David
Short Communication The nitric oxide synthase inhibitor l-NAME suppresses androgen-induced male July 2005 Abstract The synthesis of nitric oxide by the enzyme nitric oxide synthase (NOS) is involved, suggesting that the central role of nitric oxide synthesis is conserved in this species. The deficit
Facilitation of enkephalins catabolism inhibitor-induced antinociception by drugs classically used in pain management
Magdalena Mas Nieto; Jodie Wilson; Judith Walker; Jesus Benavides; Marie-Claude Fourniķ-Zaluski; Bernard P Roques; Florence Noble
The aim of this study was to investigate the facilitatory effects of subanalgesic or low doses of different drugs (acetylsalicylic acid, ibuprofen and morphine) on the antinociceptive responses induced by the endogenous opioid peptides, enkephalins, protected from their catabolism by the dual enkephalin-degrading enzymes inhibitor RB101. According to the analgesic profile of the three studied compounds different antinociceptive assays were
Endothelial Cells Inhibit Flow-Induced Smooth Muscle Cell Migration Role of Plasminogen Activator Inhibitor1
Eileen M. Redmond; John P. Cullen; Paul A. Cahill; James V. Sitzmann; Steingrimur Stefansson; Daniel A. Lawrence; S. Steve Okada
BackgroundŚThe endothelium may play a pivotal role in hemodynamic force-induced vascular remodeling. We investigated the role of endothelial cell (EC) plasminogen activator inhibitor-1 (PAI-1) in modulating flow-induced smooth muscle cell (SMC) migration. Methods and ResultsŚHuman SMCs cocultured with or without human ECs were exposed to static (0 mL\\/min) or flow (26 mL\\/min; shear stress 23 dyne\\/cm2) conditions for 24 hours
Xenobiotic-Induced Hepatocyte Proliferation Associated with Constitutive Active/Androstane Receptor (CAR) or Peroxisome Proliferator-Activated Receptor ? (PPAR?) Is Enhanced by Pregnane X Receptor (PXR) Activation in Mice
Numakura, Yuki; Kodama, Susumu; Miyata, Masaaki; Yamazoe, Yasushi; Yoshinari, Kouichi
Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR), constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor ? (PPAR?) play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPAR? activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital, or PPAR? activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16?-carbonitrile (PCN) alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPAR? is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics. PMID:23626729
Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells.
Wang, Bo; Van Veldhoven, Paul P; Brees, Chantal; Rubio, NoemĒ; Nordgren, Marcus; Apanasets, Oksana; Kunze, Markus; Baes, Myriam; Agostinis, Patrizia; Fransen, Marc
Many cellular processes are driven by spatially and temporally regulated redox-dependent signaling events. Although mounting evidence indicates that organelles such as the endoplasmic reticulum and mitochondria can function as signaling platforms for oxidative stress-regulated pathways, little is known about the role of peroxisomes in these processes. In this study, we employ targeted variants of the genetically encoded photosensitizer KillerRed to gain a better insight into the interplay between peroxisomes and cellular oxidative stress. We show that the phototoxic effects of peroxisomal KillerRed induce mitochondria-mediated cell death and that this process can be counteracted by targeted overexpression of a select set of antioxidant enzymes, including peroxisomal glutathione S-transferase kappa 1, superoxide dismutase 1, and mitochondrial catalase. We also present evidence that peroxisomal disease cell lines deficient in plasmalogen biosynthesis or peroxisome assembly are more sensitive to KillerRed-induced oxidative stress than control cells. Collectively, these findings confirm and extend previous observations suggesting that disturbances in peroxisomal redox control and metabolism can sensitize cells to oxidative stress. In addition, they lend strong support to the ideas that peroxisomes and mitochondria share a redox-sensitive relationship and that the redox communication between these organelles is not only mediated by diffusion of reactive oxygen species from one compartment to the other. Finally, these findings indicate that mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress, and this may have profound implications for our views on cellular aging and age-related diseases. PMID:23988789
Modulation of Osteoclastogenesis Induced by Nucleoside Reverse Transcriptase Inhibitors
PAN, GEORGE; KILBY, MICHAEL; McDONALD, JAY M.
Osteopenia is a common and debilitating side-effect of HAART, yet little is known concerning the effects of HAART on bone metabolism. We reported previously that zidovudine (AZT) stimulates osteoclastogenesis in vitro and causes osteopenia in mice. Here, we confirmed that the AZT-induced osteoclastogenesis is dependent on RANKL in that osteoclastogenesis is blocked by osteoprotegestin. Alendronate, which is used for the treatment of osteopenia and osteoporosis, failed to inhibit AZT-induced osteoclastogenesis in vitro. Osteoclastogenesis in vitro was not affected by tumor necrosis factor-?. Two other NRTI drugs, ddl and 3TC, also induced osteoclastogenesis in vitro and induced osteopenia in mice. The osteopenia was associated with an elevation of parameters of osteoclasts, but not with osteoblasts. Combinations of the NRTIs did not result in additive or synergistic effects in vitro or in vivo. Finally, AZT induced osteoclastogenesis of human osteoclast precursors in a RANKL-dependent manner. This in vitro osteoclastogenesis assay using human peripheral blood mononuclear cells could be useful in evaluating bone turnover and the risk of developing osteopenia in AIDS patients on HAART. PMID:17147500
PeroxisomeDB 2.0: an integrative view of the global peroxisomal metabolome.
Schl³ter, Agatha; Real-Chicharro, Alejandro; Gabald¾n, Toni; Sßnchez-Jimķnez, Francisca; Pujol, Aurora
Peroxisomes are essential organelles that play a key role in redox signalling and lipid homeostasis. They contain a highly diverse enzymatic network among different species, mirroring the varied metabolic needs of the organisms. The previous PeroxisomeDB version organized the peroxisomal proteome of humans and Saccharomyces cerevisiae based on genetic and functional information into metabolic categories with a special focus on peroxisomal disease. The new release (http://www.peroxisomeDB.org) adds peroxisomal proteins from 35 newly sequenced eukaryotic genomes including fungi, yeasts, plants and lower eukaryotes. We searched these genomes for a core ensemble of 139 peroxisomal protein families and identified 2706 putative peroxisomal protein homologs. Approximately 37% of the identified homologs contained putative peroxisome targeting signals (PTS). To help develop understanding of the evolutionary relationships among peroxisomal proteins, the new database includes phylogenetic trees for 2386 of the peroxisomal proteins. Additional new features are provided, such as a tool to capture kinetic information from Brenda, CheBI and Sabio-RK databases and more than 1400 selected bibliographic references. PeroxisomeDB 2.0 is a freely available, highly interactive functional genomics platform that offers an extensive view on the peroxisomal metabolome across lineages, thus facilitating comparative genomics and systems analysis of the organelle. PMID:19892824
The interaction between Helminthosporium carbonum and maize: Induced resistance and the role of an inhibitor
Cantone, F.A.
Helminthosporium carbonum race 1 produces large, necrotic lesions on susceptible leaves of maize, whereas race 2 causes small, chlorotic flecks. Resistance to race 1 on susceptible leaves was induced when race 2 was inoculated for at least 10 h prior to a challenge inoculation with the pathogen and was manifest as a decrease in the number of appressoria and reduced penetration by race 1 conidia. Induced resistance was prevented or reversed when HC-toxin was added to challenge race 1 inoculum. The basis for protection appears to be a volatile, inhibitory compound produced by the host. This inhibitor was always associated with treatments that resulted in resistance, whereas no inhibitory activity was detected in diffusates from susceptible reactions. The appearance of inhibitor in diffusates coincided with the appearance of protection on the leaf. In addition to race 2 of H. carbonum, other fungi (H. victoriae, H. turcicum, and Alternaria) also induced production of the inhibitor as well as resistance to race 1. The inhibitor prevented the germination of conidia of all fungi tested. The growth of two phytopathogenic bacteria was also completely inhibited. Incorporation of {sup 3}H-leucine and {sup 14}C-uridine into protein and RNA, respectively, by conidia of H. carbonum was prevented within 15 min of exposure to inhibitor. In addition, respiration of conidia in inhibitor was reduced within 90 min to just 25% of the rate of conidia germinated in water. However, inhibitory activity of the diffusates was readily reversed when conidia were rinsed with water or when organic or amino acids were added to inhibited conidia. The addition of sodium acetate to race 2 and race 1 inocula resulted in lesion enlargement and also nullified inhibitory activity in vitro.
Paradoxical Reaction to Golimumab: Tumor Necrosis Factor ? Inhibitor Inducing Psoriasis Pustulosa
Soto Lopes, Marien Siqueira; Trope, Beatriz Moritz; Rochedo Rodriguez, Maria Paula Rua; Grynszpan, Rachel Lima; Cuzzi, Tullia; Ramos-e-Silva, Marcia
Importance Golimumab is a human monoclonal antibody, used for rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis. Adverse reactions are increasing with this class of medication (tumor necrosis factor ? inhibitors). Observations The authors present a case of a female patient who presented with psoriasis pustulosa after the use of golimumab for rheumatoid arthritis. Conclusions and Relevance Paradoxically, in this case, golimumab, which is used for psoriasis, induced the pustular form of this disease. We are observing an increasing number of patients who develop collateral effects with tumor necrosis factor ? inhibitors, and the understanding of the mechanism of action and how these adverse reactions occur may contribute to avoid these sometimes severe situations. PMID:24348382
Effect of inhibitors of proteolysis and arachidonic acid metabolism on burn-induced protein breakdown.
Odessey, R
A rat model has been developed to study the local effects of burn injury on the underlying muscle tissue. Protein turnover was measured in soleus muscle incubated in vitro in which both tyrosine release and protein synthesis was measured. A scald injury (3 seconds) to a small area of one hindlimb produces an increase in muscle proteolysis and is without effect on the soleus muscle of the contralateral leg. A very high concentration of indomethacin (40 mumol/L) had no effect on proteolysis in the control muscle but specifically inhibited burn-induced protein breakdown. However, since other cyclooxygenase inhibitors (aspirin and ibuprofen), lipoxygenase inhibitors (ETYA, NDGA, and esculetin), and mepacrine (a phospholipase inhibitor) had no effect on protein breakdown, it is unlikely that a product of arachidonic acid metabolism maintains the increased proteolysis in vitro. In addition, endogenous production of prostaglandin E2 (PGE2) was not different in muscles from burned and control legs. Probes of the proteolytic pathway using inhibitors show that the burn-induced stimulation of proteolysis is consistent with the stimulation of lysosomal protease activity. These results are supported by the observation of increased acid protease activity in muscle homogenates from the burned leg. The best hypothesis that explains these data is that a lysosomal pathway of protein degradation may be enhanced by burn. Products of arachidonic acid metabolism do not appear to maintain burn-induced proteolysis in muscle, although their role in initiating the pathological changes in vivo cannot be excluded. PMID:3925289
Treatment of hypertension and renal injury induced by the angiogenesis inhibitor sunitinib: preclinical study.
Lankhorst, Stephanie; Kappers, Mariļtte H W; van Esch, Joep H M; Smedts, Frank M M; Sleijfer, Stefan; Mathijssen, Ron H J; Baelde, Hans J; Danser, A H Jan; van den Meiracker, Anton H
Common adverse effects of angiogenesis inhibition are hypertension and renal injury. To determine the most optimal way to prevent these adverse effects and to explore their interdependency, the following drugs were investigated in unrestrained Wistar Kyoto rats exposed to the angiogenesis inhibitor sunitinib: the dual endothelin receptor antagonist macitentan; the calcium channel blocker amlodipine; the angiotensin-converting enzyme inhibitor captopril; and the phosphodiesterase type 5 inhibitor sildenafil. Mean arterial pressure was monitored telemetrically. After 8 days, rats were euthanized and blood samples and kidneys were collected. In addition, 24-hour urine samples were collected. After sunitinib start, mean arterial pressure increased rapidly by ?30 mm Hg. Coadministration of macitentan or amlodipine largely prevented this rise, whereas captopril or sildenafil did not. Macitentan, captopril, and sildenafil diminished the sunitinib-induced proteinuria and endothelinuria and glomerular intraepithelial protein deposition, whereas amlodipine did not. Changes in proteinuria and endothelinuria were unrelated. We conclude that in our experimental model, dual endothelin receptor antagonism and calcium channel blockade are suitable to prevent angiogenesis inhibition-induced hypertension, whereas dual endothelin receptor antagonism, angiotensin-converting enzyme inhibitor, and phosphodiesterase type 5 inhibition can prevent angiogenesis inhibition-induced proteinuria. Moreover, the variable response of hypertension and renal injury to different antihypertensive agents suggests that these side effects are, at least in part, unrelated. PMID:25185126
Ibulocydine is a novel prodrug Cdk inhibitor that effectively induces apoptosis in hepatocellular carcinoma cells.
Cho, Seung-Ju; Kim, Young-Jong; Surh, Young-Joon; Kim, B Moon; Lee, Seung-Ki
Hepatocellular carcinoma (HCC) is frequently associated with abnormalities in cell cycle regulation, leading to increased activity of cyclin-dependent kinases (Cdks) due to the loss, or low expression of, Cdk inhibitors. In this study, we showed that ibulocydine (an isobutyrate prodrug of the specific Cdk inhibitor, BMK-Y101) is a candidate anti-cancer drug for HCC. Ibulocydine has high activity against Cdk7/cyclin H/Mat1 and Cdk9/cyclin T. Ibulocydine inhibited the growth of HCC cells more effectively than other Cdk inhibitors, including olomoucine and roscovitine, whereas ibulocydine as well as the other Cdk inhibitors and BMK-Y101 minimally influenced the growth of normal hepatocyte cells. Ibulocydine induced apoptosis in HCC cells, most likely by inhibiting Cdk7 and Cdk9. In vitro treatment of HCC cells with ibulocydine rapidly blocked phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, a process mediated by Cdk7/9. Anti-apoptotic gene products such as Mcl-1, survivin, and X-linked IAP (XIAP) are crucial for the survival of many cell types, including HCC. Following the inhibition of RNA polymerase II phosphorylation, ibulocydine caused rapid down-regulation of Mcl-1, survivin, and XIAP, thus inducing apoptosis. Furthermore, ibulocydine effectively induced apoptosis in HCC xenografts with no toxic side effects. These results suggest that ibulocydine is a strong candidate anti-cancer drug for the treatment of HCC. PMID:21478145
A family of cytokine-inducible inhibitors of signalling
Robyn Starr; Tracy A. Willson; Elizabeth M. Viney; Leecia J. L. Murray; John R. Rayner; Brendan J. Jenkins; Thomas J. Gonda; Warren S. Alexander; Donald Metcalf; Nicos A. Nicola; Douglas J. Hilton
Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of theSTAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT
Titchenell, Paul M.; Lin, Cheng-Mao; Keil, Jason M.; Sundstrom, Jeffrey M.; Smith, Charles D.; Antonetti, David A.
SYNOPSIS Pro-inflammatory cytokines and growth factors such as vascular endothelial growth factor (VEGF) contribute to the loss of the blood-retinal barrier (BRB) and subsequent macular edema in various retinal pathologies. VEGF signaling requires conventional PKC (PKC?) activity; however, PKC? inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability suggesting the involvement of alternative signaling pathways. Here, we provide evidence for the involvement of atypical protein kinase C (aPKC) signaling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small molecule inhibitors and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. These data suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis and the blood-brain barrier (BBB) in the presence of brain tumors. PMID:22721706
Lucanthone Is a Novel Inhibitor of Autophagy That Induces Cathepsin D-mediated Apoptosis*
Carew, Jennifer S.; Espitia, Claudia M.; Esquivel, Juan A.; Mahalingam, Devalingam; Kelly, Kevin R.; Reddy, Guru; Giles, Francis J.; Nawrocki, Steffan T.
Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53+/+ and p53?/? HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models. PMID:21148553
Bile acids: the role of peroxisomes
Ferdinandusse, Sacha; Denis, Simone; Faust, Phyllis L.; Wanders, Ronald J. A.
It is well established that peroxisomes play a crucial role in de novo bile acid synthesis. Studies in patients with a peroxisomal disorder have been indispensable for the elucidation of the precise role of peroxisomes. Several peroxisomal disorders are associated with distinct bile acid abnormalities and each disorder has a characteristic pattern of abnormal bile acids that accumulate, which is often used for diagnostic purposes. The patients have also been important for determining the pathophysiological consequences of defects in bile acid biosynthesis. In this review, we will discuss all the peroxisomal steps involved in bile acid synthesis and the bile acid abnormalities in patients with peroxisomal disorders. We will show the results of bile acid measurements in several tissues from patients, including brain, and we will discuss the toxicity and the pathological effects of the abnormal bile acids. PMID:19357427
A Case of Tumor Necrosis Factor-? Inhibitors-induced Pustular Psoriasis
Park, Jae-Jeong
Anti-tumor necrosis factor (TNF)-? agents promise better disease control for the treatment of ankylosing spondylitis resistant to classical disease-modifying treatments. Etanercept, a recombinant human TNF receptor fusion protein, is used to treat a variety of TNF-?-mediated diseases by inhibiting the biological activity of TNF-?. We experienced a case of pustular psoriasis in a 32-year-old man during anti-TNF-? therapy with etanercept. He had a history of ankylosing spondylitis for 2 years. Two years after treatment of etanercept, erythematous pustules developed on his palms and soles. He had no previous history of pustular psoriasis. The skin lesion improved as the etanercept therapy was stopped, but pustular skin eruption recurred as adalimumab, a different TNF-? inhibitor, was administered to manage his ankylosing spondylitis. Several TNF-? inhibitors have different molecular structures, but these inhibitors might have a similar potency to induce pustular psoriasis from this case. PMID:20548918
Aromatase Inhibitor-Induced Erythrocytosis in a Patient Undergoing Hormonal Treatment for Breast Cancer
Yeruva, Sri Lakshmi Hyndavi; Ogbonna, Onyekachi Henry; Oneal, Patricia
Aromatase inhibitors (AIs) are most commonly used for breast cancer patients with hormone receptor positive disease. Although the side effect profile of aromatase inhibitors is well known, including common side effects like arthralgia, bone pain, arthritis, hot flashes, and more serious problems like osteoporosis, we present a case of an uncommon side effect of these medications. We report the case of a postmenopausal woman on adjuvant hormonal therapy with anastrozole after completing definitive therapy for stage IIIB estrogen receptor-positive breast cancer, who was referred to hematology service for evaluation of persistent erythrocytosis. Primary and known secondary causes of polycythemia were ruled out. On further evaluation, we found that her erythrocytosis began after initiation of anastrozole and resolved after it was discontinued. We discuss the pathophysiology of aromatase inhibitor-induced erythrocytosis and reference of similar cases reported in the literature.
A novel histone deacetylase inhibitor Chidamide induces apoptosis of human colon cancer cells
Liu, Lin [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China)] [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China); Chen, Baoan, E-mail: email@example.com [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China)] [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China); Qin, Shukui [Chinese PLA Cancer Center, The 81st PLA Hospital, Nanjing 210002, Jiangsu (China)] [Chinese PLA Cancer Center, The 81st PLA Hospital, Nanjing 210002, Jiangsu (China); Li, Suyi; He, Xiangming [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China)] [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China); Qiu, Shaomin; Zhao, Wei; Zhao, Hong [Department of Internal Medicine, Nanjing Municipal Cancer Hospital, Nanjing 210003, Jiangsu (China)] [Department of Internal Medicine, Nanjing Municipal Cancer Hospital, Nanjing 210003, Jiangsu (China)
Many studies have demonstrated that histone deacetylase (HDAC) inhibitors induce various tumor cells to undergo apoptosis, and such inhibitors have been used in different clinical trials against different human cancers. In this study, we designed and synthesized a novel HDAC inhibitor, Chidamide. We showed that Chidamide was able to increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, which resulted in arresting colon cancer cells at the G1 phase of the cell cycle and promoting apoptosis. As a result, the proliferation of colon cancer cells was suppressed in vitro. Our data support the potential application of Chidamide as an anticancer agent in treating colon cancer. Future studies are needed to demonstrate its in vivo efficacy.
An aqueous extract of Salacia oblonga root, a herb-derived peroxisome proliferator-activated receptor-alpha activator, by oral gavage over 28 days induces gender-dependent hepatic hypertrophy in rats.
Rong, Xianglu; Kim, Moon Sun; Su, Ning; Wen, Suping; Matsuo, Yukimi; Yamahara, Johji; Murray, Michael; Li, Yuhao
Activation of peroxisome proliferator-activated receptor (PPAR)-alpha by natural and synthetic chemicals induces hepatic hypertrophy. An aqueous extract of Salacia oblonga root (SOW) is an Ayurvedic medicine with anti-diabetic and anti-obesity properties. In the present study, it was found that SOW (100, 300 and 900mg/kg, once daily by oral gavage over a 28 day period) elicited dose-related increases in liver weight (LW) by 1.6%, 13.4% and 42.5%, respectively, and in the ratio of LW to body weight by 8.8%, 16.7% and 40.2%, respectively, in male rats. These effects were less pronounced in females. SOW selectively increased liver mass in male rats but Sudan red staining was not different, which indicates that hepatic lipid accumulation was similar in both genders. However, SOW even at the highest dosage did not influence serum ALT and AST activities in male or female rats. Moreover, SOW was found to activate PPAR-alpha in human hepatoma-derived HepG2 cells, as evidenced by the upregulation of PPAR-alpha and acyl-CoA oxidase mRNA expression. Thus, SOW-dependent PPAR-alpha activation may precede the development of the gender difference in hepatic hypertrophy; this process may be influenced by sex hormone status. PMID:18397819
The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals
We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PER1p contains both a carboxy- (PTS1) and an amino- terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle. In wild-type H. polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support. PMID:7962056
Peroxisome biogenesis disorders: The role of peroxisomes and metabolic dysfunction in developing brain
P. L. Faust; D. Banka; R. Siriratsivawong; V. G. Ng; T. M. Wikander
Peroxisome biogenesis disorders, of which Zellweger syndrome is the most severe, result in severe neurological dysfunction associated with abnormal CNS neuronal migrations due to the lack of functional peroxisomes. The PEX2-\\/- mouse model for Zellweger syndrome has enabled us to evaluate the role of peroxisomes in the development and functioning of the nervous system. These studies have shown that, in
A novel role for the apoptosis inhibitor ARC in suppressing TNF?-induced regulated necrosis
Kung, G; Dai, P; Deng, L; Kitsis, R N
TNF? signaling can promote apoptosis or a regulated form of necrosis. ARC (apoptosis repressor with CARD (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. We discovered that ARC blocks not only apoptosis but also necrosis. TNF?-induced necrosis was abrogated by overexpression of wild-type ARC but not by a CARD mutant that is also defective for inhibition of apoptosis. Conversely, knockdown of ARC exacerbated TNF?-induced necrosis, an effect that was rescued by reconstitution with wild-type, but not CARD-defective, ARC. Similarly, depletion of ARC in vivo exacerbated necrosis caused by infection with vaccinia virus, which elicits severe tissue damage through this pathway, and sensitized mice to TNF?-induced systemic inflammatory response syndrome. The mechanism underlying these effects is an interaction of ARC with TNF receptor 1 that interferes with recruitment of RIP1, a critical mediator of TNF?-induced regulated necrosis. These findings extend the role of ARC from an apoptosis inhibitor to a regulator of the TNF? pathway and an inhibitor of TNF?-mediated regulated necrosis. PMID:24440909
The use of salt-inducible kinase inhibitors to treat autoimmune and inflammatory diseases: evaluation of WO2013136070.
Norman, Peter
Novel methods for the treatment of inflammatory and autoimmune diseases comprising the administration of salt-inducible kinase inhibitors are claimed. One novel inhibitor (HG-9-91-01) and the use of 2,4-diaminopyrimidine and 2,6-diaminopyrimidine derivatives are claimed. The use of such inhibitors upregulates the level of the anti-inflammatory cytokine IL-10 in macrophages. PMID:24745372
Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells
When epithelial cell cultures are transferred from a medium with a normal extracellular calcium concentration (1-2 mM) to a medium with a low extracellular calcium concentration (LC, less than 50 microM free Ca2+) cell-cell contacts are disrupted, and the tight junction- dependent transepithelial resistance drops. In this study, I used MDCK epithelial cells to investigate the effects of LC on the localization of the tight junction protein cingulin, and the role of protein kinases in the events induced by LC. Immunofluorescence analysis showed that within 15 min of incubation of confluent monolayers in LC, cingulin labeling was dislocated from the cell periphery, as an array of granules forming a ring-like structure. At later times after calcium removal, cingulin labeling appeared mostly cytoplasmic, in a diffuse and granular pattern, and cells appeared rounded and smaller. These events were not influenced by lack of serum, or by preincubation with 10 mM sodium azide or 6 mg/ml of cycloheximide. However, the disruption of cell-cell contacts, the cell shape changes, and the redistribution of cingulin and other junctional proteins induced by LC were inhibited when cells were pretreated with the protein kinase inhibitor H-7 (greater than or equal to 30 microM). The inhibitors H-8 and, to a lesser degree, staurosporine were also effective, whereas HA-1004 and ML-7 showed essentially no activity, suggesting a specificity of action of different inhibitors. Measurement of the transepithelial resistance showed that the kinase inhibitors that could prevent junction disassembly could also reduce the drop in transepithelial resistance induced by LC. Dose-response curves demonstrated that H-7 is the most effective among the inhibitors, and the transepithelial resistance was 70% of control up to 1 h after calcium removal. These results suggest that low extracellular calcium modulates junctional integrity and cytoskeletal organization through an effector system involving protein kinases. PMID:1556151
Neuroprotective effect of cyclooxygenase inhibitors in ICV-STZ induced sporadic Alzheimer's disease in rats.
Dhull, Dinesh Kumar; Jindal, Ankur; Dhull, Rakesh K; Aggarwal, Saurabh; Bhateja, Deepak; Padi, Satyanarayana S V
Sporadic Alzheimer's disease is an age-related neurological and psychiatric disorder characterized by impaired energy metabolism. Oxidative stress and neuroinflammation have been implicated in pathophysiology of sporadic type of dementia. The central streptozotocin administration induces behavioral and biochemical alterations resembling those in sporadic type of Alzheimer's patients. The present study was designed to investigate the effects of chronic pretreatment with cyclooxygenase-1 or cyclooxygenase-2 or cyclooxygenase-3 selective inhibitors on cognitive dysfunction and oxidative stress markers in intracerebroventricular streptozotocin-treated rats. Chronic treatment with valeryl salicylate (5 and 10 mg/kg, i.p.) and etoricoxib (5 and 10 mg/kg, i.p.) on a daily basis for a period of 21 days, beginning 1 h prior to first intracerebroventricular streptozotocin injection, significantly improved streptozotocin-induced cognitive impairment. However, phenacetin (20 and 40 mg/kg, i.p.) failed to restore the cognitive performances of streptozotocin-treated rats. Besides, improving cognitive dysfunction, chronic administration of highly selective cyclooxygenase-1 and/or cyclooxygenase-2 inhibitors (valeryl salicylate and etoricoxib, respectively), but not cyclooxygenase-3 inhibitor (phenacetin), significantly reduced elevated malondialdehyde, nitrite levels, and restored reduced glutathione and superoxide dismutase levels. Furthermore, cyclooxygenase-1 and/or cyclooxygenase-2 inhibitors significantly increased the survival of pyramidal neurons. In summary, we demonstrate for the first time that both cyclooxygenase-1 and cyclooxygenase-2 isoforms, but not cyclooxygenase-3, are involved in the progression of neuronal damage in intracerebroventricular streptozotocin-treated rats. PMID:21701788
Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells
Obeng, Esther A.; Carlson, Louise M.; Gutman, Delia M.; Harrington, William J.; Lee, Kelvin P.; Boise, Lawrence H.
Multiple myeloma (MM) is an incurable plasma cell malignancy. The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in MM cells; however, the nature of its selectivity remains unknown. Here we demonstrate that 5 different MM cell lines display similar patterns of sensitivity to 3 proteasome inhibitors (PIs) but respond differently to specific NF-?B inhibition. We further show that PIs initiate the unfolded protein response (UPR), a signaling pathway activated by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). Consistent with reports that prosurvival/physiologic UPR components are required for B-cell differentiation into antibody-secreting cells, we found that MM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. However, bortezomib rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress¢specific eIF-2? kinase; ATF4, an ER stress¢induced transcription factor; and its proapoptotic target, CHOP/GADD153. Consistent with our hypothesis that PIs induce the accumulation of misfolded ER-processed proteins, we found that the amount of immunoglobulin subunits retained within MM cells correlated with their sensitivity to PIs. These findings suggest that MM cells have a lower threshold for PI-induced UPR induction and ER stress¢induced apoptosis because they constitutively express ER stress survival factors to function as secretory cells. PMID:16507771
The effect of topically applied prostaglandin inhibitors on the laser-induced disruption of the blood aqueous barrier
W. Schrems; H. P. Dorp; S. Mager; G. K. Krieglstein
The therapeutic effect of topically applied prostaglandin inhibitors on the laser-induced disruption of the blood-aqueous barrier was investigated in six series of five rabbits each. One series was not coagulated and served as baseline, and in a reference group laser coagulation was performed without pretreatment with a prostaglandin inhibitor. In four series the iris laser coagulation of the left eyes
The STAT3 inhibitor WP1066 reverses the resistance of chronic lymphocytic leukemia cells to histone deacetylase inhibitors induced by interleukin-6.
Lu, Kang; Fang, Xiao-sheng; Feng, Li-li; Jiang, Yu-jie; Zhou, Xiang-xiang; Liu, Xin; Li, Pei-pei; Chen, Na; Ding, Mei; Wang, Na; Zhang, Jie; Wang, Xin
Interleukin-6 (IL-6) is a pleiotropic cytokine produced by a variety of cell types, including fibroblasts, endothelial cells, lymphocytes, and bone marrow stromal cells (BMSCs). Levels of IL-6 are increased in serum of CLL patients and correlated with adverse clinical features and short survival. In our study, we observed that IL-6 induced the resistance of CLL cells to pan-histone deacetylase (HDAC) inhibitors vorinostat (SAHA) and panobinostat (LBH589). Furthermore, low concentrations of SAHA and LBH589 enhanced the activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway induced by IL-6 in CLL cells. All of these effects were blocked by the STAT3-selective inhibitor, WP1066. Meanwhile, WP1066 decreased the expressions of Mcl-1 and Bcl-xL protein induced by IL-6 with or without low concentrations of HDAC inhibitors. Co-culture of CLL cells with BMSCs could also facilitate the activation of STAT3 and protected CLL cells from apoptosis when treated with HDAC inhibitors, and this cytoprotection was reversed by WP1066. The present study indicated that IL-6 or co-culture with BMSCs prevented HDAC inhibitor-induced apoptosis of CLL cells. This prevention was mediated by activation of the STAT3 signaling pathway. Moreover, WP1066 reversed the resistance of CLL cells to SAHA and LBH589 induced by either IL-6 or co-culture with BMSCs. Our findings suggest that targeting the STAT3 pathway may be a novel way to improve the efficacy of the HDAC inhibitor in CLL patients by overcoming antiapoptotic signaling of the microenvironment. PMID:25636517
Peroxisome Proliferator-Binding Protein: Identification and Partial Characterization of Nafenopin-, Clofibric Acid, and Ciprofibrate-Binding Proteins from Rat Liver
Narendra D. Lalwani; Keith Alvares; M. Kumudavalli Reddy; M. Narahari Reddy; Indu Parikh; Janardan K. Reddy
Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid
Small Molecular Inhibitor of Transforming Growth Factor-? Protects Against Development of Radiation-Induced Lung Injury
Mitchell S. Anscher; Bradley Thrasher; Larisa Zgonjanin; Zahid N. Rabbani; Michael J. Corbley; Kai Fu; Lihong Sun; Wen-Cherng Lee; Leona E. Ling; Zeljko Vujaskovic
Purpose: To determine whether an anti-transforming growth factor- (TGF-) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. Methods and Materials: One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g\\/kg or 0.15 g\\/kg) beginning 7 days before RT. The rats were
ACE genotype and ACE inhibitors induced renoprotection in chronic proteinuric nephropathies1
Annalisa Perna; Piero Ruggenenti; Alessandra Testa; Belinda Spoto; Roberto Benini; Valerio Misefari; Giuseppe Remuzzi; Carmine Zoccali
ACE genotype and ACE induced renoprotection in chronic proteinuric nephropathies.BackgroundWhether angiotensin-converting enzyme (ACE) gene polymorphism affects disease progression and response to ACE inhibitor therapy in nondiabetic proteinuric nephropathies is not clearly established.MethodsThe relationship between insertion\\/deletion (I\\/D) genotypes and proteinuria, rate of glomerular filtration rate decline (?GFR)Ścentrally evaluated by repeated measures of iohexol plasma clearanceŚand incidence of end-stage renal disease (ESRD)
Protein tyrosine kinase inhibitors prevent didemnin B-induced apoptosis in HL60 cells
Karina L. Johnson; Franńois Vaillant; Lawen Alfons
Didemnin B induces rapid apoptosis in human promyeloid HL-60 cells with an optimal concentration of 1 ?M (Grubb et al. (1995) Biochem. Biophys. Res. Commun. 215, 1130¢1136), but little is known about how it does so. In order to determine whether protein tyrosine phosphorylation is involved in this rapid induction of apoptosis, HL-60 cells were pre-treated with tyrosine kinase inhibitors
Serotonin reuptake inhibitors reduce conditioned fear stress-induced freezing behavior in rats
S. Hashimoto; T. Inoue; T. Koyama
Conditioned fear stress (CFS)-induced freezing behavior has been proposed as an animal model of anxiety. In the present study, freezing was used to determine the anxiolytic activity of selective serotonin reuptake inhibitors (SSRIs), which are reported to be clinically effective in anxiety disorders. The duration of freezing behavior was reduced by acute treatment with the SSRIs citalopram (1¢10 mg\\/kg) and
Novel selective Cox-2 inhibitors induce apoptosis in Caco-2 colorectal carcinoma cell line.
Entezari Heravi, Reza; Hadizadeh, Farzin; Sankian, Mojtaba; Tavakol Afshari, Jalil; Taghdisi, Seyed Mohammad; Jafarian, Hanieh; Behravan, Javad
The cyclooxygenase-2 (COX-2) inhibitors including celecoxib inhibit cell growth and induce apoptosis in cancer cells. As COX-2 is over expressed in solid tumors such as colorectal cancer, it can be a suitable target for cancer treatment studies. In this study we designed and synthesized 4,5-bisaryl imidazolyl imidazoles as novel COX-2 inhibitors and evaluated their apoptosis inducing activities. The ability of our synthetic compounds to inhibit ovine COX-1 and COX-2 was determined using a colorimetric method. The effects of these COX-2 inhibitors and celecoxib on the proliferation of Caco-2 cells were evaluated by MTT assay. Cell apoptosis was determined by flow cytometry and DNA fragmentation assay. cDNA microarray technique was used to evaluate the effects of these synthetic compounds on 112 genes involved in apoptosis pathways. The expression of five apoptosis-related genes Bak-1, Bcl-x, BIRC (Survivin), TNFSF10 and CASP3 were evaluated by quantitative real-time PCR. Among our synthetic compounds (3a-c), 4,5-bis(4-methoxyphenyl)-1H-imidazol-2-yl derivative (compound 3c) exhibited the highest COX-1/COX-2 selectivity index (SI=262.9) and lowest growth inhibitory concentration (IC(50)=21.20?M). In addition, compounds 3a-c could up-regulate pro-apoptotic genes and down-regulate anti-apoptotic genes. So, these synthetic compounds seem to be inducers of apoptosis in Caco-2 cell line. This study indicates that 4,5-bisaryl imidazolyl imidazole is a suitable scaffold to design COX-2 inhibitors and 4,5-bis(4-methoxyphenyl)-1H-imidazol-2-yl derivative exhibited highly COX-2 inhibitory potency and selectivity even more than celecoxib. It seems that it could induce apoptosis in Caco-2 colorectal carcinoma cell line. PMID:21939759
An endogenous sleep-inducing compound is a novel competitive inhibitor of fatty acid amide hydrolase
Matthew P. Patricelli; Jean E. Patterson; Dale L. Boger; Benjamin F. Cravattb
2-Octyl ?-bromoacetoacetate (O?Br), an endogenous compound originally isolated from human cerebrospinal fluid (CSF), has previously been demonstrated to increase REM sleep duration in cats. Based on the chemical structure of O?Br and its reported sleep-inducing effects, we synthesized O?Br along with chemically related analogs and tested these compounds as inhibitors of fatty acid amide hydrolase (FAAH), a brain enzyme that
Effect of Ethylene Action Inhibitors upon Wound-Induced Gene Expression in Tomato Pericarp.
Henstrand, J M; Handa, A K
The contribution of wound-ethylene to wound-induced gene expression was investigated in unripe tomato pericarp using inhibitors of ethylene action. Wounded unripe tomato pericarp was treated with 2,5-norbornadiene or silver thiosulfate to inhibit specifically the induction of ethylene-dependent mRNA species. Poly(A)(+) RNAs isolated from these tissues after 12 hours of wounding were translated in vitro in a rabbit reticulocyte lysate system and [(35)S]methionine-labeled polypeptides were compared to unwounded controls after separation by one and two-dimensional polyacrylamide gel electrophoresis. Results show that mechanical wounding induces a dramatic shift in gene expression (over 50 mRNA species) but expression of less than 15% of these genes is affected by the treatment with ethylene action inhibitors. A selective decrease in mRNAs coding for a 37 kilodalton doublet and 75 kilodalton polypeptides is observed in 2,5-norbornadiene and silver thiosulfate treated wounded pericarp. Levels of hydroxyproline-rich glycoprotein mRNAs induced in wounded tissue were not influenced by inhibitors of ethylene action. PMID:16666989
Effect of Ethylene Action Inhibitors upon Wound-Induced Gene Expression in Tomato Pericarp 1
Henstrand, John M.; Handa, Avtar K.
The contribution of wound-ethylene to wound-induced gene expression was investigated in unripe tomato pericarp using inhibitors of ethylene action. Wounded unripe tomato pericarp was treated with 2,5-norbornadiene or silver thiosulfate to inhibit specifically the induction of ethylene-dependent mRNA species. Poly(A)+ RNAs isolated from these tissues after 12 hours of wounding were translated in vitro in a rabbit reticulocyte lysate system and [35S]methionine-labeled polypeptides were compared to unwounded controls after separation by one and two-dimensional polyacrylamide gel electrophoresis. Results show that mechanical wounding induces a dramatic shift in gene expression (over 50 mRNA species) but expression of less than 15% of these genes is affected by the treatment with ethylene action inhibitors. A selective decrease in mRNAs coding for a 37 kilodalton doublet and 75 kilodalton polypeptides is observed in 2,5-norbornadiene and silver thiosulfate treated wounded pericarp. Levels of hydroxyproline-rich glycoprotein mRNAs induced in wounded tissue were not influenced by inhibitors of ethylene action. Images Figure 2 Figure 3 Figure 4 PMID:16666989
Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves.
Farmer, E E; Ryan, C A
Inducible defensive responses in plants are known to be activated locally and systemically by signaling molecules that are produced at sites of pathogen or insect attacks, but only one chemical signal, ethylene, is known to travel through the atmosphere to activate plant defensive genes. Methyl jasmonate, a common plant secondary compound, when applied to surfaces of tomato plants, induces the synthesis of defensive proteinase inhibitor proteins in the treated plants and in nearby plants as well. The presence of methyl jasmonate in the atmosphere of chambers containing plants from three species of two families, Solanaceae and Fabaceae, results in the accumulation of proteinase inhibitors in leaves of all three species. When sagebrush, Artemisia tridentata, a plant shown to possess methyl jasmonate in leaf surface structures, is incubated in chambers with tomato plants, proteinase inhibitor accumulation is induced in the tomato leaves, demonstrating that interplant communication can occur from leaves of one species of plant to leaves of another species to activate the expression of defensive genes. PMID:11607107
Interferon-? and cyclooxygenase-2 inhibitor cooperatively mediates TRAIL-induced apoptosis in hepatocellular carcinoma.
Zuo, Chaohui; Qiu, Xiaoxin; Liu, Nianli; Yang, Darong; Xia, Man; Liu, Jingshi; Wang, Xiaohong; Zhu, Haizhen; Xie, Hailong; Dan, Hanguo; Li, Qinglong; Wu, Qunfeng; Burns, Michael; Liu, Chen
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Interferon-alpha (IFN-?) has recently been recognized to harbor therapeutic potential in the prevention and treatment of HCC, but it remains controversial as to whether IFN-? exerts direct cytotoxicity against HCC. Cyclooxygenase-2 (COX-2) is overexpressed in HCC and is considered to play a role in hepatocarcinogenesis. Therefore, we aimed to elucidate the combined effect of a COX-2 inhibitor, celecoxib, and IFN-? on in vitro growth suppression of HCC using the hepatoma cell line HLCZ01 and the in vivo nude mouse xenotransplantation model using HLCZ01 cells. Treatment with celecoxib and IFN-? synergistically inhibited cell proliferation in a dose- and time-dependent manner. Apoptosis was identified by 4?,6-diamidino-2-phenylindole dihydrochloride and fluorescent staining. IFN-? upregulated the expression of TRAIL, while celecoxib increased the expression of TRAIL receptors. The combined regimen with celecoxib and IFN-? reduced the growth of xenotransplanted HCCs in nude mice. The regulation of IFN-?- and COX-2 inhibitor-induced cell death is impaired in a subset of TRAIL-resistant cells. The molecular mechanisms of HCC cells resistant to TRAIL-induced apoptosis were explored using molecular biological and immunological methods. Interferon-? and the COX-2 inhibitor celecoxib synergistically increased TRAIL-induced apoptosis in hepatocellular carcinoma. These data suggest that IFN-? and celecoxib may offer a novel role with important implications in designing new therapeutics for TRAIL-resistant tumors. PMID:25724899
Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae
The goal of this research is to identify and characterize the protein machinery that functions in the intracellular translocation and assembly of peroxisomal proteins in Saccharomyces cerevisiae. Several genes encoding proteins that are essential for this process have been identified previously by Kunau and collaborators, but the mutant collection was incomplete. We have devised a positive selection procedure that identifies new mutants lacking peroxisomes or peroxisomal function. Immunofluorescence procedures for yeast were simplified so that these mutants could be rapidly and efficiently screened for those in which peroxisome biogenesis is impaired. With these tools, we have identified four complementation groups of peroxisome biogenesis mutants, and one group that appears to express reduced amounts of peroxisomal proteins. Two of our mutants lack recognizable peroxisomes, although they might contain peroxisomal membrane ghosts like those found in Zellweger syndrome. Two are selectively defective in packaging peroxisomal proteins and moreover show striking intracellular clustering of the peroxisomes. The distribution of mutants among complementation groups implies that the collection of peroxisome biogenesis mutants is still incomplete. With the procedures described, it should prove straightforward to isolate mutants from additional complementation groups. PMID:7902359
Further studies of auxin and ACC induced feminization in the cucumber plant using ethylene inhibitors
NASA Technical Reports Server (NTRS)
Takahashi, H.; Jaffe, M. J.
The present study was designed to establish the role of an essential hormone controlling sex expression in cucumber. A potent anti-ethylene agent, AgNO3, completely inhibited pistillate flower formation caused by IAA, ACC or ethephon. Inhibitors of ethylene biosynthesis, AVG and CoCl2 also suppressed feminization due to exogenous IAA or ACC. Though AVG also suppressed ethephon-induced feminization, this may be due to the second effect of AVG rather than the effect on ACC biosynthesis. These results confirm that ethylene is a major factor regulating feminization and that exogenous auxin induces pistillate flower formation through its stimulation of ethylene production, rather than ACC production.
Effects of inhibitors on 1-methyladenine induced maturation of starfish oocytes
NASA Astrophysics Data System (ADS)
Lee, Harold H.; Xu, Quanhan
1-methladenine (1-MA) induces starfish oocytes maturation via surface reaction followed by the appearance of a cytoplasmic maturation factor which in turn induces germinal vesicle breakdown (GVBD) to resume meiosis. Cellular mechanisms involved in GVBD were investigated by microinjection of metabolic inhibitors. Colchicine (Co) inhibited maturation, cytochalasin-B (CB) delayed GVBD and actinomycin-D-(Act-D) and puromycin (Pu) had no effect. It appears that the microtubule and the microfilament systems are associated with the nuclear membrane dissolution during the process of oocyte maturation of starfish.
Inhibition of peroxisome-proliferator-activated receptor (PPAR)alpha by MK886.
Kehrer, J P; Biswal, S S; La, E; Thuillier, P; Datta, K; Fischer, S M; Vanden Heuvel, J P
Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may also indicate that PPAR alpha is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPAR alpha, making it the first compound identified to have such an effect. PMID:11389700
Histone Deacetylase Inhibitors Facilitate Dihydroartemisinin-Induced Apoptosis in Liver Cancer In Vitro and In Vivo
Zhang, Chris Zhiyi; Pan, Yinghua; Cao, Yun; Lai, Paul B. S.; Liu, Lili; Chen, George Gong; Yun, Jingping
Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy. PMID:22761917
Translation inhibitors induce formation of cholesterol ester-rich lipid droplets.
Suzuki, Michitaka; Ohsaki, Yuki; Tatematsu, Tsuyako; Shinohara, Yuki; Maeda, Takashi; Cheng, Jinglei; Fujimoto, Toyoshi
Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy. PMID:22879956
Factors Affecting Development of Peroxisomes and Glycolate Metabolism among Algae of Different Evolutionary Lines of the Prasinophyceae.
Kehlenbeck, P.; Goyal, A.; Tolbert, N. E.
Leaf-type peroxisomes are not present in the primitive unicellular Prasinophycean line of algae but are present in the multicellular algae Mougeotia, Chara, and Nitella, which are in the one evolutionary line, Charophyceae, that led to higher plants. Processes related to glycolate metabolism that may have been modified or induced with the appearance of peroxisomes have been examined. The algal dissolved inorganic carbon-concentrating mechanism and alkalization of the medium during photosynthesis were not lost when peroxisomes appeared in the members of the Charophycean line of algae. Therefore, it is unlikely that lowering of the CO2 concentration in the environment was a major factor in the evolutionary appearance of peroxisomes. Multicellular Mougeotia, early members of the Charophycean line of algae, have peroxisomes, but they excrete excess glycolate into the medium. The cytosolic pyruvate reductase for D-lactate synthesis and the glycolate dehydrogenase activity almost disappeared when peroxisomal glycolate oxidase, which also oxidizes L-lactate, appeared. These biochemical changes do not indicate what caused the induction of leaf-type peroxisomes in this evolutionary line of algae. The oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and glycolate oxidase require about 200 to 400 [mu]M O2 for 0.5 Vmax. These high-O2-requiring steps in glycolate metabolism would have functioned faster with increasing atmospheric O2, which might have been the causative factor in the induction of peroxisomes. PMID:12228674
Proton pump inhibitor alone vs proton pump inhibitor plus mucosal protective agents for endoscopic submucosal dissection-induced ulcer: a systematic review and meta-analysis
Nishizawa, Toshihiro; Suzuki, Hidekazu; Kanai, Takanori; Yahagi, Naohisa
Mucosal protective agents may improve healing of patients with endoscopic submucosal dissection-induced ulcers. The present study systematically evaluated published clinical trials to determine whether combined therapeutic use of mucosal protective agents and proton pump inhibitors can improve the outcome of patients with endoscopic submucosal dissection-induced ulcers compared to treatment with proton pump inhibitors alone. PubMed, the Cochrane Library, and the Igaku-Chuo-Zasshi database were searched to identify eligible randomized trials for systematic review. We identified 11 randomized trials for inclusion in our study (1,160 patients). Pooled endoscopic submucosal dissection-induced ulcer healing rates were 45.8% and 34.4% for patients with or without mucosal protective agents, respectively. The odds ratio was 2.28 (95% confidence interval, 1.57¢3.31) with no significant study heterogeneity. In conclusion, the systematic review and meta-analysis showed that the combined therapeutic use of proton pump inhibitors and mucosal protective agents improved healing rates of endoscopic submucosal dissection-induced ulcers compared to treatment with proton pump inhibitor monotherapy. PMID:25759512
HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: A potential mechanism for protease inhibitor-induced hyperlipidemia
Jun-Shan Liang; Oliver Distler; David A. Cooper; Haris Jamil; Richard J. Deckelbaum; Henry N. Ginsberg; Stephen L. Sturley
Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins.
cPLA2 Is Protective Against COX Inhibitor¢Induced Intestinal Damage
Montrose, David C.; Kadaveru, Krishna; Ilsley, Jillian N. M.; Root, Sierra H.; Rajan, Thiruchanduri V.; Ramesh, Manish; Nichols, Frank C.; Liang, Bruce T.; Sonin, Dmitry; Hand, Arthur R.; Zarini, Simona; Murphy, Robert C.; Belinsky, Glenn S.; Nakanishi, Masako; Rosenberg, Daniel W.
Cytosolic phospholipase A2 (cPLA2) is the rate-limiting enzyme responsible for the generation of prostaglandins (PGs), which are bioactive lipids that play critical roles in maintaining gastrointestinal (GI) homeostasis. There has been a long-standing association between administration of cyclooxygenase (COX) inhibitors and GI toxicity. GI injury is thought to be induced by suppressed production of GI-protective PGs as well as direct injury to enterocytes. The present study sought to determine how pan-suppression of PG production via a genetic deletion of cPLA2 impacts the susceptibility to COX inhibitor¢induced GI injury. A panel of COX inhibitors including celecoxib, rofecoxib, sulindac, and aspirin were administered via diet to cPLA2? / ? and cPLA2+ / + littermates. Administration of celecoxib, rofecoxib, and sulindac, but not aspirin, resulted in acute lethality (within 2 weeks) in cPLA2? / ? mice, but not in wild-type littermates. Histomorphological analysis revealed severe GI damage following celecoxib exposure associated with acute bacteremia and sepsis. Intestinal PG levels were reduced equivalently in both genotypes following celecoxib exposure, indicating that PG production was not likely responsible for the differential sensitivity. Gene expression profiling in the small intestines of mice identified drug-related changes among a panel of genes including those involved in mitochondrial function in cPLA2? / ? mice. Further analysis of enterocytic mitochondria showed abnormal morphology as well as impaired ATP production in the intestines from celecoxib-exposed cPLA2? / ? mice. Our data demonstrate that cPLA2 appears to be an important component in conferring protection against COX inhibitor¢induced enteropathy, which may be mediated through affects on enterocytic mitochondria. PMID:20562220
Oppositional Regulation of Noxa by JNK1 and JNK2 during Apoptosis Induced by Proteasomal Inhibitors
Pietkiewicz, Sabine; Sohn, Dennis; Piekorz, Roland P.; Grether-Beck, Susanne; Budach, Wilfried; Sabapathy, Kanaga; Jõnicke, Reiner U.
Proteasome inhibitors (PIs) potently induce apoptosis in a variety of tumor cells, but the underlying mechanisms are not fully elucidated. Comparing PI-induced apoptosis susceptibilities of various mouse embryonic fibroblast (MEF) lines differing in their c-jun N-terminal kinase (JNK) 1 and 2 status, we show that several hallmarks of apoptosis were most rapidly detectable in JNK2?/? cells, whereas they appeared only delayed and severely reduced in their intensities in cells expressing JNK2. Consistent with our finding that PI-induced apoptosis requires de novo protein synthesis, the proteasomal inhibitor MG-132 induced expression of the BH3-only protein Noxa at the transcriptional level in a JNK1-dependent, but JNK2-opposing manner. As the knockdown of Noxa blocked only the rapid PI-induced apoptosis of JNK2?/? cells, but not the delayed death occurring in JNK1?/? and JNK1+/+ cells, our data uncover a novel PI-induced apoptosis pathway that is regulated by the JNK1/2-dependent expression of Noxa. Furthermore, several transcription factors known to modulate Noxa expression including ATF3, ATF4, c-Jun, c-Myc, HIF1?, and p53 were found upregulated following MG-132 exposure. From those, only knockdown of c-Myc rescued JNK2?/? cells from PI-induced apoptosis, however, without affecting expression of Noxa. Together, our data not only show that a rapid execution of PI-induced apoptosis requires JNK1 for upregulation of Noxa via an as yet unknown transcription factor, but also that JNK2 controls this event in an oppositional manner. PMID:23593480
Interleukin-4-induced macrophage fusion is prevented by inhibitors of mannose receptor activity.
McNally, A. K.; DeFife, K. M.; Anderson, J. M.
A potential role for the macrophage mannose receptor in human monocyte-derived macrophage fusion was explored by testing the effects of previously described inhibitors of its activity on the formation of interleukin-4-induced foreign body giant cells in vitro Giant cell formation was prevented or reduced in the presence of alpha-man-nan and synthetic neoglycoprotein conjugates according to the following pattern of relative inhibition: mannose-bovine serum albumin (BSA) > N-acetylgucosamine-BSA congruent to glucose-BSA. Laminarin (beta-glucan) or galactose-BSA were not inhibitory. Swainsonine and castanospermine, inhibitors of glycoprotein processing that interfere with the arrival of newly synthesized mannose receptors at the cell surface, also attenuated macrophage fusion and the formation of giant cells, whereas another glycosidase inhibitor, deoxymannojirimycin, was without effect. Mannose receptors were confirmed to be specifically up-regulated by interleukin-4 in this culture system and also demonstrated to be present and concentrated at macrophage fusion interfaces. These data suggest that the macrophage mannose receptor may be an essential participant in the mechanism of interleukin-4-induced macrophage fusion and implicate a novel function for this endocytic/phagocytic receptor in mediating foreign body giant cell formation at sites of chronic inflammation. Images Figure 1 Figure 4 PMID:8780401
Grape seed proanthocyanidin extracts ameliorate podocyte injury by activating peroxisome proliferator-activated receptor-? coactivator 1? in low-dose streptozotocin-and high-carbohydrate/high-fat diet-induced diabetic rats.
Bao, Lei; Cai, Xiaxia; Dai, Xiaoqian; Ding, Ye; Jiang, Yanfei; Li, Yujie; Zhang, Zhaofeng; Li, Yong
Podocytes are part of the glomerular filtration membrane in kidney and serve to prevent the filtration of protein from the blood. Several evidences suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of diabetic nephropathy and it is an early event in podocyte injury. Mitochondrial dysfunction promotes oxidative stress that can favor the development of podocyte injury. Peroxisome proliferator-activated receptor-? coactivator 1? (PGC-1?) was considered to be a major regulator of metabolic homeostasis and mitochondrial function. Some studies indicated that polyphenols may improve mitochondrial dysfunction, maintain the podocyte integrity and have therapeutic effects on glomerular diseases by promoting PGC-1? expression. Our study investigated whether grape seed proanthocyanidin extracts (GSPE), a strong antioxidant, ameliorate podocyte injury by activating PGC-1? in low-dose streptozotocin-and high-carbohydrate/high-fat diet-induced diabetic rats. After 16 weeks of GSPE treatment, GSPE slightly increased the body weight and decreased plasma glucose, food intake, water intake and urine volume in diabetic rats. Further, GSPE significantly decreased 24 h albumin levels and increased the expression of nephrin and podocalyxin. The antioxidant levels were improved and the cellular damage of kidney in diabetic rats was also relieved effectively after the treatment. Moreover, GSPE increased the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content. Finally, GSPE activated the expression of PGC-1?, silent mating type information regulation 2 homolog 1 (SIRT1) and AMP-activated protein kinase (AMPK). These results suggest that GSPE ameliorate podocyte injury in diabetic nephropathy by the activation of AMPK-SIRT1-PGC-1? signalling, which appears to inhibit oxidative stress and mitochondrial dysfunction in the kidney. PMID:24941909
Neuroprotective effects of peroxisome proliferator-activated receptor alpha and gamma agonists in model of parkinsonism induced by intranigral 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine.
Barbiero, JanaĒna K; Santiago, Ronise M; Persike, Daniele Suzete; da Silva Fernandes, Maria Josķ; Tonin, Fernanda S; da Cunha, Claudio; Lucio Boschen, Suelen; Lima, Marcelo M S; Vital, Maria A B F
A large body of evidence suggests that peroxisome proliferator-activated receptor (PPAR) agonists may improve some of the pathological features of Parkinson's disease (PD). In the present study, we evaluated the effects of the PPAR-? agonist fenofibrate (100mg/kg) and PPAR-? agonist pioglitazone (30mg/kg) in a rat model of parkinsonism induced by intranigral 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP). Male Wistar rats were pretreated with both drugs for 5 days and received an infusion of MPTP. The experiments were divided into two parts. First, 1, 7, 14, and 21 days after surgery, the animals were submitted to the open field test. On days 21 and 22, the rats were subjected to the forced swim test and two-way active avoidance task. In the second part of the study, 24h after neurotoxin administration, immunohistochemistry was performed to assess tyrosine hydroxylase activity. The levels of dopamine and its metabolites in the striatum were determined using high-performance liquid chromatography, and fluorescence detection was used to assess caspase-3 activation in the substantia nigra pars compacta (SNpc). Both fenofibrate as pioglitazone protected against hypolocomotion, depressive-like behavior, impairment of learning and memory, and dopaminergic neurodegeneration caused by MPTP, with dopaminergic neuron loss of approximately 33%. Fenofibrate and pioglitazone also protected against the increased activation of caspase-3, an effector enzyme of the apoptosis cascade that is considered one of the pathological features of PD. Thus, PPAR agonists may contribute to therapeutic strategies in PD. PMID:25127682
Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins
As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy- terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals. PMID:2901422
Phosphodiesterase IV inhibitors as therapy for eosinophil-induced lung injury in asthma.
Torphy, T J; Barnette, M S; Hay, D W; Underwood, D C
Asthma is a complex, multifactorial disease that is underpinned by airway inflammation. A variety of cytotoxic substances are released into the airway from infiltrating inflammatory cells, especially the eosinophil. These cytotoxic substances, including reactive oxygen metabolites, produce damage to the airway epithelium, a histologic feature of chronic asthma. Damage to the airway epithelium, in turn, is thought to be a major factor responsible for the development of airway hyperreactivity, a hallmark of asthma. One notable molecular target for novel antiasthmatic drugs is the cyclic AMP-specific phosphodiesterase (PDE) or PDE IV. This isozyme is the predominant form of cyclic nucleotide PDE activity in inflammatory cells. Thus, in view of the putative role of cyclic AMP as an inhibitory second messenger in these cells, PDE IV inhibitors have been shown to suppress inflammatory cell activity. The purpose of the present experiments was to examine the effect of the PDE IV inhibitor, R-rolipram, on three key functions of the guinea pig eosinophil: a) superoxide anion (O2-) production, b) adhesion to human umbilical vein endothelial cells (HUVECs), and c) infiltration into the airway. R-rolipram-elevated eosinophil cyclic AMP content (EC50 = 1.7 microM) and inhibited fMLP-induced O2- production in a concentration-dependent manner (IC50 = 0.3 microM). In contrast, neither siguazodan, a PDE III inhibitor, nor zaprinast, a PDE V inhibitor, had an appreciable effect. R-rolipram (30 microM) also reduced by 25 to 40% the adhesion of eosinophils to HUVECs stimulated with phorbol myristate acetate or tumor necrosis factor-alpha, particularly under conditions in which both cell types were simultaneously exposed to the PDE IV inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7705312
Topical Review: Molecular and Neurologic Findings of Peroxisome Biogenesis Disorders
Nobuyuki Shimozawa; Tomoko Nagase; Yasuhiko Takemoto; Michinori Funato; Naomi Kondo; Yasuyuki Suzuki
Peroxisomal disorders, an expanding group of genetic disorders in humans, can be grouped into three categories: peroxisome biogenesis disorders, single peroxisomal enzyme deficiencies, and contiguous gene syndrome. At present, 13 complementation groups of peroxisome biogenesis disorders and their responsible genes have been identified, including our newly identified group with a PEX14 defect. We describe neuronal abnormalities related to deficiencies in
Topical Review: Molecular and Neurologic Findings of Peroxisome Biogenesis Disorders
Nobuyuki Shimozawa; Tomoko Nagase; Yasuhiko Takemoto; Michinori Funato; Naomi Kondo; Yasuyuki Suzuki
Peroxisomal disorders, an expanding group of genetic disorders in humans, can be grouped into three categories: peroxisome biogenesis disorders, single peroxisomal enzyme deficiencies, and contiguous gene syndrome. At present, 13 complementation groups of peroxisome biogenesis disorders and their responsible genes have been identified, including our newly identified group with a PEX14 defect. We describe neuronal abnormalities related to deficiencies in
Angiotensin II induces secretion of plasminogen activator inhibitor 1 and a tissue metalloprotease inhibitor-related protein from rat brain astrocytes
Olson, J.A. Jr.; Shiverick, K.T.; Ogilvie, S.; Buhi, W.C.; Raizada, M.K. (Univ. of Florida College of Medicine, Gainesville (USA))
The present study investigates angiotensin (Ang) II effects on secretory protein synthesis in brain astrocytes cultured from neonatal and 21-day-old rats. Ang II-induced changes in the de novo synthesis of (35S)methionine-labeled secretory proteins were visualized using two-dimensional NaDodSO4/PAGE. Astrocytes from 21-day-old rat brain possess specific high-affinity receptors for Ang II. These cells express two Ang II-induced secretory proteins with Mr 55,000 (AISP-55K) and Mr 30,000 (AISP-30K), which were time- and dose-dependent (EC50, 1 nM). (Sar1, Ile8)Ang II (where Sar is sarcosine) inhibited Ang II-induced secretion of AISP-55K but not AISP-30K. N-terminal amino acid sequencing indicates that AISP-55K is identical to rat plasminogen activator inhibitor 1, whereas AISP-30K exhibits 72-81% identity to three closely related proteins: human tissue inhibitor of metalloproteases, a rat phorbol ester-induced protein, and the murine growth-responsive protein 16C8. Immunofluorescent staining with rat plasminogen activator inhibitor 1 antibody was induced in the majority of cells in culture after Ang II treatment of astrocytes from 21-day-old rat brains. Absence of this response to Ang II in astrocytes from neonatal rat brain provides evidence that this action of Ang II on astrocytes is developmentally regulated.
Peroxisome proliferator-activated receptor (PPAR) alpha-regulated growth responses and their importance to hepatocarcinogenesis
N. H James; J. H Gill; R Brindle; N. J Woodyatt; N Macdonald; M Rolfe; S. C Hasmall; J. D Tugwood; P. R Holden; R. A Roberts
Peroxisome proliferators (PPs) are a class of non-genotoxic rodent hepatocarcinogens that act by perturbing liver growth regulation. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-?1 (TGF?1). More recently, we have demonstrated that PPs can suppress apoptosis induced by more diverse stimuli such as DNA damage
p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells
Huang, Shi-Wei [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Wu, Chun-Ying [Division of Gastroenterology and Hepatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, Yen-Ting [Department of Medical Research and Education, Cheng Hsin General Hospital, Taipei, Taiwan (China); Kao, Jun-Kai [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Pediatrics, Children's Hospital, Changhua Christian Hospital, Changhua, Taiwan (China); Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chiu, Husan-Wen [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chang, Chuan-Hsun [Department of Surgical Oncology, Cheng Hsin General Hospital, Taipei, Taiwan (China); Department of Nutrition Therapy, Cheng Hsin General Hospital, Taipei, Taiwan (China); School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan (China); Liang, Shu-Mei [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chen, Yi-Ju [Department of Dermatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Huang, Jau-Ling [Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan (China); Shieh, Jeng-Jer, E-mail: firstname.lastname@example.com [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan (China)
Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ? Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ? Compound C can upregulate p53 expression and induce p53 activation. ? Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ? p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ? Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.
Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica.
Ito, Takashi; Fujimura, Shuki; Matsufuji, Yoshimi; Miyaji, Tatsuro; Nakagawa, Tomoyuki; Tomizuka, Noboru
In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA. PMID:17506110
hCLCA2 is a p53-inducible inhibitor of breast cancer cell proliferation
Walia, Vijay; Ding, Ming; Kumar, Sumit; Nie, Daotai; Premkumar, Louis; Elble, Randolph C.
hCLCA2 is frequently downregulated in breast cancer and is a candidate tumor suppressor gene. We show here that the hCLCA2 gene is strongly induced by p53 in response to DNA damage. Adenoviral expression of p53 induces hCLCA2 in a variety of breast cell lines. Further, we find that p53 binds to consensus elements in the hCLCA2 promoter and mutation of these sites abolishes p53-responsiveness and induction by DNA damage. Adenoviral transduction of hCLCA2 into immortalized cells induces p53, CDK inhibitors p21 and p27, and cell cycle arrest by 24 hours, and caspase induction and apoptosis by 40 hours post-infection. Transduction of the malignant tumor cell line BT549 on the other hand does not induce p53, p21, or p27 but instead induces apoptosis directly and more rapidly. Knockout and knockdown studies indicate that growth inhibition and apoptosis are signaled via multiple pathways. Conversely, suppression of hCLCA2 by RNA interference enhances proliferation of MCF10A and reduces sensitivity to doxorubicin. Gene expression profiles indicate that hCLCA2 levels are strongly predictive of tumor cell sensitivity to doxorubicin and other chemotherapeutics. Because certain Cl- channels are proposed to promote apoptosis by reducing intracellular pH, we tested whether, and established that, hCLCA2 enhances Cl- current in breast cancer cells and reduces pH to ?6.7. These results reveal hCLCA2 as a novel p53-inducible growth inhibitor, explain how its downregulation confers a survival advantage to tumor cells, and suggest both prognostic and therapeutic applications. PMID:19654313
Histone deacetylase inhibitor treatment induces 'BRCAness' and synergistic lethality with PARP inhibitor and cisplatin against human triple negative breast cancer cells.
Ha, Kyungsoo; Fiskus, Warren; Choi, Dong Soon; Bhaskara, Srividya; Cerchietti, Leandro; Devaraj, Santhana G T; Shah, Bhavin; Sharma, Sunil; Chang, Jenny C; Melnick, Ari M; Hiebert, Scott; Bhalla, Kapil N
There is an unmet need to develop new, more effective and safe therapies for the aggressive forms of triple negative breast cancers (TNBCs). While up to 20% of women under 50 years of age with TNBC harbor germline mutations in BRCA1, and these tumors are sensitive to treatment with poly(ADP) ribose polymerase inhibitors, a majority of TNBCs lack BRCA1 mutations or loss of expression. Findings presented here demonstrate that by attenuating the levels of DNA damage response and homologous recombination proteins, pan-histone deacetylase inhibitor (HDI) treatment induces 'BRCAness' and sensitizes TNBC cells lacking BRCA1 to lethal effects of PARP inhibitor or cisplatin. Treatment with HDI also induced hyperacetylation of nuclear hsp90. Similar effects were observed following shRNA-mediated depletion of HDAC3, confirming its role as the deacetylase for nuclear HSP90. Furthermore, cotreatment with HDI and ABT-888 induced significantly more DNA strand breaks than either agent alone, and synergistically induced apoptosis of TNBC cells. Notably, co-treatment with HDI and ABT-888 significantly reduced in vivo tumor growth and markedly improved the survival of mice bearing TNBC cell xenografts. These findings support the rationale to interrogate the clinical activity of this novel combination against human TNBC, irrespective of its expression of mutant BRCA1. PMID:25026298
Cyclooxygenase-2 inhibitor nimesulide blocks ultraviolet B-induced photocarcinogenesis in SKH-1 hairless mice.
Tang, Xiuwei; Kim, Arianna L; Kopelovich, Levy; Bickers, David R; Athar, Mohammad
Cyclooxygenase-2 (COX-2) inhibition can inhibit UVB-induced carcinogenesis in the skin. We have shown that COX-2 is overexpressed in UVB-induced squamous cell carcinomas (SCCs). Celecoxib, a specific inhibitor of COX-2, blocks UVB-induced papillomas and carcinomas in murine skin. However, as COX-2 inhibitors of this type are associated with an increased risk of adverse cardiovascular events, we decided to study nimesulide, a different class of COX-2 inhibitor, an N-arylmethanesulfonamide derivative not known to have these untoward effects. To assess the antitumor-promoting effects of nimesulide, 90 mice were equally divided into three groups. Group I animals received no test agent or UVB and served as age-matched controls; group II animals were irradiated with UVB (180 mJ cm(-2), twice weekly for 35 weeks) and group III animals received 300 p.p.m. nimesulide in drinking water and were irradiated with UVB as described for group-II. Nimesulide treatment reduced the growth of UVB-induced tumors both in terms of tumor number and tumor volume. By weeks 25, 30 and 35, the tumor numbers in the nimesulide-treated group were 79%, 49% and 53% less than the number occurring in UVB-treated animals whereas tumor volume was reduced 69%, 54% and 53%, respectively, compared to the UVB-irradiated control group. Nimesulide also inhibited the malignant progression of SCCs. The reduction in tumorigenesis was paralleled by a decrease in cell cycle regulatory proteins (cyclins A, B1, D1, E, CDK2/4/6) and the antiapoptotic protein (Bcl2); concomitantly there was an increase in proapoptotic markers, poly (ADP-ribose) polymerase (PARP) and caspase-3. Nimesulide also decreased ornithine decarboxylase expression and the nuclear accumulation of nuclear factor kappa B transcriptionally active protein complexes. These results show that alternative classes of COX-2 inhibitors may likely be efficacious as cancer chemopreventive agents and may have an improved therapeutic index. PMID:18266822
Administration of an aldose reductase inhibitor induces a decrease of collagen fluorescence in diabetic rats.
Sußrez, G; Rajaram, R; Bhuyan, K C; Oronsky, A L; Goidl, J A
As a consequence of an increased flux through the sorbitol pathway fructose levels rise in various tissues in diabetes. Also, in vitro nonenzymatic fructosylation of protein induces the generation of fluorescence at a rate 10 times greater than glucosylation. The administration of sorbinil, an aldose reductase inhibitor known to lower tissue fructose concentration, to experimental diabetic rats led to a decrease in the fluorescence related to advanced Maillard products in their skin collagen. This effect is consistent with the in vivo occurrence of nonenzymatic fructosylation of collagen. A potential pathogenetic role for this posttranslational modification in diabetic complications should be considered. PMID:3136193
Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells
Suzuki, Nobutaka; Urano, Jun; Tamanoi, Fuyuhiko
Farnesyltransferase inhibitors (FTIs) represent a new class of anticancer drugs that show promise in blocking the growth of tumors. Here, we report that FTIs are capable of inducing apoptosis of transformed but not untransformed cells. Treatment of v-K-ras-transformed normal rat kidney (KNRK) cells with FTIs leads to the induction of apoptotic cell morphology, chromatin condensation and DNA fragmentation. In addition, fluorescence-activated cell sorter analysis of FTI-treated KNRK cells shows a sub-G1 apoptotic peak (chromosome content of <2 N). This FTI-induced apoptosis is evident only when the cells are grown in low serum conditions (0.1% fetal calf serum) and is observed selectively with transformed KNRK cells and not with untransformed NRK cells. Further analysis of the mechanism underlying this apoptosis has shown that FTI treatment of KNRK cells results in the activation of caspase 3 but not caspase 1. Moreover, the addition of Z-DEVD-fmk, an agent that interferes with caspase 3 activity, can inhibit FTI-induced apoptosis in a dose-dependent manner. Introduction of the CASP-3 gene into MCF7 cells, which lack caspase 3 activity, results in a significant increase of FTI-induced apoptosis. Furthermore, FTI induces the release of cytochrome c into the cytosol. This release is an important feature of caspase 3-mediated apoptosis. These results suggest that FTIs induce apoptosis through the release of cytochrome c from the mitochondria resulting in caspase 3 activation. PMID:9860973
Role of plasminogen activator inhibitor-1 in glucocorticoid-induced diabetes and osteopenia in mice.
Tamura, Yukinori; Kawao, Naoyuki; Yano, Masato; Okada, Kiyotaka; Okumoto, Katsumi; Chiba, Yasutaka; Matsuo, Osamu; Kaji, Hiroshi
Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC. PMID:25552599
Serine protease inhibitors modulate smoke-induced chemokine release from human lung fibroblasts.
Numanami, Hiroki; Koyama, Sekiya; Nelson, Dan K; Hoyt, Jeffrey C; Freels, Jon L; Habib, Michael P; Amano, Jun; Haniuda, Masayuki; Sato, Etsuro; Robbins, Richard A
Smoking is associated with lung inflammation and a protease-antiprotease imbalance. We previously reported that cigarette smoke extract (CSE) stimulates human lung fibroblasts to release chemotactic cytokines. We hypothesized that serine protease inhibitors might modulate lung fibroblast release of chemotactic cytokines in response to CSE. To test this hypothesis, serine protease inhibitors (FK706, alpha1-antitrypsin, methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone, or Nalpha-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) from human fetal lung fibroblasts by the blind-well chemotactic chamber. Metalloproteinases and cysteine proteinases were not examined in this study. Similarly, the release and gene expression of chemokines and nuclear factor-kappaB (NF-kappaB) activation were measured by means of enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Release of NCA, MCA, chemotactic chemokines including interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor, and the expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA were attenuated by FK706. Furthermore, FK706 suppressed NF-kappaB activation. These data suggest that serine protease inhibitors attenuate the CSE-induced release of NCA and MCA from human fetal lung fibroblasts and that the inhibitory action of antiproteases might depend on NF-kappaB signaling pathway. PMID:12738688
VEGF-induced HUVEC migration and proliferation are decreased by PDE2 and PDE4 inhibitors.
Favot, Laure; Keravis, ThķrĶse; Holl, Vincent; Le Bec, Alain; Lugnier, Claire
Migration and proliferation of endothelial cells in response to VEGF play an important role in angiogenesis associated to pathologies such as atherosclerosis, diabetes and tumor development. Elevation of cAMP in endothelial cells has been shown to inhibit growth factor-induced proliferation. Our hypothesis was that inactivation of cAMP-specific phosphodiesterases (PDEs) would inhibit angiogenesis. The purpose of this study was to evaluate the effect of PDE inhibitors on in vitro and in vivo angiogenesis, using human umbilical vein endothelial cell (HUVEC) and chick chorioallantoic membrane (CAM) models respectively. Here, we report that: 1) PDE2, PDE3, PDE4 and PDE5 are expressed in HUVEC; 2) EHNA (20 microM), PDE2 selective inhibitor, and RP73401 (10 microM), PDE4 selective inhibitor, are able to increase the intracellular cAMP level in HUVEC; 3) EHNA and RP73401 are able to inhibit proliferation, cell cycle progression and migration of HUVEC stimulated by VEGF; 4) these in vitro effects can be mimic by treating HUVEC with the cAMP analogue, 8-Br-cAMP (600 microM); 5) only the association of EHNA and RP73401 inhibits in vivo angiogenesis, indicating that both migration and proliferation must be inhibited. These data strongly suggest that PDE2 and PDE4 represent new potential therapeutic targets in pathological angiogenesis. PMID:12888882
Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, protects dopaminergic neurons from neurotoxin-induced damage
Chen, SH; Wu, HM; Ossola, B; Schendzielorz, N; Wilson, BC; Chu, CH; Chen, SL; Wang, Q; Zhang, D; Qian, L; Li, X; Hong, JS; Lu, RB
BACKGROUND AND PURPOSE Prevention or disease-modifying therapies are critical for the treatment of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. However, no such intervention is currently available. Growing evidence has demonstrated that administration of histone deacetylase (HDAC) inhibitors ameliorates a wide range of neurologic and psychiatric disorders in experimental models. Suberoylanilide hydroxamic acid (SAHA) was the first HDAC inhibitor approved by the Food and Drug Administration for the sole use of cancer therapy. The purpose of this study was to explore the potential new indications of SAHA for therapy of neurodegenerative diseases in in vitro Parkinson's disease models. EXPERIMENTAL APPROACH Mesencephalic neuron¢glia cultures and reconstituted cultures were used to investigate neurotrophic and neuroprotective effects of SAHA. We measured toxicity in dopaminergic neurons, using dopamine uptake assay and morphological analysis and expression of neurotrophic substances by enzyme-linked immunosorbent assay and real-time RT PCR. KEY RESULTS In mesencephalic neuron¢glia cultures, SAHA displayed dose- and time-dependent prolongation of the survival and protection against neurotoxin-induced neuronal death of dopaminergic neurons. Mechanistic studies revealed that the neuroprotective effects of SAHA were mediated in part by promoting release of neurotrophic factors from astroglia through inhibition of histone deacetylation. CONCLUSION AND IMPLICATIONS The novel neurotrophic and neuroprotective effects of SAHA demonstrated in this study suggest that further study of this HDAC inhibitor could provide a new therapeutic approach to the treatment of neurodegenerative diseases. PMID:21726209
Topically applied Hsp90 inhibitor 17AAG inhibits UVR-induced cutaneous squamous cell carcinomas.
Singh, Anupama; Singh, Ashok; Sand, Jordan M; Bauer, Samuel J; Hafeez, Bilal Bin; Meske, Louise; Verma, Ajit K
We present here that heat-shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits UVR-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500?nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8?kJ?m(-2)). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and protein kinase C epsilon (PKC?)-overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by a decrease in UVR-induced (1) hyperplasia, (2) Hsp90?-PKC? interaction, and (3) expression levels of Hsp90?, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473, and matrix metalloproteinase (MMP). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR-exposed or organ transplant population. PMID:25337691
The PI3K inhibitor GS-1101 synergistically potentiates HDAC inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and ERK pathways
Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T.; Portell, Craig A.; Lannutti, Brian J.; Almasan, Alexandru; Hsi, Eric D.
Previously, we showed that inhibition of the protein kinase C ? (PKC?)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines and primary Non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic. PMID:23889282
Eribulin synergizes with Polo-like kinase 1 inhibitors to induce apoptosis in rhabdomyosarcoma.
Stehle, Angelika; Hugle, Manuela; Fulda, Simone
Eribulin, a novel microtubule-interfering drug, was recently shown to exhibit high antitumor activity in vivo against various pediatric cancers. Here, we identify a novel synthetic lethal interaction of Eribulin together with Polo-like kinase 1 (PLK1) inhibitors against rhabdomyosarcoma (RMS) in vitro and in vivo. Eribulin and the PLK1 inhibitor BIĀ2536 at subtoxic concentrations synergize to induce apoptosis in RMS cells as confirmed by calculation of combination index (CI). Also, Eribulin/BIĀ2536 co-treatment is significantly more effective than monotherapy to reduce cell viability and inhibit colony formation of RMS cells. Similarly, Eribulin and BIĀ2536 act in concert to trigger apoptosis in a primary, patient-derived ARMS culture, underscoring the clinical relevance of this combination. Importantly, Eribulin and BI 2536 cooperate to suppress tumor growth in an in vivo model of RMS. On molecular grounds, Eribulin/BIĀ2536 co-treatment causes profound mitotic arrest, which is critically required for synergism, since inhibition of mitotic arrest by CDK1 inhibitor RO-3306 abolishes Eribulin/BIĀ2536-mediated apoptosis. Eribulin and BI 2536 cooperate to activate caspase-9, -3 and -8, which is necessary for apoptosis induction, since the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) reduces Eribulin/BIĀ2536-induced apoptosis significantly, yet partially. Intriguingly, knockdown of endonuclease G (ENDOG) also significantly inhibits Eribulin/BIĀ2536-triggered apoptosis, demonstrating the involvement of both caspase-dependent and -independent effector pathways. Synergistic induction of apoptosis is similarly found for Eribulin/BI 2536 co-treatment in neuroblastoma cells and for the combination of vincristine (another antimicrotubule chemotherapeutic) with Poloxin (another PLK1 inhibitor), thus pointing to a broader significance of this concomitant microtubule- and PLK1-targeting strategy for pediatric oncology. In conclusion, the identification of a novel synthetic lethality by dual targeting of mitosis using microtubule-interfering and PLK1-targeted drugs, i.e. Eribulin and BI 2536, has important implications for the development of more effective treatment strategies for RMS. PMID:25917079
The p38 MAPK inhibitor, PD169316, inhibits transforming growth factor ?-induced Smad signaling in human ovarian cancer cells
Yangxin Fu; Laura M OÆConnor; Trevor G Shepherd; Mark W Nachtigal
Transforming growth factor ? (TGF?) can signal through a variety of Smad-independent pathways, including the p38 MAPK pathway. Recent work has shown that inhibitors of p38 MAPK, such as SB203580 and SB202190, can inhibit signaling induced by TGF?. Here we show that another p38 MAPK inhibitor, PD169316, abrogates signaling initiated by both TGF? and Activin A, but not bone morphogenetic
Changes of trypsin in activity and secondary structure induced by complex with trypsin inhibitors and tea polyphenol
Huihua Huang; Mouming Zhao
To reveal the relationships between the activity of trypsin and its structural change, changes of trypsin in biological activity\\u000a induced by complex with Bowman-Birk trypsin inhibitor (BBTI), Kunitz soybean trypsin inhibitor (KSTI, type I-S) and tea polyphenol\\u000a (TP) were detected and their relationship with the secondary structure changes were studied by far-UV circular dichroism (CD)\\u000a spectra measurement. BBTI and KSTI
The MEK inhibitor PD184352 enhances BMS-214662-induced apoptosis in CD34+ CML stem\\/progenitor cells
F Pellicano; P Ŗimara; A Sinclair; G V Helgason; M Copland; S Grant; T L Holyoake
The cytotoxic farnesyl transferase inhibitor BMS-214662 has been shown to potently induce mitochondrial apoptosis in primitive CD34+ chronic myeloid leukaemia (CML) stem\\/progenitor cells. Here, to enhance the BMS-214662 apoptotic effect, we further targeted the extracellular signal-regulated kinase (ERK) pathway, downstream of BCR¢ABL, by treating CD34+ CML stem\\/progenitor cells with a highly selective adenosine triphosphate (ATP) non-competitive MEK inhibitor, PD184352. PD184352
Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated receptor {delta} in human keratinocytes.
Jiang, Yan J; Uchida, Yoshikazu; Lu, Biao; Kim, Peggy; Mao, Cungui; Akiyama, Masashi; Elias, Peter M; Holleran, Walter M; Grunfeld, Carl; Feingold, Kenneth R
ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise approximately 50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C(2)-Cer and C(6)-Cer), but not C(8)-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C(6)-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARdelta expression (but not other PPARs or liver X receptors), whereas PPARdelta knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARdelta is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARdelta-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter. PMID:19429679
Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation¢Induced Cell Death
Sharlow, Elizabeth R.; Epperly, Michael W.; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M.; Wipf, Peter; Greenberger, Joel S.
Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated ?IR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of ?IR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after ?IR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased ?IR-induced DNA damage as measured by ?H2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death. PMID:24068833
Peroxisomal alterations in AlzheimerÆs disease
Jianqiu Kou; Gabor G. Kovacs; Romana H÷ftberger; Willem Kulik; Alexander Brodde; Sonja Forss-Petter; Selma H÷nigschnabl; Andreas Gleiss; Britta Br³gger; Ronald Wanders; Wilhelm Just; Herbert Budka; Susanne Jungwirth; Peter Fischer; Johannes Berger
In AlzheimerÆs disease (AD), lipid alterations are present early during disease progression. As some of these alterations\\u000a point towards a peroxisomal dysfunction, we investigated peroxisomes in human postmortem brains obtained from the cohort-based,\\u000a longitudinal Vienna-Transdanube Aging (VITA) study. Based on the neuropathological Braak staging for AD on one hemisphere,\\u000a the patients were grouped into three cohorts of increasing severity (stages
The peroxisome deficient PEX2 zellweger mouse
Phyllis L. Faust; Hui-Min Su; Ann Moser; Hugo W. Moser
Zellweger syndrome is the prototypic human peroxisomal biogenesis disorder that results in abnormal neuronal migration in\\u000a the central nervous system and severe neurologic dysfunction. A murine model for this disorder was previously developed by\\u000a targeted deletion of the PEX2 peroxisomal gene. By labeling neuronal precursor cells in vivo with a mitotic marker, we can demonstrate a delay in neuronal\\u000a migration
Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor Ā Expression and Transactivation
Chenguang Wang; Nagarajan Pattabiraman; Jian Nian Zhou; Maofu Fu; Toshiyuki Sakamaki; Chris Albanese; Zhiping Li; Kongming Wu; James Hulit; Peter Neumeister; Phyllis M. Novikoff; Michael Brownlee; Philipp E. Scherer; Joan G. Jones; Kathleen D. Whitney; Lawrence A. Donehower; Emily L. Harris; Thomas Rohan; David C. Johns; Richard G. Pestell
The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumor- igenesis. Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR induces hepatic steatosis, and liganded PPAR promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR function, transactivation, expres- sion, and promoter activity. PPAR transactivation induced by
Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.
Davis, S T; Benson, B G; Bramson, H N; Chapman, D E; Dickerson, S H; Dold, K M; Eberwein, D J; Edelstein, M; Frye, S V; Gampe Jr, R T; Griffin, R J; Harris, P A; Hassell, A M; Holmes, W D; Hunter, R N; Knick, V B; Lackey, K; Lovejoy, B; Luzzio, M J; Murray, D; Parker, P; Rocque, W J; Shewchuk, L; Veal, J M; Walker, D H; Kuyper, L F
Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients. PMID:11141566
The natural product honokiol inhibits calcineurin inhibitor-induced and Ras-mediated tumor promoting pathways.
Banerjee, Pallavi; Basu, Aninda; Arbiser, Jack L; Pal, Soumitro
Although calcineurin inhibitors (CNIs) are very useful in preventing allograft rejection, they can mediate a rapid progression of post-transplantation malignancies. The CNI cyclosporine A (CsA) can promote renal tumor growth through activation of the proto-oncogene ras and over-expression of the angiogenic cytokine VEGF; the ras activation also induces over-expression of the cytoprotective enzyme HO-1, which promotes survival of renal cancer cells. Here, we show that the natural product honokiol significantly inhibited CsA-induced and Ras-mediated survival of renal cancer cells through the down-regulations of VEGF and HO-1. Thus, honokiol treatment may help to prevent tumor-promoting effects of CsA in transplant patients. PMID:23752066
A novel PKC-? inhibitor abrogates cell proliferation and induces apoptosis in neuroblastoma.
Pillai, Prajit; Desai, Shraddha; Patel, Rekha; Sajan, Mini; Farese, Robert; Ostrov, David; Acevedo-Duncan, Mildred
Protein Kinase C-iota (PKC-?), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-? confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-? inhibitor in neuroblastoma cell proliferation and apoptosis. ICA-1 specifically inhibits the activity of PKC-? but not that of PKC-zeta (PKC-?), the closely related atypical PKC family member. The IC(50) for the kinase activity assay was approximately 0.1?M which is 1000 times less than that of aurothiomalate, a known PKC-? inhibitor. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-? is an in vitro Cdk7 kinase and the phosphorylation of Cdk7 by PKC-? was potently inhibited by ICA-1. Furthermore, our data shows that neuroblastoma cells proliferate via a PKC-?/Cdk7/cdk2 cell signaling pathway and ICA-1 mediates its antiproliferative effects by inhibiting this pathway. ICA-1 (0.1?M) inhibited the in vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-? inhibitor and chemotherapeutic agent for neuroblastoma. PMID:21315177
Efficacy of Rho kinase inhibitor on cognitive impairment induced by chronic cerebral hypoperfusion in rats.
Zhang, Qiang; Zhang, Jun-Jian; Han, Zhong-Mou
This work aims to explore the efficacy of Rho kinase inhibitor Fasudil on cognitive impairment induced by chronic cerebral hypoperfusion in rats. A total of 32 male adult Sprague Dawley (SD) rats were randomly divided into three groups: treatment group, control group and sham-operated group for severe carotid artery stenosis model. After two weeks, 8.35 mg/kg Fasudil and physiological saline were intraperitoneally applied twice per day in treatment group and control group, respectively. Morris water maze test was performed in each group to detect the changes of cognitive function and observe the hippocampal pathomorphology in rats after eight weeks. The average escape latency distinctly shortened (P < 0.01) and the percentage of swimming distance in the platform quadrant significantly increased (P < 0.01) in treatment group compared with those at corresponding time points in control group. The rate of carotid artery stenosis in rats had no statistical difference between treatment and control groups (P > 0.05). Fasudil effectively improved hippocampal pathomorphology. Rho kinase inhibitor obviously ameliorated cognitive impairment induced by chronic cerebral hypoperfusion in rats. PMID:25932185
Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain
Barbato, G.; Cicero, D.O.; Cordier, F.; Narjes, F.; Gerlach, B.; Sambucini, S.; Grzesiek, S.; Matassa, V.G.; De Francesco, R.; Bazzo, R.
Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N¢terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His¢Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His¢Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes. PMID:10716920
Glycine transporters type 1 inhibitor promotes brain preconditioning against NMDA-induced excitotoxicity.
Pinto, Mauro Cunha Xavier; Lima, Isabel Vieira de Assis; da Costa, Flßvia Lage Pessoa; Rosa, Daniela ValadŃo; Mendes-Goulart, VŌnia Aparecida; Resende, Rodrigo Ribeiro; Romano-Silva, Marco Aurķlio; de Oliveira, Ant¶nio Carlos Pinheiro; Gomez, Marcus VinĒcius; Gomez, Renato Santiago
Brain preconditioning is a protective mechanism, which can be activated by sub-lethal stimulation of the NMDA receptors (NMDAR) and be used to achieve neuroprotection against stroke and neurodegenerative diseases models. Inhibitors of glycine transporters type 1 modulate glutamatergic neurotransmission through NMDAR, suggesting an alternative therapeutic strategy of brain preconditioning. The aim of this work was to evaluate the effects of brain preconditioning induced by NFPS, a GlyT1 inhibitor, against NMDA-induced excitotoxicity in mice hippocampus, as well as to study its neurochemical mechanisms. C57BL/6 mice (male, 10-weeks-old) were preconditioned by intraperitoneal injection of NFPS at doses of 1.25, 2.5 or 5.0 mg/kg, 24 h before intrahippocampal injection of NMDA. Neuronal death was evaluated by fluoro jade C staining and neurochemical parameters were evaluated by gas chromatography-mass spectrometry, scintillation spectrometry and western blot. We observed that NFPS preconditioning reduced neuronal death in CA1 region of hippocampus submitted to NMDA-induced excitotoxicity. The amino acids (glycine and glutamate) uptake and content were increased in hippocampus of animals treated with NFPS 5.0 mg/kg, which were associated to an increased expression of type-2 glycine transporter (GlyT2) and glutamate transporters (EAAT1, EAAT2 and EAAT3). The expression of GlyT1 was reduced in animals treated with NFPS. Interestingly, the preconditioning reduced expression of GluN2B subunits of NMDAR, whereas did not change the expression of GluN1 or GluN2A in all tested doses. Our study suggests that NFPS preconditioning induces resistance against excitotoxicity, which is associated with neurochemical changes and reduction of GluN2B-containing NMDAR expression. PMID:25312280
Farnesyltransferase inhibitor, tipifarnib, prevents galactosamine/lipopolysaccharide-induced acute liver failure.
Shirozu, Kazuhiro; Hirai, Shuichi; Tanaka, Tomokazu; Hisaka, Shinsuke; Kaneki, Masao; Ichinose, Fumito
Acute liver failure (ALF) is a fatal syndrome associated with massive hepatocyte death. There is no cure for ALF except liver transplantation. Protein farnesylation is a lipid modification of cysteine residues that is catalyzed by farnesyltransferase (FTase) and has been proposed as an integral component of acute inflammation. Previously, we have demonstrated that FTase inhibitors improve survival in mouse models of endotoxemia and sepsis. Here we studied the effects of FTase inhibitor, tipifarnib, on galactosamine (GalN)/lipopolysaccharide (LPS)-induced ALF. The effects of tipifarnib (10 mg/kg, i.p.) were studied in GalN (400 mg/kg, i.p.)- and LPS (3 ?g/kg)-challenged mice by histological and biochemical analyses. Galactosamine/LPS administration caused prominent liver injury characterized by the increased plasma alanine aminotransferase and aspartic aminotransferase levels, leading to significant mortality in mice. Tipifarnib inhibited GalN/LPS-induced caspase 3 activation, inflammatory cytokine production, and c-Jun N-terminal kinase phosphorylation in the liver. On the other hand, tipifarnib upregulated antiapoptotic protein, Bcl-xL, in the liver after GalN/LPS challenge. Tipifarnib also protected primary hepatocytes from GalN/tumor necrosis factor ?-induced cell death by inhibiting caspase 3 activation and upregulating antiapoptotic proteins. Galactosamine/LPS-induced liver injury was associated with increased protein farnesylation in the liver. Tipifarnib prevented protein farnesylation in the liver and markedly attenuated liver injury and mortality in GalN/LPS-challenged mice. These results suggest that protein farnesylation is a novel potential molecular target to prevent hepatocyte death and acute inflammatory liver failure in fulminant hepatitis. PMID:25046541
Pre-treatment with ACE Inhibitor Attenuates Doxorubicin Induced Cardiomyopathy via Preservation of Mitochondrial Function
Hiona, Asimina; Lee, Andrew Stephen; Nagendran, Jayan; Xie, Xiaoyan; Connolly, Andrew J.; Robbins, Robert C.; Wu, Joseph C.
Aims Doxorubicin is a widely used chemotherapy drug, but its application is associated with cardiotoxicity. Free radical generation and mitochondrial dysfunction are thought to contribute to doxorubicin-induced cardiac failure. Angiotensin-converting enzyme (ACE) inhibitors are commonly used as cardioprotective agents and have recently been shown in clinical studies to be efficacious in the prevention of anthracycline induced heart failure. Here we evaluated a mechanism for these protective effects by testing the ability of the ACE inhibitor enalapril to preserve mitochondrial function in a model of chronic doxorubicin treatment in rats. Methods Sprague Dawley rats were divided into three groups and followed for a total of 10 weeks: a) control-untreated, b) Doxorubicin treated (Dox), and c) Doxorubicin + Enalapril treated (DE). Doxorubicin was administered via intraperitoneal injection at weekly intervals from week 2 through week 7. Enalapril was administered in the drinking water of the DE group for the study duration. Results Doxorubicin treatment produced a significant loss in left ventricular contractility (P< 0.05), decrease in mitochondrial function via impairment of state-3 respiration, decrease in the cytosolic fraction of ATP, and up-regulation of free radical production. Enalapril significantly attenuated the decrease in percent fractional shortening (P< 0.05) and prevented the doxorubicin-associated reduction in respiratory efficiency and cytosolic ATP content (P< 0.05). Importantly, enalapril also abolished the robust doxorubicin-induced increase in free radical formation. Conclusions Administration of enalapril attenuates doxorubicin-induced cardiac dysfunction via preservation of mitochondrial respiratory efficiency and reduction in doxorubicin-associated free radical generation. PMID:21094500
Bowman-Birk protease inhibitor from soybeans enhances cisplatin-induced cytotoxicity in human mesothelioma cells
Malignant mesothelioma (MM) is an aggressive cancer with no effective treatment options. Enforced expression of the gap junction (GJ) component connexin 43 (Cx43) increases the sensitivity of MM cells to cisplatin. Bowman-Birk protease inhibitor (BBI) induces the restoration of Cx43 in several types of tumor cells. In this study, we examined the capability of BBI to enhance the cytotoxic effect of cisplatin in MM cells via the induction of Cx43. Human MM H28 cells were used. Cell viability was evaluated by a WST-1 assay and proteasomal activity was determined by fluorometric analysis. Protein and mRNA levels were determined by immunoblot analysis and real-time RT-PCR, respectively. GJ function mediated by Cx43 was evaluated using the scrape-loading method. BBI effectively inhibited H28 cell growth in a dose-dependent manner (200¢400 ?g/ml). In parallel with the growth inhibition, Cx43 levels (mRNA and protein) and GJ function were elevated by BBI treatment. Knockdown of BBI-induced Cx43 by an antisense nucleotide treatment almost cancelled the growth inhibition. BBI enhanced cisplatin-induced cytotoxicity in H28 cells, and down-regulation of Cx43 by the antisense nucleotide treatment abrogated the enhancing effect of BBI. The induction of Cx43 by BBI contributed to Src inactivation and subsequent induction of Bax. Furthermore, an Src inhibitor (SU6656) also enhanced cisplatin-induced cytotoxicity in H28 cells. These results suggest that BBI improves the cytotoxic efficacy of cisplatin in H28 cells via the inhibition of Src signaling. PMID:22977565
The ER-peroxisome connection in plants: Development of the ōER semi-autonomous peroxisome maturation and replicationö model for plant peroxisome biogenesis
Robert T. Mullen; Richard N. Trelease
The perceived role of the ER in the biogenesis of plant peroxisomes has evolved significantly from the original ōER vesiculationö model, which portrayed co-translational import of proteins into peroxisomes originating from the ER, to the ōER semi-autonomous peroxisomeö model wherein membrane lipids and post-translationally acquired peroxisomal membrane proteins (PMPs) were derived from the ER. Results from more recent studies of
The Cytoskeleton and the Peroxisomal-Targeted SNOWY COTYLEDON3 Protein Are Required for Chloroplast Development in Arabidopsis[W
Albrecht, Ver¾nica; Ŗimkovß, Klßra; Carrie, Chris; Delannoy, Etienne; Giraud, Estelle; Whelan, Jim; Small, Ian David; Apel, Klaus; Badger, Murray R.; Pogson, Barry James
Here, we describe the snowy cotyledon3 (sco3-1) mutation, which impairs chloroplast and etioplast development in Arabidopsis thaliana seedlings. SCO3 is a member of a largely uncharacterized protein family unique to the plant kingdom. The sco3-1 mutation alters chloroplast morphology and development, reduces chlorophyll accumulation, impairs thylakoid formation and photosynthesis in seedlings, and results in photoinhibition under extreme CO2 concentrations in mature leaves. There are no readily apparent changes to chloroplast biology, such as transcription or assembly that explain the disruption to chloroplast biogenesis. Indeed, SCO3 is actually targeted to another organelle, specifically to the periphery of peroxisomes. However, impaired chloroplast development cannot be attributed to perturbed peroxisomal metabolic processes involving germination, fatty acid ?-oxidation or photorespiration, though there are so far undescribed changes in low and high CO2 sensitivity in seedlings and young true leaves. Many of the chloroplasts are bilobed, and some have persistent membranous extensions that encircle other cellular components. Significantly, there are changes to the cytoskeleton in sco3-1, and microtubule inhibitors have similar effects on chloroplast biogenesis as sco3-1 does. The localization of SCO3 to the periphery of the peroxisomes was shown to be dependent on a functional microtubule cytoskeleton. Therefore, the microtubule and peroxisome-associated SCO3 protein is required for chloroplast development, and sco3-1, along with microtubule inhibitors, demonstrates an unexpected role for the cytoskeleton and peroxisomes in chloroplast biogenesis. PMID:20978221
An inventory of peroxisomal proteins and pathways in Drosophila melanogaster
Faust, Joseph E.; Verma, Avani; Peng, Chengwei; McNew, James A.
Peroxisomes are ubiquitous organelles housing a variety of essential biochemical pathways. Peroxisome dysfunction causes a spectrum of human diseases known as peroxisome biogenesis disorders (PBD). While much is known regarding the mechanism of peroxisome biogenesis, it is still unclear how peroxisome dysfunction leads to the disease state. Several recent studies have shown that mutations in Drosophila peroxin genes cause phenotypes similar to those seen in humans with PBDs suggesting that Drosophila might be a useful system to model PBDs. We have analyzed the proteome of Drosophila to identify the proteins involved in peroxisomal biogenesis and homeostasis as well as metabolic enzymes that function within the organelle. The subcellular localization of five of these predicted peroxisomal proteins was confirmed. Similar to C. elegans, Drosophila appears to only utilize the peroxisome targeting signal (PTS) type 1 system for matrix protein import. This work will further our understanding of peroxisomes in Drosophila and add to the usefulness of this emerging model system. PMID:22758915
Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency.
Ahlemeyer, Barbara; Vogt, Julia-Franziska; Michel, Vera; Hahn-Kohlberger, Petra; Baumgart-Vogt, Eveline
The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine(«) 2000, FuGENE 6, HiPerFect(«), INTERFERinÖ, RiboJuiceÖ) as well as microporation using the NeonÖ Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0-70%), to the reduction of PEX5 mRNA (by 90 vs. 0-50%) and PEX5 protein levels (by 70 vs. 0-50%). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3' fragment (15%) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies. PMID:25224142
PredPlantPTS1: A Web Server for the Prediction of Plant Peroxisomal Proteins
Reumann, Sigrun; Buchwald, Daniela; Lingner, Thomas
Prediction of subcellular protein localization is essential to correctly assign unknown proteins to cell organelle-specific protein networks and to ultimately determine protein function. For metazoa, several computational approaches have been developed in the past decade to predict peroxisomal proteins carrying the peroxisome targeting signal type 1 (PTS1). However, plant-specific PTS1 protein prediction methods have been lacking up to now, and pre-existing methods generally were incapable of correctly predicting low-abundance plant proteins possessing non-canonical PTS1 patterns. Recently, we presented a machine learning approach that is able to predict PTS1 proteins for higher plants (spermatophytes) with high accuracy and which can correctly identify unknown targeting patterns, i.e., novel PTS1 tripeptides and tripeptide residues. Here we describe the first plant-specific web server PredPlantPTS1 for the prediction of plant PTS1 proteins using the above-mentioned underlying models. The server allows the submission of protein sequences from diverse spermatophytes and also performs well for mosses and algae. The easy-to-use web interface provides detailed output in terms of (i) the peroxisomal targeting probability of the given sequence, (ii) information whether a particular non-canonical PTS1 tripeptide has already been experimentally verified, and (iii) the prediction scores for the single C-terminal 14 amino acid residues. The latter allows identification of predicted residues that inhibit peroxisome targeting and which can be optimized using site-directed mutagenesis to raise the peroxisome targeting efficiency. The prediction server will be instrumental in identifying low-abundance and stress-inducible peroxisomal proteins and defining the entire peroxisomal proteome of Arabidopsis and agronomically important crop plants. PredPlantPTS1 is freely accessible at ppp.gobics.de. PMID:22969783
Nitric oxide synthase inhibitors exert differential time-dependent effects on LPS-induced uveitis.
Allen, J B; McGahan, M C; Ferrell, J B; Adler, K B; Fleisher, L N
Nitric oxide (NO) is a highly reactive radical which plays an integral role in physiological and pathophysiological processes. NO is produced endogenously in small amounts by a constitutive NO synthase (cNOS) as a regulator of vascular tone and neurotransmission. NO can also be produced in large amounts by an inducible NOS (iNOS) in response to endotoxin and cytokines, and has been reported to be a mediator of lipopolysaccharide (LPS)-induced uveitis in rats. The purpose of the present study was to investigate the effects of NOS inhibitors with different NOS isoform specificities in the rabbit model of endotoxin-induced ocular inflammation. LPS and/or inhibitors of NOS. NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG), were injected intravitreally and the eyes observed by slit lamp for 24 hr. Coinjection of LPS with L-NAME inhibited anterior inflammation in rabbits. Iridal hyperemia (IH) and aqueous flare (AF) were completely abolished in eight out of nine rabbits in a dose-dependent manner. In addition, total cell counts were significantly suppressed (7393 +/- 697 vs. 325 +/- 188, P < 0.05) and aqueous protein levels were reduced to near control levels (25 +/- 0.75 vs. 1.72 +/- 0.36, P < 0.05). Similar suppression was seen with AG (cell counts = 351 +/- 246 and proteins = 3.1 +/- 1.2). Administration of L-NAME 0.5 hr after LPS injection suppressed inflammation to a lesser extent than coinjection. In contrast, administration of L-NAME 6 hr after LPS injection was not inhibitory, and in fact significantly increased cellular infiltration. However, AG given 6 hr after LPS had a remarkably different effect, since it significantly decreased both protein extravasation and cellular infiltration into the aqueous humor. In fact, our results suggest that cNOS may play a greater role in the earlier stages of this developing inflammatory response. These results extend others' observations that NO is a key mediator in uveitis, that induction of iNOS plays a critical role in experimental uveitis, and suggest that NO has a complex role in the ocular inflammatory process. Inhibitors of NOS can abort the LPS-induced inflammatory response if administered early enough, but could potentially exacerbate an established inflammatory episode. PMID:8674509
Effect of the Angiotensin I Converting Enzyme Inhibitor, MK-421, on Experimentally Induced Drinking
NASA Technical Reports Server (NTRS)
Fregley, Melvin J.; Fater, Dennis C.; Greenleaf, John E.
MK-421, the ethyl ester maleate salt of N-(S)-1-(ethoxycarbonyl)-3-phenyl-propyl- Ala-L-Pro, is an angiotensin I converting enzyme inhibitor. An initial objective was to determine whether MK-421, administered at 0, 2.5, 5.0, 10.0, 20.0 and 40.0 mg/kg, ip to 96 female rats 15 min prior to administration of the beta-adrenergic agonist, isoproterenol (25 microgram/kg, ip), would inhibit the drinking induced by isoproterenol during 2 h after its administration. The water intake induced by isoproterenol was inhibited significantly by 2.5 mg MK-421/kg. When a similar experiment was performed using Angiotensin I (AI) (200 microgram/kg, ip) as the dipsogenic agent, MK-421 (5 mg/kg, ip), administered 15 min prior to AI, inhibited significantly both the dipsogenic and the diuretic effect of AI. However, administration of angiotensin II (AII, 200 microgram/kg, ip) 15 min after MK-421 (5mg/kg) was accompanied by a water intake that did not differ from AII alone. The drink induced by ip administration of 1.0 m NaCl solution (1% of body wt, ip) was not inhibited by administration of MK-421 (5 mg/kg) 15 min prior to allowing access to water while the drink induced by a 24 h dehydration was partially inhibited. Thus, the drinks induced by administraition of either isoproterenol or AI are dependent on formation of AII. That induced by dehydration is partially dependent, while that induced by hypertonic siilinc is independent of the formation of AII.
A rat retinal damage model predicts for potential clinical visual disturbances induced by Hsp90 inhibitors
Zhou, Dan, E-mail: email@example.com [Synta Pharmaceuticals Corp., 45 Hartwell Avenue, Lexington, MA 02421 (United States); Liu, Yuan; Ye, Josephine; Ying, Weiwen; Ogawa, Luisa Shin; Inoue, Takayo; Tatsuta, Noriaki; Wada, Yumiko; Koya, Keizo [Synta Pharmaceuticals Corp., 45 Hartwell Avenue, Lexington, MA 02421 (United States); Huang, Qin [Department of Pathology and Laboratory Medicine, Veterans Affairs Boston Healthcare System, 1400 VFW Parkway, West Roxbury, MA 02132 (United States); Bates, Richard C.; Sonderfan, Andrew J. [Synta Pharmaceuticals Corp., 45 Hartwell Avenue, Lexington, MA 02421 (United States)
In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24 h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30 min post-injection were retained in the retina 6 h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6 h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential. - Highlights: Ģ In human trials some Hsp90 inhibitors cause visual disorders, others do not. Ģ Prolonged inhibition of Hsp90 in the rat eye results in photoreceptor cell death. Ģ Retina/plasma ratio and retinal elimination rate are linked to toxicity potential. Ģ Rat retinotoxic responses to individual Hsp90 inhibitors reflect clinical profiles. Ģ Rodent modeling may be used to assess ocular risks of targeted Hsp90 compounds.
[Cell senescence induced by histone deacetylase inhibitor sodium butyrate in rodent transformed cells resistant to apoptosis].
Shitikova, Zh V; Aksenov, N D; Pospelov, V A; Pospelov, T V
The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts transformed by E1A+E1B19 kDa oncogenes has been studied. These transformants are resistant to apoptosis in response to gamma-irradiation and growth factor deprivation. The process of cell senescence was investigated by the analysis of cell growth curves, G1/S and G2/M cell cycle arrest, and senescent associated beta-galactosidase expression. The irreversibility of sodium butyrate antiproliferative activity was analyzed by clonogenic assay. We show that sodium butyrate suppresses proliferation and induces senescence in the E1A+E1B19 kDa transformed cells. Interestingly, NaB induces growth arrest due to accumulation of cells in G2/M phase, these cells are not tetraploid but mainly binuclear. Thus, in case of NaB induced senescence in E1A+E1B19 kDa transformed fibroblasts, the observed suppression of cell proliferation may be the result of cytokinesis failure leading to formation of binuclear and multinuclear cells incapable to proliferate. PMID:21598691
Treatment with the hyaluronic Acid synthesis inhibitor 4-methylumbelliferone suppresses LPS-induced lung inflammation.
McKallip, Robert J; Ban, Hao; Uchakina, Olga N
Exposure to bacterial endotoxins, such as lipopolysaccharide (LPS), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for LPS-induced inflammation. In the current study, we investigated the potential use of the hyaluronic acid (HA) synthesis inhibitor 4-methylumbelliferone (4-MU) on LPS-induced acute lung inflammation. Culturing LPS-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production, and an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from LPS-induced lung injury. Specifically, 4-MU treatment led to a reduction in LPS-induced hyaluronic acid synthase (HAS) messenger RNA (mRNA) levels, reduction in lung permeability, and reduction in proinflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target HA production may be an effective treatment for the inflammatory response following exposure to LPS. PMID:25537799
Targeting ER stress¢induced autophagy overcomes BRAF inhibitor resistance in melanoma
Ma, Xiao-Hong; Piao, Sheng-Fu; Dey, Souvik; Mcafee, Quentin; Karakousis, Giorgos; Villanueva, Jessie; Hart, Lori S.; Levi, Samuel; Hu, Janice; Zhang, Gao; Lazova, Rossitza; Klump, Vincent; Pawelek, John M.; Xu, Xiaowei; Xu, Wei; Schuchter, Lynn M.; Davies, Michael A.; Herlyn, Meenhard; Winkler, Jeffrey; Koumenis, Constantinos; Amaravadi, Ravi K.
Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAFV600E melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAFV600E melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway. PMID:24569374
Tissue specificity and species differences in the distribution of urate oxidase in peroxisomes.
Usuda, N; Reddy, M K; Hashimoto, T; Rao, M S; Reddy, J K
The localization of urate oxidase in different tissues of rat and in the livers of selected mammalian species was investigated by immunoblot analysis and protein A-gold immunoelectron microscopy. Urate oxidase was purified from rat liver and used as an antigen to generate polyclonal antibodies in the rabbit. The antibodies were found to be monospecific by immunodiffusion and immunoblot analyses. By immunoblot analysis, urate oxidase was detected in the livers of rat, two strains of mice, hamster, dog, cat, and cow, but not in the Cynomolgus monkey and human liver. Urate oxidase was not detected by immunoblot method in rat kidney, jejunal mucosa, adrenal gland, testis, and pancreas. The subcellular localization of urate oxidase was ascertained by the protein A-gold immunocytochemical staining of the Lowicryl K4M embedded tissues. Urate oxidase was localized exclusively in the crystalloid core of the peroxisome in hepatic parenchymal cells of rat, mouse, hamster, dog, cat, and cow. The limiting membrane and the matrix of hepatic peroxisomes in these species were negative for the staining. The marginal plates of feline, canine, and bovine hepatic peroxisomes were also negative for urate oxidase. This enzyme was also not detected within the peroxisomes of human and monkey livers by the immunocytochemical technique. Peroxisomes (microperoxisomes) in extrahepatic rat tissues did not stain positively for urate oxidase by the protein A-gold immunocytochemical method, although they were positive for catalase. Fatty acyl-CoA oxidase was present in peroxisomes of jejunal mucosa, Leydig cells of test-is and pancreas but not in adrenal gland. Administration of a hepatic peroxisome proliferator, ciprofibrate or Wy-14643, failed to induce urate oxidase in rat liver. These results indicate that urate oxidase is a liver specific protein in rat and its localization within the liver peroxisomes of six mammals, excluding man and a nonhuman primate, and that its localization is limited exclusively to the crystalloid core. Unlike fatty acyl-CoA oxidase, urate oxidase does not appear to be inducible significantly by peroxisome proliferator treatment in the rat liver. PMID:3336202
bis(2,6-dioxopiperaxine) derivatives, topoisomerase II inhibitors which do not form a DNA cleavable complex, induce thymocyte apoptosis.
Onishi, Y; Azuma, Y; Kizaki, H
Internucleosomal DNA fragmentation and cell death were induced dose- and time-dependently by incubation of mouse thymocytes with bis(2,6-dioxopiperazine) derivatives, ICRF-154 and MST-16, inhibitors of topoisomerase II, which do not induce cleavable complex formation. The process was inhibited by actinomycin D and cycloheximide, indicating that the process was an active apoptotic process. Bis(2,6-dioxopiperazine) derivatives have been known to inhibit the etoposide-induced DNA cleavage, but ICRF-154 did not inhibit etoposide-induced apoptosis in thymocytes at 6 h incubation, suggesting that DNA cleavage is not essential for induction of apoptosis by topoisomerase II inhibitors. The alteration of DNA helicity induced by a subtle inhibition of topoisomerase II activity may have an important role in the induction of apoptosis in thymocytes, since topoisomerase II is a major component of the nuclear matrix that can regulate gene expression. PMID:8012276
Isoflurane-Induced Spatial Memory Impairment in Mice is Prevented by the Acetylcholinesterase Inhibitor Donepezil
Wang, Beilei; Xu, Huan; Li, Wen; Chen, Jie; Wang, Xiangrui
Although many studies have shown that isoflurane exposure impairs spatial memory in aged animals, there are no clinical treatments available to prevent this memory deficit. The anticholinergic properties of volatile anesthetics are a biologically plausible cause of cognitive dysfunction in elderly subjects. We hypothesized that pretreatment with the acetylcholinesterase inhibitor donepezil, which has been approved by the Food and Drug Administration (FDA) for the treatment of Alzheimer's disease, prevents isoflurane-induced spatial memory impairment in aged mice. In present study, eighteen-month-old mice were administered donepezil (5 mg/kg) or an equal volume of saline by oral gavage with a feeding needle for four weeks. Then the mice were exposed to isoflurane (1.2%) for six hours. Two weeks later, mice were subjected to the Morris water maze to examine the impairment of spatial memory after exposure to isoflurane. After the behavioral test, the mice were sacrificed, and the protein expression level of acetylcholinesterase (AChE), choline acetylase (ChAT) and ?7 nicotinic receptor (?7-nAChR) were measured in the brain. Each group consisted of 12 mice. We found that isoflurane exposure for six hours impaired the spatial memory of the mice. Compared with the control group, isoflurane exposure dramatically decreased the protein level of ChAT, but not AChE or ?7-nAChR. Donepezil prevented isoflurane-induced spatial memory impairments and increased ChAT levels, which were downregulated by isoflurane. In conclusions, pretreatment with the AChE inhibitor donepezil prevented isoflurane-induced spatial memory impairment in aged mice. The mechanism was associated with the upregulation of ChAT, which was decreased by isoflurane. PMID:22114680
Fatty acid-induced differential regulation of the genes encoding peroxisome proliferator-activated receptor-? coactivator-1? and -1? in human skeletal muscle cells that have been differentiated in vitro
H. Staiger; K. Staiger; C. Haas; M. Weisser; F. Machicao; H.-U. Hõring
Aims\\/hypothesisĀĀThe transcriptional coactivator peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) enhances metabolically relevant pathways, such as gluconeogenesis, fatty acid oxidation, thermogenesis, oxidative phosphorylation and mitochondrial biogenesis. Since regulation of the expression of the gene encoding PGC-1 (PPARGC1A) by nutrients\\/metabolites has not been assessed in detail, the aim of this study was to determine whether PPARGC1A (and PPARGC1B) expression is modulated by common
Redox interplay between mitochondria and peroxisomes
Lismont, Celien; Nordgren, Marcus; Van Veldhoven, Paul P.; Fransen, Marc
Reduction-oxidation or ōredoxö reactions are an integral part of a broad range of cellular processes such as gene expression, energy metabolism, protein import and folding, and autophagy. As many of these processes are intimately linked with cell fate decisions, transient or chronic changes in cellular redox equilibrium are likely to contribute to the initiation and progression of a plethora of human diseases. Since a long time, it is known that mitochondria are major players in redox regulation and signaling. More recently, it has become clear that also peroxisomes have the capacity to impact redox-linked physiological processes. To serve this function, peroxisomes cooperate with other organelles, including mitochondria. This review provides a comprehensive picture of what is currently known about the redox interplay between mitochondria and peroxisomes in mammals. We first outline the pro- and antioxidant systems of both organelles and how they may function as redox signaling nodes. Next, we critically review and discuss emerging evidence that peroxisomes and mitochondria share an intricate redox-sensitive relationship and cooperate in cell fate decisions. Key issues include possible physiological roles, messengers, and mechanisms. We also provide examples of how data mining of publicly-available datasets from ōomicsö technologies can be a powerful means to gain additional insights into potential redox signaling pathways between peroxisomes and mitochondria. Finally, we highlight the need for more studies that seek to clarify the mechanisms of how mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress. The outcome of such studies may open up exciting new avenues for the community of researchers working on cellular responses to organelle-derived oxidative stress, a research field in which the role of peroxisomes is currently highly underestimated and an issue of discussion.
Redox interplay between mitochondria and peroxisomes.
Lismont, Celien; Nordgren, Marcus; Van Veldhoven, Paul P; Fransen, Marc
Reduction-oxidation or "redox" reactions are an integral part of a broad range of cellular processes such as gene expression, energy metabolism, protein import and folding, and autophagy. As many of these processes are intimately linked with cell fate decisions, transient or chronic changes in cellular redox equilibrium are likely to contribute to the initiation and progression of a plethora of human diseases. Since a long time, it is known that mitochondria are major players in redox regulation and signaling. More recently, it has become clear that also peroxisomes have the capacity to impact redox-linked physiological processes. To serve this function, peroxisomes cooperate with other organelles, including mitochondria. This review provides a comprehensive picture of what is currently known about the redox interplay between mitochondria and peroxisomes in mammals. We first outline the pro- and antioxidant systems of both organelles and how they may function as redox signaling nodes. Next, we critically review and discuss emerging evidence that peroxisomes and mitochondria share an intricate redox-sensitive relationship and cooperate in cell fate decisions. Key issues include possible physiological roles, messengers, and mechanisms. We also provide examples of how data mining of publicly-available datasets from "omics" technologies can be a powerful means to gain additional insights into potential redox signaling pathways between peroxisomes and mitochondria. Finally, we highlight the need for more studies that seek to clarify the mechanisms of how mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress. The outcome of such studies may open up exciting new avenues for the community of researchers working on cellular responses to organelle-derived oxidative stress, a research field in which the role of peroxisomes is currently highly underestimated and an issue of discussion. PMID:26075204
Fusicoccin-induced catalase inhibitor is produced independently of H+-ATPase activation and behaves as an organic acid.
Beffagna, Nicoletta; Riva, Marzia Alessandra
The phytotoxin fusicoccin (FC) was found to induce an increase in apoplastic H?O? content in Arabidopsis thaliana cells, apparently linked to the presence of an as yet unidentified catalase inhibitor detectable even in the external medium of FC-treated cells. This study, aimed to further characterize the inhibitor's features, shows that (1) FC-induced H?O? accumulation increases as a function of FC concentration and correlates to the amount of inhibitor released at apoplastic level. The pattern of H+ efflux, conversely, does not fit with that of these two parameters, suggesting that neither the production nor the release of the catalase inhibitor is linked to the main role of FC in activating the plasma membrane (PM) H+-ATPase; (2) treatment with 10 ĄM erythrosine B (EB) early and totally inhibits net H+ and K+ fluxes across the PM, indicative of the H+ pump activity; nevertheless, also in these conditions a huge FC-induced H?O? accumulation occurs, confirming that this effect is not related to the FC-induced PM H+-ATPase activation; (3) the inhibitor's release increases with time in all conditions tested and is markedly affected by extracellular pH (a higher pH value being associated to a larger efflux), in agreement with a weak acid release; and (4) the inhibitor can be almost completely recovered in a CH?C?-soluble fraction extracted from the incubation medium by sequential acid-base partitioning which contains nearly all of the organic acids released. These final results strongly suggest that the metabolite responsible for the FC-induced catalase inhibition belongs to the organic acid class. PMID:21320127
Peroxisome proliferators (PP) are a large class of structurally diverse chemicals that mediate their effects in the liver mainly through the PP-activated receptor ┐ (PPAR?). Development of PP induced hepatocarcinogenesis in mouse liver is known to be dependent on PPAR...
Peroxisome proliferator-activated receptorĀ?-dependent and -independent growth inhibition of gastrointestinal tumour cells
M. Azharul Karim Rumi; Shunji Ishihara; Yasunori Kadowaki; C. F. Ortega-Cava; Hideaki Kazumori; Kousaku Kawashima; Nagisa Yoshino; Takafumi Yuki; Norihisa Ishimura; Yoshikazu Kinoshita
Peroxisome proliferator-activated receptor ? ? ? ? (PPAR? ? ? ? ) acts as a ligand-activated transcription factor. Although ligand-induced cellular differentiation and growth inhibition have been mostly studied on human cancers expressing PPAR? ? ? ? , it is unclear if the transcriptional activation of PPAR? ? ? ? is the main mechanism of growth inhibition. In this study,
Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism
Wang, Hung-Jung; Hsu, Kai-Cheng; Lin, Shuang-Chih; Chen, Tzu-Jung; Yang, Jinn-Moon; Wang, Wen-Ching
Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure¢activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSKĘSO4, R57A, and HpSKĢshikimate-3-phosphateĢADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114AĢ162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism. PMID:22438938
Cyclooxygenase-2 Inhibitor Reduces Simvastatin-Induced Bone Morphogenetic Protein-2 and Bone Formation In Vivo
Bradley, J. D.; Cleverly, D. G.; Burns, A. M.; Helm, N. B.; Schmid, M. J.; Marx, D. B.; Cullen, D. M.
Objectives: Simvastatin, a cholesterol-lowering drug, also stimulates oral bone growth when applied topically, without systemic side effects. However, the mechanisms involved in vivo are not known. We hypothesized that bone morphogenetic protein-2 (BMP-2), nitric oxide synthase (NOS) and cyclooxygenase (COX)-2 are involved, based on prior in vitro evidence. Material and Methods: A rat bilateral mandible model, where 0.5 mg simvastatin in methylcellulose gel (SIM) was placed on one side and gel alone (GEL) on the other, was used to quantitate NO, COX-2 and BMP-2 (via tissue extraction, enzyme activity or immunoassay), and bone formation rate (BFR; via undecalcified histomorphometry). COX-2 and NOS inhibitors (N-398 and L-NAME, respectively) also were administered intraperitoneally. Results: SIM was found to stimulate local BMP-2, NO and regional BFR (p < 0.05), while NS-398 inhibited BMP-2 and BFR (p ? 0.05). Conclusion: These data suggest an association between simvastatin-induced BMP-2 and bone formation in the mandibular microenvironment, and the negative effect of COX-2 inhibitors on bone growth. PMID:17451547
S156C Mutation in Tissue Inhibitor of Metalloproteinases-3 Induces Increased Angiogenesis*
Qi, Jian Hua; Dai, Ganying; Luthert, Philip; Chaurasia, Shyam; Hollyfield, Joe; Weber, Bernhard H. F.; St÷hr, Heidi; Anand-Apte, Bela
Tissue Inhibitor of metalloproteinases-3 (TIMP-3) is a potent matrix-bound angiogenesis inhibitor. Mutations in TIMP-3 cause Sorsby Fundus Dystrophy, a dominant inherited, early onset macular degenerative disease, with choroidal neovascularization causing a loss of vision in the majority of patients. Here we report that expression of S156C TIMP-3 mutation in endothelial cells results in an abnormal localization of the protein, increased gly co sy la tion, decreased matrix metalloproteinase inhibitory activity, and increased vascular endothelial growth factor (VEGF) binding with a consequent increase in VEGF-de pend ent migration and tube formation. These enhanced signaling events appear to be mediated as a consequence of a post-transcriptionally regulated increase in the expression of membrane-associated VEGFR-2 in endothelial cells of Timp-3156/156 mutant mice as well as in human Sorsby fundus dystrophy eyes. Understanding the mechanism(s) by which mutant TIMP-3 can induce abnormal neovascularization provides important insight into the pathophysiology of a number of diseases with increased angiogenesis. PMID:19478078
Small molecule XIAP inhibitors enhance TRAIL-induced apoptosis and antitumor activity in preclinical models of pancreatic carcinoma.
Vogler, Meike; Walczak, Henning; Stadel, Dominic; Haas, Tobias L; Genze, Felicitas; Jovanovic, Marjana; Bhanot, Umesh; Hasel, Cornelia; M÷ller, Peter; Gschwend, J³rgen E; Simmet, Thomas; Debatin, Klaus-Michael; Fulda, Simone
Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer. PMID:19258513
Urinary trypsin inhibitor attenuated inflammatory response of patients undergoing cardiopulmonary bypass by inducing activated Treg cells.
Hao, Xing; Han, Junyan; Xing, Zhichen; Hao, Yu; Jiang, Chunjing; Zhang, Jianping; Yang, Jing; Hou, Xiaotong
The urinary trypsin inhibitor (ulinastatin) is used in the clinic to prevent inflammatory responses in patients undergoing cardiopulmonary bypass (CPB); however, the anti-inflammatory mechanism is unclear. In the current study, we recruited 40 patients undergoing selective cardiac valve replacement surgery; and these patients were randomly divided into two groups (ulinastatin group [UG] and control group [CG]). We collected peripheral blood preoperatively, at the end of CPB, and postoperative days 1 and 3 and analyzed the kinetic changes in regulatory T (Treg) cell subsets. There was no statistically significant difference in the number of CD4(+) T cells between the two groups. The number of CD4(+)CD25(+) Treg cells, especially the suppressive activated Treg (aTreg) subset, was higher in the UG than the CG 1 and 3 days postoperatively. Thus, ulinastatin alleviated the inflammatory response during CPB by inducing the expansion of aTreg cells. PMID:23765601
Inhibitors of nitric oxide synthetase prevent castor-oil-induced diarrhoea in the rat.
Mascolo, N.; Izzo, A. A.; Barbato, F.; Capasso, F.
1. Castor oil (2 ml orally) produced copious diarrhoea in rats 3 h after its administration. 2. Pretreatment (intraperitoneal, i.p.) of rats with the NO synthesis inhibitors NG-nitro-L-arginine methyl ester (L-NAME, 1-25 mg kg-1) and NG-monomethyl-L-arginine (L-NMMA, 2.5-100 mg kg-1) inhibited or prevented castor-oil-induced diarrhoea. L-Arginine (150-600 mg kg-1, i.p.) administered to rats pretreated with L-NAME 10 mg kg-1, drastically reduced the antidiarrhoeal activity of L-NAME in a dose-related manner. D-Arginine (900 mg kg-1) did not modify the protection by L-NAME. 3. Pretreatment (i.p.) of rats with L-NAME (2.5-25 mg kg-1) decreased the intestinal fluid accumulation and Na+ secretion induced by castor oil. L-Arginine (600 mg kg-1) but not D-arginine (900 mg kg-1) counteracted the inhibitory effect of L-NAME (10 mg kg-1). 4. L-NAME (10 and 25 mg kg-1) had no significant effect on the intestinal transit in normal rats or those given castor oil. 5. These results provide evidence that nitric oxide (NO) could play an important role in castor-oil-induced diarrhoea. PMID:7683565
Thrombocytopenia induced by the histone deacetylase inhibitor abexinostat involves p53-dependent and -independent mechanisms
Ali, A; Bluteau, O; Messaoudi, K; Palazzo, A; Boukour, S; Lordier, L; Lecluse, Y; Rameau, P; Kraus-Berthier, L; Jacquet-Bescond, A; LeliĶvre, H; Depil, S; Dessen, P; Solary, E; Raslova, H; Vainchenker, W; Plo, I; Debili, N
Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34+ cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated ?H2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation. PMID:23887629
Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.
Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang
Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis. PMID:19450551
Sensitization to UV-induced apoptosis by the histone deacetylase inhibitor trichostatin A (TSA)
Kim, Myoung Sook [Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Baek, Jin Hyen; Chakravarty, Devulapalli [Biochemistry and Molecular Biology Department, School of Medicine, and Greenebaum Cancer Center, University of Maryland, 108 North Greene Street, Room 330, Baltimore, MD 21201-1503 (United States); Sidransky, David [Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Carrier, France [Biochemistry and Molecular Biology Department, School of Medicine, and Greenebaum Cancer Center, University of Maryland, 108 North Greene Street, Room 330, Baltimore, MD 21201-1503 (United States)]. E-mail: email@example.com
UV-induced apoptosis is a protective mechanism that is primarily caused by DNA damage. Cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts are the main DNA adducts triggered by UV radiation. Because the formation of DNA lesions in the chromatin is modulated by the structure of the nucleosomes, we postulated that modification of chromatin compaction could affect the formation of the lesions and consequently apoptosis. To verify this possibility we treated human colon carcinoma RKO cells with the histone deacetylase inhibitor trichostatin A (TSA) prior to exposure to UV radiation. Our data show that pre-treatment with TSA increased UV killing efficiency by more than threefold. This effect correlated with increased formation of CPDs and consequently apoptosis. On the other hand, TSA treatment after UV exposure rather than before had no more effect than UV radiation alone. This suggests that a primed (opened) chromatin status is required to sensitize the cells. Moreover, TSA sensitization to UV-induced apoptosis is p53 dependent. p53 and acetylation of the core histones may thus contribute to UV-induced apoptosis by modulating the formation of DNA lesions on chromatin.
Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers
Cheung, Hiu Wing; Du, Jinyan; Boehm, Jesse S.; He, Frank; Weir, Barbara A.; Wang, Xiaoxing; Butaney, Mohit; Sequist, Lecia V.; Luo, Biao; Engelman, Jeffrey A.; Root, David E.; Meyerson, Matthew; Golub, Todd R.; Jõnne, Pasi A.; Hahn, William C.
We previously identified a region of recurrent amplification on chromosome 22q11.21 in a subset of primary lung adenocarcinomas. Here we show that CRKL, encoding for an adaptor protein, is amplified and overexpressed in non-small cell lung cancer (NSCLC) cells that harbor 22q11.21 amplifications. Overexpression of CRKL in immortalized human airway epithelial cells promoted anchorage independent growth and tumorigenicity. Oncogenic CRKL activates SOS1-RAS-RAF-ERK and SRC-C3G-RAP1 pathways. Suppression of CRKL in NSCLC cells that harbor CRKL amplifications induced cell death. Overexpression of CRKL in EGFR mutant cells induces resistance to gefitinib by activating ERK and AKT signaling. We identified CRKL amplification in an EGFR inhibitor treated lung adenocarcinoma that was not present prior to treatment. These observations show that CRKL overexpression induces cell transformation, credential CRKL as a therapeutic target for a subset of NSCLC that harbor CRKL amplifications and implicate CRKL as an additional mechanism of resistance to EGFR-directed therapy. PMID:22586683
Proteasome inhibitor bortezomib impairs both myelofibrosis and osteosclerosis induced by high thrombopoietin levels in mice.
Wagner-Ballon, Orianne; Pisani, Didier F; Gastinne, Thomas; Tulliez, Micheline; Chalignķ, Ronan; Lacout, Catherine; Auradķ, Frķdķric; Villeval, Jean-Luc; Gonin, Patrick; Vainchenker, William; Giraudier, Stķphane
Primary myelofibrosis (PMF) is the most serious myeloproliferative disorder, characterized by clonal myeloproliferation associated with cytokine-mediated bone marrow stromal reaction including fibrosis and osteosclerosis. Current drug therapy remains mainly palliative. Because the NF-kappaB pathway is implicated in the abnormal release of cytokines in PMF, the proteasome inhibitor bortezomib might be a potential therapy. To test its effect, we used the lethal murine model of myelofibrosis induced by thrombopoietin (TPO) overexpression. In this TPO(high) model, the development of the disease is related to a deregulated MPL signaling, as recently described in PMF patients. We first demonstrated that bortezomib was able to inhibit TPO-induced NF-kappaB activation in vitro in murine megakaryocytes. It also inhibited NF-kappaB activation in vivo in TPO(high) mice leading to decreased IL-1alpha plasma levels. After 4 weeks of treatment, bortezomib decreased TGF-beta1 levels in marrow fluids and impaired marrow and spleen fibrosis development. After 12 weeks of treatment, bortezomib also impaired osteosclerosis development through osteoprotegerin inhibition. Moreover, this drug reduced myeloproliferation induced by high TPO level. Finally, bortezomib dramatically improved TPO(high) mouse survival (89% vs 8% at week 52). We conclude that bortezomib appears as a promising therapy for future treatment of PMF patients. PMID:17374740
Peroxisomes Are Required for in Vivo Nitric Oxide Accumulation in the Cytosol following Salinity Stress of Arabidopsis Plants1[C][W][OA
Corpas, Francisco J.; Hayashi, Makoto; Mano, Shoji; Nishimura, Mikio; Barroso, Juan B.
Peroxisomes are unique organelles involved in multiple cellular metabolic pathways. Nitric oxide (NO) is a free radical active in many physiological functions under normal and stress conditions. Using Arabidopsis (Arabidopsis thaliana) wild type and mutants expressing green fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, this study analyzes the temporal and cell distribution of NO during the development of 3-, 5-, 8-, and 11-d-old Arabidopsis seedlings and shows that Arabidopsis peroxisomes accumulate NO in vivo. Pharmacological analyses using nitric oxide synthase (NOS) inhibitors detected the presence of putative calcium-dependent NOS activity. Furthermore, peroxins Pex12 and Pex13 appear to be involved in transporting the putative NOS protein to peroxisomes, since pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content. Additionally, we show that under salinity stress (100 mm NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO?) and in increasing protein tyrosine nitration, which is a marker of nitrosative stress. PMID:19783645
USP14 inhibitor IU1 prevents ventilator-induced lung injury in rats.
Xu, Y-H; Guo, N-L
The pathophysiology of ventilator-induced lung injury (VILI) involves multiple mechanisms including inflammation. USP14 removes the ubiquitin chain of I-?B, therefore inducing I-?B degradation and increasing cytokine release. The purpose of this study was to examine the protecting roles and mechanisms of USP14 inhibitor on I-?B expression and lung injury induced by high tidal volume ventilation in normal rat lung. Male Sprague-Dawley rats were divided into follows: Two ventilation modalities were used: rats in Groups LD (low volume + DMSO) and LI (low volume + IU1) received ventilation with a tidal volume of 8 ml/kg, while the rats in Groups HD (high volume + DMSO) and HI (high volume + IU1) were ventilated with a tidal volume of 40 ml/kg. The levels of lung wet-to-dry weight ratio were used as indicators of water metabolism in lung tissue; the detection of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid was used to indicate inflammatory response, while lung injury was assessed by injury score and morphological changes under light microscope. The USP14 and I-?B protein level was measured in lung tissue by Western blot. Our results indicated that administration of IU1 alleviated ventilator-induced lung injury which was accompanied by reduced MPO activity, wet-to-dry weight ratio, lower TNF-?, IL-1?, IL-6 and IL-8 levels and increased I-?B expression in lung tissue. IU1 could significantly alleviate ventilator-induced rat lung injury by attenuate intrapulmonary inflammatory response. PMID:25198582
mTOR inhibitor AZD8055 inhibits proliferation and induces apoptosis in laryngeal carcinoma
Zhao, Lijing; Teng, Bo; Wen, Lianji; Feng, Qingjie; Wang, Hebin; Li, Na; Wang, Yafang; Liang, Zuowen
The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, against all class I phosphatidylinositol3-kinase (PI3K) and other members of the PI3K-like kinase family. The study was to determine the effect of AZD8055 on proliferation and apoptosis on Hep-2, a human laryngeal cancer cell line and to investigate the underlying mechanism(s) of action. Hep-2 cells were treated with AZD8055 for 24, 48 or 72 h. MTT was used to determine cell proliferation. Rhodamine 123 and TUNEL staining were used to determine mitochondrial membrane potential and cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Protein expressions were examined by western blotting. Treatment with AZD8055 inhibited proliferation and induced apoptosis in Hep-2 cells in a dose- and time-dependent manner. During the prolonged treatment with AZD8055, AZD8055 inhibits the mammalian target of rapamycin mTOR. Further experiments showed which signaling cascade p-4EBP1 and substrate EIF4E as well as downstream proteins were down regulated. Furthermore, our study showed that the expression profiles of various BH3-only proteins including Bid, Bad, and Bim, apoptosis regulatory protein cleaved caspase3 was up regulated in a time-dependent manner in Hep-2 cells treated with AZD8055. Thus, in vitro, AZD8055 potently inhibits proliferation and induces apoptosis in head and neck squamous cell carcinoma. PMID:24600487
Reactive oxygen species induce a procoagulant state in endothelial cells by inhibiting tissue factor pathway inhibitor.
Cimmino, Giovanni; Cirillo, Plinio; Ragni, Massimo; Conte, Stefano; Uccello, Giuseppe; Golino, Paolo
Tissue factor pathway inhibitor (TFPI) is a serine-protease inhibitor, which modulates coagulation tissue factor-dependent (TF). It binds directly and inhibits the TF-FVII/FVIIa complex as well as FXa. Time to reperfusion of acute ischemic myocardium is essential for tissue salvage. However, reperfusion also results in a unique form of myocardial damage, such as contractile dysfunction, decreased coronary flow and altered vascular reactivity. Oxidants and reactive oxygen species (ROS) formation is increased in the post-ischemic heart and is responsible of post-ischemic injury. It has been reported that ROS promote a procoagulant state via TF expression while no data are available on the effect on TFPI. Endothelial cells were incubated with two different ROS generating systems, xanthine (X)/xanthine oxidase (XO) for 5Āmin, or H2O2 (500Ā?M) for 24Āh. TFPI activity was measured in supernatants by chromogenic assay and TFPI-mRNA analyzed by RT-PCR 2Āh after ROS exposure. Unstimulated cells and cells exposed to either X or XO served as controls. Western blot and ligand dot blot was performed to evaluate ROS effect on TFPI structure and binding to FXa. ROS generated by X/XO as well as H2O2 system resulted in decreased TFPI activity compared to unstimulated cells while X or XO alone had no effect. No differences in TFPI mRNA levels versus controls was observed. A significant degradation of TFPI was induced by ROS exposure, resulting in a decreased ability to bind FXa. ROS induce a procoagulant state in endothelial cells by altering TFPI structure, resulting in inhibition of TFPI binding to Factor Xa and loss of activity. This phenomenon might have important consequences during reperfusion of post-ischemic myocardium. PMID:25712553
The PARP inhibitor AZD2281 (Olaparib) induces autophagy/mitophagy in BRCA1 and BRCA2 mutant breast cancer cells.
Arun, Banu; Akar, Ugur; Gutierrez-Barrera, Angelica M; Hortobagyi, Gabriel N; Ozpolat, Bulent
PARP inhibitors are considered promising anticancer agents and currently being tested in clinical trials in hereditary breast cancer patients harboring mutations in BRCA1 and BRCA2 genes. In this study, we investigated the antiproliferative effects and mechanism of PARP inhibitors ABT-888 (Veliparib), BSI-201 (Iniparib) and AZD228 (Olaparib) in breast cancer cell lines with BRCA1 or BRCA2 mutations and 9 different BRCA wild-type cell lines with BRCA1 allelic loss. We found that AZD2281 was the most potent in the PARP inhibitors and induces significant growth inhibition (~95%) in BRCA1 mutant (HCC?1937, MDA-MB-436, and SUM-149PT) and BRCA2 mutant (HCC?1428) cell lines. AZD2281 treatment also resulted in growth inhibition ranging from 20 to 50% in cells with BRCA1 allelic loss, including ER(+), HER2/Neu(+) and triple-negative breast cancer (TNBC) cells, but showed no effect in cells without with type BRCA without allelic loss. Knocking down of BRCA1 or BRCA2 in TNBC cells with BRCA1 allelic loss by RNA interference significantly enhanced AZD2281-induced growth inhibition and induced significant autophagy that was associated with mitophagy in cells with BRCA mutations. Inhibition of autophagy by gene knockdown significantly diminished AZD2281-induced mitophagy and apoptosis, indicating that autophagic process mediates some of the downstream effects of PARP inhibitors. In conclusion, our data provide the first evidence of PARP inhibitor AZD2281 autophagy and mitophagy in breast cancer cell lines with BRCA mutations or BRCA-allelic loss. In addition, our results indicate that the patients with BRCA1 allelic loss may also benefit from PARP inhibitor therapy if BRCA is further inhibited. PMID:25975349
VEGF-A Induces Angiogenesis by Perturbing the Cathepsin-Cysteine Protease Inhibitor Balance in Venules, Causing Basement Membrane
Bogyo, Matthew
VEGF-A Induces Angiogenesis by Perturbing the Cathepsin-Cysteine Protease Inhibitor Balance Tumors initiate angiogenesis primarily by secreting vascular endothelial growth factor (VEGF-A164 tissues with an adenoviral vector expressing VEGF-A164 (Ad-VEGF-A164 ) or with VEGF-AŁ secreting TA3/St
3Hydroxy3-Methylglutaryl Coenzyme A Reductase and Isoprenylation Inhibitors Induce Apoptosis of Vascular Smooth Muscle Cells in Culture
Carlos Guijarro; Luis Miguel Blanco-Colio; Monica Ortego; Covadonga Alonso; Alberto Ortiz; Juan JosePlaza; Cristina Diaz; Gonzalo Hernandez; Jesus Egido
Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to induce apoptosis in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis of VSMCs, cultured rat VSMCs were treated with increasing doses of atorvastatin
Effects of L-canavanine, an inhibitor of inducible nitric oxide synthase, on endotoxin mediated shock in rats.
Fatehi-Hassanabad, Z; Burns, H; Aughey, E A; Paul, A; Plevin, R; Parratt, J R; Furman, B L
The effects of L-canavanine, an inhibitor of nitric oxide synthase, on endotoxin-induced shock was investigated in the pentobarbitone anesthetized rat. Endotoxin infusion (2.5 mg kg-1 h-1 over 6 h) produced progressive and marked hypotension and hypoglycemia. Electron microscopy showed marked changes in the kidney, comprising severe endothelial cell disruption and the accumulation of platelets in the blood vessels. In the lung, there was marked accumulation of polymorphonuclear leukocytes in small blood vessels and endothelial disruption. Treatment with L-canavanine (10 mg kg-1 by bolus injection each hour starting 70 min after endotoxin or saline infusion) significantly reduced endotoxin-induced hypotension, without any effect on the hypoglycemia. This treatment markedly reduced the endotoxin-induced electron microscopical changes in the kidneys and lungs. Although L-canavanine, like L-NAME, inhibited both cerebellar constitute and splenic inducible nitric oxide synthase in vitro, in contrast to L-NAME it did not modify either arterial blood pressure or carotid artery blood flow in control rats. The data are consistent with L-canavanine being a selective inhibitor of inducible nitric oxide synthase, at least in vivo, and suggest that inhibitors of this enzyme may be beneficial in endotoxin-induced shock. PMID:8885085
Peroxisome proliferator-activated receptors for hypertension
Usuda, Daisuke; Kanda, Tsugiyasu
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes (?, ?, ?, and ?). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPAR? and PPAR? agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPAR? agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin II receptor blockers, should be studied. This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases. PMID:25228953
Inhibition of atrial wall stretch-induced cardiac hormone secretion by lavendustin A, a potent tyrosine kinase inhibitor.
Taskinen, P; Toth, M; Vuolteenaho, O; Magga, J; Ruskoaho, H
The cellular processes linking mechanical wall stretch to atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) secretion from the heart are unclear. In the present study, a paced perfused rat heart preparation was used to study the signaling mechanisms of atrial wall stretch-induced secretion of ANP and BNP. Vehicle or drugs were infused into the perfusate for 40 min and right atrial wall stretch was superimposed for 10 min after 25-min drug infusions by elevating the level of the pulmonary artery cannula tip. Lavendustin A, a potent inhibitor of protein tyrosine kinases, at the concentrations of 0.5 and 1.3 microM decreased atrial wall stretch-induced ANP secretion (53% and 68%, respectively, P < 0.001) in the perfused rat heart preparation, whereas no difference in the hemodynamic variables (heart rate, contractile force and perfusion pressure) were noted between groups. Lavendustin A also completely abolished the wall stretch-induced secretion of BNP. Several other protein kinase inhibitors including staurosporine (protein kinase C inhibitor), ML-9 (myosin light chain kinase inhibitor), KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) and H-89 (protein kinase A inhibitor) had no significant effect on atrial wall stretch-stimulated ANP secretion. In a separate series of experiments, in which the right atria were stretched for 2 h, administration of lavendustin A (1 microM) but not staurosporine (30 nM) significantly decreased sustained wall stretch-induced ANP secretion. Okadaic acid, a potent protein phosphatase A2 (PPA2) and PP1 inhibitor, at the concentration of 100 nM had no effect on basal ANP secretion but significantly accelerated the ANP secretory response to atrial wall stretch (P < 0.05). In conclusion, the findings that inhibitors of protein tyrosine kinase and protein phosphatase selectively modulated atrial wall stretch-induced ANP secretion suggest a new mechanism involving endogenous protein tyrosine activity in the regulation of natriuretic peptide exocytosis from cardiac myocytes. PMID:10465292
Regulation of apoptosis by peroxisome proliferators.
Roberts, Ruth A; Michel, Cecile; Coyle, Beth; Freathy, Caroline; Cain, Kelvin; Boitier, Eric
Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis. Results show that some of the key steps of the LCM process had an impact on the gene profiles generated. However, this did not preclude accurate determination of a PP-specific molecular signature. Thus, the choice of appropriate controls will ensure that meaningful gene expression analyses can be performed on tissue microdissected from the foci generated in clofibric acid treated livers. These data will allow the identification of specific genes that are regulated by PPs leading to changes in apoptosis and ultimately to tumours. PMID:15093246
Myelin associated inhibitors: A link between injury-induced and experience-dependent plasticity
Feras Akbik; William B. J. Cafferty; Stephen M. Strittmatter
In the adult, both neurologic recovery and anatomical growth after a CNS injury are limited. Two classes of growth inhibitors, myelin associated inhibitors (MAIs) and extracellular matrix associated inhibitors, limit both functional recovery and anatomical rearrangements in animal models of spinal cord injury. Here we focus on how MAIs limit a wide spectrum of growth that includes regeneration, sprouting, and
RANKL Inhibitors Induce Osteonecrosis of the Jaw in Mice With Periapical Disease
Aghaloo, Tara L; Cheong, Simon; Bezouglaia, Olga; Kostenuik, Paul; Atti, Elisa; Dry, Sarah M; Pirih, Flavia Q; Tetradis, Sotirios
Antiresorptive medications are essential in treating diseases of pathologic osteoclastic bone resorption, including bone cancer and osteoporosis. Bisphosphonates (BPs) are the most commonly used antiresorptives in clinical practice. Although inhibition of bone resorption is important in regulating unwanted malignant and metabolic osteolysis, BP treatment is associated with potential side effects, including osteonecrosis of the jaws (ONJ). Recently, non-BP antiresorptive medications targeting osteoclastic function and differentiation, such as denosumab, have entered the clinical arena. Denosumab treatment results in a similar rate of ONJ as BPs. Animal models of ONJ, using high-dose BP treatment in combination with tooth extraction or dental disease, provide valuable tools and insight in exploring ONJ pathophysiology. However, the ability of other antiresorptives to induce ONJ-like lesions in animal models has not been explored. Such studies would be beneficial in providing support for the role of osteoclast inhibition in ONJ pathogenesis versus a direct BP effect on oral tissues. Here, we tested the ability of the receptor activator of NF-kB ligand (RANKL) inhibitors RANK-Fc (composed of the extracellular domain of RANK fused to the fragment crystallizable [Fc] portion of immunoglobulin G [IgG]) and OPG-Fc (composed of the RANKL-binding domains of osteoprotegerin [OPG] linked to the Fc portion of IgG) to induce ONJ in mice in the presence of periapical disease, but in the absence of dental extractions. We demonstrate radiographic evidence of ONJ in RANK-Fc¢treated and OPG-Fc¢treated mice, including inhibition of bone loss, increased bone density, lamina dura thickening, and periosteal bone deposition. These findings closely resembled the radiographic appearance of an ONJ patient on denosumab treatment. Histologic examination revealed that RANK-Fc treatment and OPG-Fc treatment resulted in absence of osteoclasts, periosteal bone formation, empty osteocytic lacunae, osteonecrosis, and bone exposure. In conclusion, we have successfully induced ONJ in mice with periapical disease, using potent osteoclast inhibitors other than BPs. Our findings, coupled with ONJ animal models using high-dose BPs, suggest that osteoclast inhibition is pivotal to the pathogenesis of ONJ. PMID:24115073
Amelioration of intracerebroventricular streptozotocin induced cognitive dysfunction and oxidative stress by vinpocetine -- a PDE1 inhibitor.
Deshmukh, Rahul; Sharma, Vivek; Mehan, Sidharth; Sharma, Nidhi; Bedi, K L
Enhancing cyclic nucleotides signaling by inhibition of phosphodiesterases (PDEs) is known to be beneficial in disorders associated with cognitive decline. The present study was designed to investigate the effect of vinpocetine (PDE1 inhibitor) on intracerebroventricular (i.c.v.) streptozotocin induced experimental sporadic dementia of Alzheimer's type. Infusion of streptozotocin impaired learning and memory, increased oxidative-nitritive stress and induced cholinergic hypofunction in rats. Chronic treatment with vinpocetine (5, 10 and 20 mg/kg i.p.) for 21 days following first i.c.v. streptozotocin infusion significantly improved learning and memory in Morris water maze and passive avoidance paradigms. Further, vinpocetine significantly reduced the oxidative-nitritive stress, as evidenced by decrease in malondialdehyde (MDA) and nitrite levels, and restored the reduced glutathione (GSH) levels. Significant increase in acetylcholinesterase activity and lactate dehydrogenase levels was observed in the present model indicating cholinergic hypofunction and increase in neuronal cell damage. Chronic treatment with vinpocetine also reduced significantly the increase in acetylcholinesterase activity and lactate dehydrogenase levels indicating restorative capacity of vinpocetine with respect to cholinergic functions and preventing the neuronal damage. The observed beneficial effects of vinpocetine on spatial memory may be due to its ability to favorably modulate cholinergic functions, prevent neuronal cell damage and possibly through its antioxidant mechanism also. PMID:19699735
Aminoguanidine, an inhibitor of inducible nitric oxide synthase, ameliorates experimental autoimmune encephalomyelitis in SJL mice.
Cross, A H; Misko, T P; Lin, R F; Hickey, W F; Trotter, J L; Tilton, R G
Previous work from our laboratory localized nitric oxide to the affected spinal cords of mice with experimental autoimmune encephalomyelitis, a prime model for the human disease multiple sclerosis. The present study shows that activated lymphocytes sensitized to the central nervous system encephalitogen, myelin basic protein, can induce nitric oxide production by a murine macrophage cell line. Induction was inhibited by amino-guanidine, a preferential inhibitor of the inducible nitric oxide synthase isoform, and by NG-monomethyl-L-arginine. Aminoguanidine, when administered to mice sensitized to develop experimental autoimmune encephalomyelitis, inhibited disease expression in a dose-related manner. At 400 mg aminoguanidine/kg per day, disease onset was delayed and the mean maximum clinical score was 0.9 +/- 1.2 in aminoguanidine versus 3.9 +/- 0.9 in placebo-treated mice. Histologic scoring of the spinal cords for inflammation, demyelination, and axonal necrosis revealed significantly less pathology in the aminoguanidine-treated group. The present study implicates excessive nitric oxide production in the pathogenesis of murine inflammatory central nervous system demyelination, and perhaps in the human disease multiple sclerosis. Images PMID:7515395
The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress
Riganti, Chiara [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Torino (Italy)], E-mail: firstname.lastname@example.com; Costamagna, Costanzo; Doublier, Sophie; Miraglia, Erica; Polimeni, Manuela; Bosia, Amalia; Ghigo, Dario [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Torino (Italy)
We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-{kappa}B and decreased intracellular level of its inhibitor IkB{alpha}. These effects, accompanied by increased production of H{sub 2}O{sub 2}, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-{kappa}B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.
JTP-103237, a novel monoacylglycerol acyltransferase inhibitor, modulates fat absorption and prevents diet-induced obesity.
Okuma, Chihiro; Ohta, Takeshi; Tadaki, Hironobu; Hamada, Hiromi; Oda, Tomohiro; Taniuchi, Hideyuki; Yamanaka, Kenji; Ishii, Yukihito; Ohe, Yasuhiro; Yata, Shinji; Nishiu, Jun; Aratsu, Yusuke; Oshida, Shinichi; Kume, Shinichi; Kakutani, Makoto
Monoacylglycerol acyltransferase 2 (MGAT2) plays an important role in intestinal fat absorption. We discovered the novel MGAT2 inhibitor, JTP-103237, and evaluated its pharmacological profile. JTP-103237 selectively inhibited MGAT2 without remarkable species differences and reduced absorbed lipids in circulation. After lipid administration, JTP-103237 slightly but significantly decreased triglyceride content in proximal small intestine and significantly increased the lipids content in the distal small intestine. In addition, JTP-103237 significantly increased MGAT substrate (monoacylglycerol and fatty acid) content in the small intestine. JTP-103237 increased plasma peptide YY levels after lipid loading and reduced food intake in a dietary fat-dependent manner. After chronic treatment, JTP-103237 significantly decreased body weight and increased O2 consumption in the early dark phase in high fat diet induced obese (DIO) mice. Moreover, JTP-103237 improved glucose tolerance and decreased fat weight and hepatic triglyceride content in DIO mice. Our findings indicate that JTP-103237 prevents diet-induced obesity by inhibiting intestinal MGAT2 and has unique properties as a drug for the treatment of obesity. PMID:25857225
The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey
Ofman, Rob; Bunse, Christian; Pawlas, Magdalena; Hayen, Heiko; Eisenacher, Martin; Stephan, Christian; Meyer, Helmut E.; Waterham, Hans R.; Erdmann, Ralf; Wanders, Ronald J.; Warscheid, Bettina
The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or ?-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD+ becomes regenerated during fatty acid ?-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease. PMID:23460848
Anti-inflammatory effect of a selective inhibitor of elastase, cathepsin G and chymotrypsin on carrageenin-induced inflammation in rats.
Nakagawa, H; Kaneko, M; Watanabe, K; Sato, K; Tsurufuji, S
The anti-inflammatory activity of a proteinase inhibitor on carrageenin-induced inflammation was studied by using N-(2,4-dinitrophenyl)-benzisothiazolinone-1,1-dioxide, a selective inhibitor of elastase, cathepsin G and chymotrypsin. The selective inhibitor suppressed leukocyte chemotaxis in vivo and in vitro, vascular permeability and development of granulation tissue. These results suggest that a selective inhibitor of elastase, cathepsin G and chymotrypsin is an effective agent for suppression of induction and development of carrageenin-induced inflammation in rats. PMID:3637227
Short-term cardiovascular effects of selective phosphodiesterase 3 inhibitor olprinone versus non-selective phosphodiesterase inhibitor aminophylline in a meconium-induced acute lung injury.
Mokra, D; Tonhajzerova, I; Pistekova, H; Visnovcova, Z; Mokry, J; Drgova, A; Repcakova, M; Calkovska, A
Various anti-inflammatory drugs have been used for treatment of neonatal meconium aspiration syndrome (MAS). As their adverse effects are poorly described, this study compared effects of selective phosphodiesterase (PDE) 3 inhibitor olprinone and non-selective PDE inhibitor aminophylline on cardiovascular parameters in animal model of MAS. Oxygen-ventilated rabbits were intratracheally instilled 4 mL/kg of meconium (25 mg/mL) or saline. Thirty minutes later, meconium-instilled animals were intravenously given olprinone (0.2 mg/kg) at a single dose at 0.5 h after meconium instillation, or aminophylline (2.0 mg/kg) at two doses at 0.5 and 2.5 h after meconium instillation, or were left without treatment. Cardiovascular changes were evaluated within 5 min of administration and 5 min after finishing the administration. Furthermore, respiratory and cardiovascular parameters were measured within 5 hours following treatment delivery. Oxidation markers (thiobarbituric acid-reactive substances (TBARS), and total antioxidant status) and markers of cardiovascular injury (aldosterone, gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT)) were determined in the plasma. Meconium instillation induced acute lung injury associated with oxidative stress, elevated aldosterone, and slightly increased GGT and AST levels. Both aminophylline and olprinone improved lung functions and reduced oxidation stress. However, the PDE inhibitors acutely increased blood pressure and heart rate, whereas heart rate variability remained higher till the end of experiment and correlated well with markers of cardiovascular injury. Considering that systemic administration of olprinone and aminophylline was accompanied by acute cardiovascular changes in the meconium-instilled animals, use of PDE inhibitors in the newborns with MAS should be carefully monitored. PMID:24388890
N-(3-(Aminomethyl)benzyl)acetamidine, an inducible nitric oxide synthase inhibitor, decreases colonic inflammation induced by trinitrobenzene sulphonic acid in rats
Luis A. Menchķn; Arturo L. Col¾n; Mar??a A. Moro; Juan C. Leza; Ignacio Lizasoain; Pedro Menchķn; Emilio Alvarez; Pedro Lorenzo
Gastrointestinal inflammation has been associated with an increased generation of nitric oxide (NO) and the expression of the inducible NO synthase (iNOS). Using an experimental model of colitis induced by trinitrobenzene sulphonic acid (TNBS), we sought to determine whether the administration of N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a specific inhibitor of iNOS, has a beneficial action on the colonic injury. 1400W (0.4 and
Mitochondrial Alterations Caused by Defective Peroxisomal Biogenesis in a Mouse Model for Zellweger Syndrome (PEX5 Knockout Mouse)
Baumgart, Eveline; Vanhorebeek, Ilse; Grabenbauer, Markus; Borgers, Marcel; Declercq, Peter E.; Fahimi, H. Dariush; Baes, Myriam
Zellweger syndrome (cerebro-hepato-renal syndrome) is the most severe form of the peroxisomal biogenesis disorders leading to early death of the affected children. To study the pathogenetic mechanisms causing organ dysfunctions in Zellweger syndrome, we have recently developed a knockout-mouse model by disrupting the PEX5 gene, encoding the targeting receptor for most peroxisomal matrix proteins (M Baes, P Gressens, E Baumgart, P Carmeliet, M Casteels, M Fransen, P Evrard, D Fahimi, PE Declercq, D Collen, PP van Veldhoven, GP Mannaerts: A mouse model for Zellweger syndrome. Nat Genet 1997, 17:49¢57 1 ). In this study, we present evidence that the absence of functional peroxisomes, causing a general defect in peroxisomal metabolism, leads to proliferation of pleomorphic mitochondria with severe alterations of the mitochondrial ultrastructure, changes in the expression and activities of mitochondrial respiratory chain complexes, and an increase in the heterogeneity of the mitochondrial compartment in various organs and specific cell types (eg, liver, proximal tubules of the kidney, adrenal cortex, heart, skeletal and smooth muscle cells, neutrophils). The changes of mitochondrial respiratory chainenzymes are accompanied by a marked increase ofmitochondrial manganese-superoxide dismutase, asrevealed by in situ hybridization and immunocytochemistry, suggesting increased production of reactive oxygen species in altered mitochondria. This increased oxidative stress induced probably by defective peroxisomal antioxidant mechanisms combined with accumulation of lipid intermediates of peroxisomal ?-oxidation system could contribute significantly to the pathogenesis of multiple organ dysfunctions in Zellweger syndrome. PMID:11583975
Simvastatin suppresses dexamethasone-induced secretion of plasminogen activator inhibitor-1 in human bone marrow adipocytes
Background Osteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1) is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship between PAI-1 and steroid-induced osteonecrosis of the femoral head, and the preventive effects of lipid-lowering agents (statins) against steroid-induced osteonecrosis of the femoral head. We previously reported that adipokines and dexamethasone induced PAI-1 secretion from bone marrow adipocytes. The purpose of the present study is to examine the effects of simvastatin on PAI-1 secretion from human bone marrow adipocytes in vitro. Methods Primary bone marrow adipocytes were extracted from collagenase-treated bone marrow fluid obtained from the femoral necks of 40 patients (6 men, 34 women; age range, 52-81 years) undergoing hip joint replacement surgery. After suspended culture with or without dexamethasone or simvastatin, PAI-1 mRNA expression was assessed by real-time RT-PCR. Total PAI-1 protein secretion in culture medium was assessed by enzyme-linked immunosorbent assay. Results PAI-1 mRNA expression was up-regulated by 388% (P = 0.002) with dexamethasone, and down-regulated by 45% (P = 0.002) with simvastatin, as compared to control levels. Dexamethasone increased total PAI-1 secretion by 166% (P = 0.001) and simvastatin decreased total PAI-1 secretion by 64% (P = 0.002). No significant changes were observed in adiponectin mRNA expression and secretion by dexamethasone and simvastatin, while pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion by 89%, as compared to control levels. Conclusion The present study confirmed the suppressive effects of simvastatin on PAI-1 expression and secretion from bone marrow adipocytes. Furthermore, pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion. Simvastatin may thus exhibit preventive effects against steroid-induced osteonecrosis of the femoral head by suppressing PAI-1 secretion. PMID:21524281
The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production.
Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie
In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our inĀvitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. InĀvivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation inĀvitro and inĀvivo, and ceramide production might be the key mechanism responsible for its actions. PMID:26026677
MET inhibitor PHA-665752 suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells.
Liu, Tongxin; Li, Qi; Sun, Quanquan; Zhang, Yuqin; Yang, Hua; Wang, Rong; Chen, Longhua; Wang, Wei
Although ionizing radiation (IR) has provided considerable improvements in nasopharyngeal carcinoma (NPC), in subsets of patients, radioresistance is still a major problem in the treatment. In this study, we demonstrated that irradiation induced MET overexpression and activation, and the aberrant MET signal mediated by hepatocyte growth factor (HGF) induced radioresistance. We also found that MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. Further investigation indicated that PHA-665752 suppressed the phosphorylation of the Akt, ERK1/2, and STAT3 proteins in a dose-dependent manner. Our data indicated that the combination of IR with a MET inhibitor, such as PHA-665752, might be a promising therapeutic strategy for NPC. PMID:24802404
Tyrosine kinase inhibitor tyrphostin AG490 reduces liver injury in LPS-induced shock.
Gyurkovska, Valeriya; Ivanovska, Nina
Sepsis remains a serious clinical problem despite continuous efforts to increase survival. Experimental animal models of sepsis are pointed to a great extent on blocking the activity of cytokines. A number of signal-transducing molecules are associated with the occurrence of excessive tissue inflammation. Through inhibition of tyrosine phosphorilation and thereby changing cell signaling, tyrosine kinase inhibitors can influence multiple inflammatory pathways. The purpose of the present investigation was to evaluate the effect of tyrosine kinase inhibitor tyrphostin AG490 in a mouse LPS-induced shock. Cytokine and chemokine blood levels were determined by ELISA assays. CD11b(+) Ly6C(+), CD3(+)CD69(+) and C5aR positive cell populations in the peritoneal exudate were detected by flow cytometry. The expression of iNOS and Signal Transducer and Activator of Transcription (STAT) in the liver were observed by immunohistochemistry. We found that tyrphostin AG490 inhibited Regulated upon activation normal T cell expressed and secreted (RANTES), IL-6 and IL-12 serum levels, decreased the number of CD11b(+)Ly6C(+) and CD3(+)CD69(+) subpopulations in the peritoneal exudate and prevented the decrease of cells expressing C5a receptor and TNF-alpha receptor. Tyrphostin ameliorated liver injury associated with suppressed iNOS, STAT3 and pSTAT3 expression. Our data suggest that tyrphostin AG490 diminished the degree of inflammation starting in peritoneal cavity and minimized liver dysfunction thus representing one approach for better outcome of sepsis conditions. PMID:25666385
Model boiler testing to evaluate inhibitors for caustic induced stress corrosion cracking of Alloy 600 tubes
Daret, J. [Commissariat a l`Energie Atomique, Hague (France); Paine, J.P.N. [Electric Power Research Inst., Palo Alto, CA (United States); Partridge, M.J. [Dominion Engineering Inc., McLean, VA (United States)
A series of model boiler tests, using a mixture of precracked and non-precracked (virgin) tube-to-tube support plate intersections was performed. The testing supported the qualification of inhibitors for mitigating the secondary side corrosion of alloy 600 steam generator tubes. Many utilities suspect that the caustic impurities come from the feedwater. Candidate inhibitors included boric acid (as a reference), cerous acetate, and two forms of titanium dioxide: a laboratory produced titania-silica sol-gel, and manometer sized anatase The latter was combined with a 150 C pre-soaking with a titanium lactate, and was tested with and without a zeta potential treatment by sodium aluminate. Effectiveness of boric acid to prevent and retard caustic induced intergranular corrosion was confirmed in all crevice configurations (open and packed). The cerous acetate treatment multiplied by two to four the time necessary to detect a primary-to-secondary leak on virgin tubes, and reduced the propagation rate on precracked tubes. Cerium was found intimately mixed, as cerianite, with the free span and crevice deposits, when the crevices were sufficiently accessible. Due to its very low solubility and large particle size, the titania-silica sol-gel was unable to penetrate the crevices and had no effect on the degradation process. The nanometric particle size titania treatment and/or the preceding soaking with soluble titanium lactate drastically increased the titanium concentration in free span and open crevice deposit (with no added sodium aluminate, titania reacted with magnetite to form ilmenite) and showed undeniable capacity to prevent tubing degradation. Its effectiveness, in the case of packed crevices and for arresting cracks, was not so conclusive.
Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation
Kim, Sang-pil [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of)] [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)] [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Chung, Sung Woon [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of)] [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Hong, Ki Whan; Kim, Chi Dae [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)] [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Bae, Sun Sik, E-mail: email@example.com [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)
Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.
Xanthine crystals induced by topiroxostat, a xanthine oxidoreductase inhibitor, in rats, cause transitional cell tumors.
Shimo, Takeo; Moto, Mitsuyoshi; Ashizawa, Naoki; Matsumoto, Koji; Iwanaga, Takashi; Saito, Kazuhiro
The present study was performed to elucidate the underlying mechanism of transitional cell tumors found in the carcinogenicity testing of topiroxostat, a xanthine oxidoreductase inhibitor, in which topiroxostat was orally given to F344 rats at 0.3, 1, and 3Āmg/kg for 2Āyears. In the urinary bladder, transitional cell papillomas and/or carcinomas were seen in males receiving 0.3, 1, and 3Āmg/kg (1/49, 3/49, and 10/50, respectively). In the kidney, transitional cell papillomas and/or carcinomas in the pelvis were seen in 2/50 males and 1/50 females receiving 3Āmg/kg. In the mechanistic study by 52-week oral treatment with topiroxostat at 3Āmg/kg to F344 male rats, with and without citrate, simple and papillary transitional cell hyperplasias of the urinary bladder epithelium were observed in 5/17 in the topiroxostat-alone treatment group, along with xanthine-induced nephropathy, in contrast to neither xanthine crystals nor lesions in urinary organs by co-treatment group with citrate. As for sex differences of urinary bladder tumors, the BrdU labeling index for epithelial cells of the urinary bladder by 5-week oral treatment with topiroxostat at 10Āmg/kg to F344 rats was increased in males only, showing consistency with histopathological findings. Therefore, the present study indicates that transitional cell tumors induced by topiroxostat in rats were due to physical stimulation to transitional cells of xanthine crystals/calculi and provides that other factors were not implicated in this tumorigenesis. Furthermore, the present study suggests that such tumors do not predict for humans since topiroxostat-induced xanthine deposition is a rodent-specific event. PMID:24448833
Substrate and Inhibitor-induced Dimerization and Cooperativity in Caspase-1 but Not Caspase-3*
Datta, Debajyoti; McClendon, Christopher L.; Jacobson, Matthew P.; Wells, James A.
Caspases are intracellular cysteine-class proteases with aspartate specificity that is critical for driving processes as diverse as the innate immune response and apoptosis, exemplified by caspase-1 and caspase-3, respectively. Interestingly, caspase-1 cleaves far fewer cellular substrates than caspase-3 and also shows strong positive cooperativity between the two active sites of the homodimer, unlike caspase-3. Biophysical and kinetic studies here present a molecular basis for this difference. Analytical ultracentrifugation experiments show that mature caspase-1 exists predominantly as a monomer under physiological concentrations that undergoes dimerization in the presence of substrate; specifically, substrate binding shifts the KD for dimerization by 20-fold. We have created a hemi-active site-labeled dimer of caspase-1, where one site is blocked with the covalent active site inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. This hemi-labeled enzyme is about 9-fold more active than the apo-dimer of caspase-1. These studies suggest that substrate not only drives dimerization but also, once bound to one site in the dimer, promotes an active conformation in the other monomer. Steady-state kinetic analysis and modeling independently support this model, where binding of one substrate molecule not only increases substrate binding in preformed dimers but also drives the formation of heterodimers. Thus, the cooperativity in caspase-1 is driven both by substrate-induced dimerization as well as substrate-induced activation. Substrate-induced dimerization and activation seen in caspase-1 and not in caspase-3 may reflect their biological roles. Whereas caspase-1 cleaves a dramatically smaller number of cellular substrates that need to be concentrated near inflammasomes, caspase-3 is a constitutively active dimer that cleaves many more substrates located diffusely throughout the cell. PMID:23386603
Defining the Plant Peroxisomal Proteome: From Arabidopsis to Rice
Kaur, Navneet; Hu, Jianping
Peroxisomes are small subcellular organelles mediating a multitude of processes in plants. Proteomics studies over the last several years have yielded much needed information on the composition of plant peroxisomes. In this review, the status of peroxisome proteomics studies in Arabidopsis and other plant species and the cumulative advances made through these studies are summarized. A reference Arabidopsis peroxisome proteome is generated, and some unique aspects of Arabidopsis peroxisomes that were uncovered through proteomics studies and hint at unanticipated peroxisomal functions are also highlighted. Knowledge gained from Arabidopsis was utilized to compile a tentative list of peroxisome proteins for the model monocot plant, rice. Differences in the peroxisomal proteome between these two model plants were drawn, and novel facets in rice were expounded upon. Finally, we discuss about the current limitations of experimental proteomics in decoding the complete and dynamic makeup of peroxisomes, and complementary and integrated approaches that would be beneficial to defining the peroxisomal metabolic and regulatory roadmaps. The synteny of genomes in the grass family makes rice an ideal model to study peroxisomes in cereal crops, in which these organelles have received much less attention, with the ultimate goal to improve crop yield. PMID:22645559
Protein phosphatase 2A holoenzyme is targeted to peroxisomes by piggybacking and positively affects peroxisomal ?-oxidation.
Kataya, Amr R A; Heidari, Behzad; Hagen, Lars; Kommedal, Roald; Slupphaug, Geir; Lillo, Cathrine
The eukaryotic, highly conserved serine (Ser)/threonine-specific protein phosphatase 2A (PP2A) functions as a heterotrimeric complex composed of a catalytic (C), scaffolding (A), and regulatory (B) subunit. In Arabidopsis (Arabidopsis thaliana), five, three, and 17 genes encode different C, A, and B subunits, respectively. We previously found that a B subunit, B'?, localized to peroxisomes due to its C-terminal targeting signal Ser-Ser-leucine. This work shows that PP2A C2, C5, andA2 subunits interact and colocalize with B'? in peroxisomes. C and A subunits lack peroxisomal targeting signals, and their peroxisomal import depends on B'? and appears to occur by piggybacking transport. B'? knockout mutants were impaired in peroxisomal ?-oxidation as shown by developmental arrest of seedlings germinated without sucrose, accumulation of eicosenoic acid, and resistance to protoauxins indole-butyric acid and 2,4-dichlorophenoxybutyric acid. All of these observations strongly substantiate that a full PP2A complex is present in peroxisomes and positively affects ?-oxidation of fatty acids and protoauxins. PMID:25489022
Catalase degradation in sunflower cotyledons during peroxisome transition from glyoxysomal to leaf peroxisomal function. [Helianthus annuus
Eising, R.; Gerhardt, B.
First order rate constant for the degradation (degradation constants) of catalase in the cotyledons of sunflower (Helianthus annuus L.) were determined by measuring the loss of catalase containing /sup 14/C-labeled heme. During greening of the cotyledons, a period when peroxisomes change from glyoxysomal to leaf peroxisomal function, the degradation of glyoxysomal catalase is significantly slower than during all other stages of cotyledon development in light or darkness. The degradation constant during the transition stage of peroxisome function amounts to 0.205 day/sup -1/ in contrast to the constants ranging from 0.304 day/sup -1/ to 0.515 day/sup -1/ during the other developmental stages. Density labeling experiments comprising labeling of catalase with /sup 2/H/sub 2/O and its isopycnic centrifugation on CsCl gradients demonstrated that the determinations of the degradation constants were not substantially affected by reutilization of /sup 14/C-labeled compounds for catalase synthesis. The degradation constants for both glyoxysomal catalase and catalase synthesized during the transition of peroxisome function do not differ. This was shown by labeling the catalases with different isotopes and measuring the isotope ratio during the development of the cotyledons. The results are inconsistent with the concept that an accelerated and selective degradation of glyoxysomes underlies the change in peroxisome function. The data suggest that catalase degradation is at least partially due to an individual turnover of catalase and does not only result from a turnover of the whole peroxisomes.
Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells
Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)] [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates); Galadari, Sehamuddin, E-mail: email@example.com [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)] [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)
Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.
A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response.
McConnell, Jeanette R; Alexander, Leslie A; McAlpine, Shelli R
When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways. PMID:24360559
A comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model Aurora kinase inhibitors.
Gollapudi, P; Hasegawa, L S; Eastmond, D A
Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements. PMID:24680981
Histone deacetylase inhibitors are potent inducers of gene expression in latent EBV and sensitize lymphoma cells to nucleoside antiviral agents
Ghosh, Sajal K.; Perrine, Susan P.; Williams, Robert M.
Induction of EBV lytic-phase gene expression, combined with exposure to an antiherpes viral drug, represents a promising targeted therapeutic approach to EBV-associated lymphomas. Short-chain fatty acids or certain chemotherapeutics have been used to induce EBV lytic-phase gene expression in cultured cells and mouse models, but these studies generally have not translated into clinical application. The recent success of a clinical trial with the pan-histone deacetylase (pan-HDAC) inhibitor arginine butyrate and the antiherpes viral drug ganciclovir in the treatment of EBV lymphomas prompted us to investigate the potential of several HDAC inhibitors, including some new, highly potent compounds, to sensitize EBV+ human lymphoma cells to antiviral agents in vitro. Our study included short-chain fatty acids (sodium butyrate and valproic acid); hydroxamic acids (oxamflatin, Scriptaid, suberoyl anilide hydroxamic acid, panobinostat [LBH589], and belinostat [PXD101]); the benzamide MS275; the cyclic tetrapeptide apicidin; and the recently discovered HDAC inhibitor largazole. With the exception of suberoyl anilide hydroxamic acid and PXD101, all of the other HDAC inhibitors effectively sensitized EBV+ lymphoma cells to ganciclovir. LBH589, MS275, and largazole were effective at nanomolar concentrations and were 104 to 105 times more potent than butyrate. The effectiveness and potency of these HDAC inhibitors make them potentially applicable as sensitizers to antivirals for the treatment of EBV-associated lymphomas. PMID:22160379
Plasminogen Activator Inhibitor-1 Is Involved in Streptozotocin-Induced Bone Loss in Female Mice
Tamura, Yukinori; Kawao, Naoyuki; Okada, Kiyotaka; Yano, Masato; Okumoto, Katsumi; Matsuo, Osamu; Kaji, Hiroshi
In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1¢deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism. PMID:23715621
CD8+ T Cell-Induced Expression of Tissue Inhibitor of Metalloproteinses-1 Exacerbated Osteoarthritis
Hsieh, Jeng-Long; Shiau, Ai-Li; Lee, Che-Hsin; Yang, Shiu-Ju; Lee, Bih-O; Jou, I-Ming; Wu, Chao-Liang; Chen, Shun-Hua; Shen, Po-Chuan
Despites the fact that T cells are involved in the pathogenesis of osteoarthritis (OA) little is known about the roles of CD8+ T cells in this disease. We investigated the effects of CD8+ T cells and the expression of tissue inhibitor of metalloproteinases 1 (TIMP-1) on joint pathology. Using anterior cruciate ligament-transection (ACLT), OA was induced in mice. The knee joints were histologically assessed for manifestations of OA. The CD8+ T cells from splenocytes and synovium were flow-cytometrically and immunochemically evaluated, respectively. Local expression of TIMP-1, matrix metalloproteinase (MMP)-13, and VEGF were examined. Cartilage degeneration was slower in CD8+ T cell knockout mice than in control mice. CD8+ T cells were activated once OA was initiated and expanded during OA progression. More CD8+ T cells from splenocytes expressed TIMP-1 in ACLT-group mice than in Sham-group mice. The number of TIMP-1-expressing CD8+ T cells in OA mice correlated with the disease severity. TIMP-1 expression in cartilage was co-localized with that of MMP-13 and VEGF. TIMP-1 protein was detected in synovium in which angiogenesis occurred. During the pathogenesis of OA, the expression of TIMP-1, VEGF and MMP-13 accompanying with CD8+ T cells activation were increased. Furthermore, inhibiting the expression of TIMP-1 in joints could retard the progression of OA. PMID:24108368
Protease inhibitor reduces airway response and underlying inflammation in cockroach allergen-induced murine model.
Saw, Sanjay; Arora, Naveen
Protease(s) enhances airway inflammation and allergic cascade. In the present study, effect of a serine protease inhibitor was evaluated in mouse model of airway disease. Mice were sensitized with cockroach extract (CE) or Per a 10 and treated with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) 1 h before or after challenge to measure airway response. Mice were euthanized to collect bronchoalveolar lavage fluid (BALF), blood, and lung to evaluate inflammation. AEBSF treatment significantly reduced the AHR in allergen-challenged mice in dose-dependent manner (p??0.01). IgE (p?0.05) and Th2 cytokines (p?0.05) were significantly reduced in treated mice. AEBSF treatment lowered total cell (p?0.05), eosinophil (p?0.05), and neutrophil (p?0.05) in BALF and lung tissue. Oxidative stress parameters were impaired on treatment in allergen-challenged mice (p?0.05). AEBSF had therapeutic effect in allergen-induced airway resistance and underling inflammation and had potential for combination or as add-on therapy for respiratory diseases. PMID:25052477
Forodesine, an inhibitor of purine nucleoside phosphorylase, induces apoptosis in chronic lymphocytic leukemia cells
Balakrishnan, Kumudha; Nimmanapalli, Ramadevi; Ravandi, Farhad; Keating, Michael J.; Gandhi, Varsha
Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine, a potent inhibitor of PNP, was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK), we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at Ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data, a phase 2 clinical trial of forodesine has been initiated for CLL patients. PMID:16778146
Thrombin-activatable fibrinolysis inhibitor deficiency attenuates bleomycin-induced lung fibrosis.
Fujimoto, Hajime; Gabazza, Esteban C; Taguchi, Osamu; Nishii, Yoichi; Nakahara, Hiroki; Bruno, Nelson E; D'Alessandro-Gabazza, Corina N; Kasper, Michael; Yano, Yutaka; Nagashima, Mariko; Morser, John; Broze, George J; Suzuki, Koji; Adachi, Yukihiko
Decreased fibrinolytic function favors the development of pulmonary fibrosis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a strong suppressor of fibrinolysis, but its role in lung fibrosis is unknown. Therefore, we compared bleomycin-induced lung fibrosis in TAFI-deficient, heterozygous, and wild-type mice. The animals were sacrificed 21 days after bleomycin administration, and markers of lung fibrosis and inflammation were measured. The bronchoalveolar lavage fluid levels of total protein, neutrophil proteases (elastase, myeloperoxidase), cytokines (tumor necrosis factor-alpha, interleukin-13), chemokine (monocyte chemoattractant protein-1), coagulation activation marker (thrombin-antithrombin complex), total soluble collagen, and growth factors (platelet-derived growth factor, transforming growth factor-beta1, granulocytic-macrophage growth factor) were significantly decreased in knockout mice compared to wild-type mice. Further, histological findings of fibrosis, fibrin deposition, and hydroxyproline and collagen content in the lung were significantly decreased in knockout mice compared to wild-type mice. Depletion of fibrinogen by ancrod treatment led to equalization in the amount of fibrosis and collagen deposition in the lungs of knockout and wild-type mice. No difference was detected in body temperature or arterial pressure between the different mouse phenotypes. These results suggest that the anti-fibrinolytic activity of TAFI promotes lung fibrosis by hindering the rate at which fibrin is degraded. PMID:16565485
Interaction between Nitric Oxide Synthase Inhibitor Induced Oscillations and the Activation Flow Coupling Response
Ances, Beau M.; Greenberg, Joel. H.; Detre, John A.
The role of nitric oxide (NO) in the activation-flow coupling (AFC) response to periodic electrical forepaw stimulation was investigated using signal averaged laser Doppler (LD) flowmetry. LD measures of calculated cerebral blood flow (CBF) were obtained both prior and after intra-peritoneal administration of the non-selective nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA) (40 mg/kg). Characteristic baseline low frequency vasomotion oscillations (0.17 Hz) were observed after L-NNA administration. These LDCBF oscillations were synchronous within but not between hemispheres. L-NNA reduced the magnitude of the AFC response (p< 0.05) for longer stimuli (1 minute) with longer inter-stimulus intervals (2 minutes). In contrast, the magnitude of the AFC response for short duration stimuli (4 seconds) with short inter-stimulus intervals (20 seconds) was augmented (p < 0.05) after L-NNA. An interaction occurred between L-NNA induced vasomotion oscillations and the AFC response with the greatest increase occurring at the stimulus harmonic closest to the oscillatory frequency. Nitric oxide may therefore modulate the effects of other vasodilators involved in vasomotion oscillations and the AFC response. PMID:19900416
Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents
Suzuki, Nobutaka; Del Villar, Keith; Tamanoi, Fuyuhiko
Farnesyltransferase inhibitors (FTIs) exhibit the remarkable ability to inhibit transformed phenotypes of a variety of human cancer cell lines and to block the growth of cancer cells in a number of animal model systems. In this paper, we report that the addition of FTI to v-K-ras- transformed NRK cells (KNRK) results in dramatic morphological changes. Within 24 h after the addition of FTI, the round morphology of KNRK cells was changed to an elongated (flattened and spread out) morphology resembling those of untransformed NRK cells. No morphological effects were seen when similar concentrations of FTI were added to NRK cells. Phalloidin staining showed that FTI treatment did not restore the disrupted actin cytoskeleton in KNRK cells. In contrast, FTI addition resulted in the appearance of extensive microtubule networks in KNRK cells. The addition of a low concentration (1.2 nM) of vincristine or vinblastine, agents that interfere with microtubule dynamics, blocked the FTI-induced morphological changes in KNRK cells. In contrast, cytochalasin B, which interferes with actin polymerization, did not block the morphological changes. The FTI-induced morphological changes were associated with a decrease in the percentage of cells in S-phase, and the addition of 1.2 nM vincristine did not have additional effects on cell cycle progression. A higher concentration (12 nM) of vincristine caused synergistic effect with FTI to enrich dramatically KNRK cells in G2/M phase. These results suggest that FTI affects cell morphology and that microtubule dynamics are involved in these processes. PMID:9724732
Exogenous cannabinoids as substrates, inhibitors, and inducers of human drug metabolizing enzymes: a systematic review.
Stout, Stephen M; Cimino, Nina M
Exogenous cannabinoids are structurally and pharmacologically diverse compounds that are widely used. The purpose of this systematic review is to summarize the data characterizing the potential for these compounds to act as substrates, inhibitors, or inducers of human drug metabolizing enzymes, with the aim of clarifying the significance of these properties in clinical care and drug interactions. In vitro data were identified that characterize cytochrome P-450 (CYP-450) enzymes as potential significant contributors to the primary metabolism of several exogenous cannabinoids: tetrahydrocannabinol (THC; CYPs 2C9, 3A4); cannabidiol (CBD; CYPs 2C19, 3A4); cannabinol (CBN; CYPs 2C9, 3A4); JWH-018 (CYPs 1A2, 2C9); and AM2201 (CYPs 1A2, 2C9). CYP-450 enzymes may also contribute to the secondary metabolism of THC, and UDP-glucuronosyltransferases have been identified as capable of catalyzing both primary (CBD, CBN) and secondary (THC, JWH-018, JWH-073) cannabinoid metabolism. Clinical pharmacogenetic data further support CYP2C9 as a significant contributor to THC metabolism, and a pharmacokinetic interaction study using ketoconazole with oromucosal cannabis extract further supports CYP3A4 as a significant metabolic pathway for THC and CBD. However, the absence of interaction between CBD from oromucosal cannabis extract with omeprazole suggests a less significant role of CYP2C19 in CBD metabolism. Studies of THC, CBD, and CBN inhibition and induction of major human CYP-450 isoforms generally reflect a low risk of clinically significant drug interactions with most use, but specific human data are lacking. Smoked cannabis herb (marijuana) likely induces CYP1A2 mediated theophylline metabolism, although the role of cannabinoids specifically in eliciting this effect is questionable. PMID:24160757
Oxaceprol, an atypical inhibitor of inflammation, reduces leukocyte adherence in mouse antigen-induced arthritis.
Veihelmann, A; Hofbauer, A; Refior, H J; Messmer, K
Oxaceprol (N-acetyl-L-hydroxyproline), an atypical inhibitor of inflammation, is an established drug forjoint disease without serious side-effects. Recent studies have emphasized that oxaceprol has an effect on the microcirculation. Since the exact mechanism of action remains unclear, the aim of our study was to investigate the leukocyte-endothelial cell interactions in oxaceprol-treated mice with antigen-induced arthritis (AiA) using intravital microscopy. In our study, Balb/c mice were allocated to 4 groups (n 7, 8, 8, 8): 2 control groups with saline or oxaceprol and 2 groups of arthritic animals which received saline or oxaceprol (100 mg/kg twice a day intraperitoneally). The severity of arthritis was quantified by the transverse knee joint diameter. For the intravital fluorescence microscopy measurements on day 10 after inducing arthritis, the patella tendon was partily resected to visualize the intraarticular synovial tissue of the knee joint. The number of rolling and adherent leukocytes as well as RBC velocity and functional capillary density (FCD) were quantified in synovial microvessels. Furthermore, leukocyte infiltration was determined in the histological sections with an established score. No significant changes in mean arterial blood pressure or functional capillary density were found in any of the groups. However, the leukocyte rolling fraction and number of leukocytes adherent to the endothelium were increased in postcapillary venules of the synovium in arthritic animals (0.16 to 0.31, 78 cells/mm2 to 220 cells/mm2). In animals with AiA treated with oxaceprol, leukocyte adherence and swelling were significantly reduced in comparison to the arthritic animals treated with saline. Furthermore, the histological score showed less leukocyte infiltration in the oxaceprol treated arthritic animals. Thus, oxaceprol reduces leukocyte adherence in vivo and leukocyte infiltration in mouse AiA, indicating an effect on synovial microcirculation. PMID:11480608
A possible antineoplastic potential of selective, irreversible proteasome inhibitor, carfilzomib on chemically induced hepatocarcinogenesis in rats.
Mansour, Mahmoud A; Aljoufi, Mohammed A; Al-Hosaini, Khaled; Al-Rikabi, Ammar C; Nagi, Mahmoud N
The antineoplastic effect of carfilzomib (CFZ) against chemically induced hepatocarcinogenesis was studied. A total of 60 male Wistar albino rats were divided into six groups with 10 animals in each group. Rats in group 1 (control group) were given dimethylsulphoxide (DMSO) (0.4 mL/kg i.p) twice a week for 3Āweeks from weekĀ8 to weekĀ10. Animals in groups 2 and 3 were given CFZ (2 and 4 mg/kg i.p) twice a week from weekĀ8 to weekĀ10, respectively. Rats in group 4 were given diethylnitrosamine (DENA) at a dose of 0.01% in drinking water for 10 weeks and received a DMSO (0.4 mL/kg i.p) twice a week from weekĀ8 to weekĀ10. Animals in groups 5 and 6 were given DENA at a dose of 0.01% in drinking water for 10Āweeks and treated with CFZ (2 and 4 mg/kg i.p) twice a week from weekĀ8 to weekĀ10, respectively. CFZ succeeded in suppressing the elevated serum tumor marker ?-fetoprotein and carcinoembryonic antigen. The antineoplastic effect of CFZ was also accompanied by normalization of elevated hepatic tissue growth factors, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1, and augmentation of hepatic endostatin and metallothionein. A histopathological examination of liver samples treated with CFZ after DENA intoxication correlated with the biochemical observation. Treatment with CFZ confers an antineoplastic activity against chemically induced hepatocarcinogenesis. These findings suggest that CFZ plays a pivotal role in the treatment of hepatocarcinogenesis. PMID:24861196
Fumagillin, a potent angiogenesis inhibitor, induces Kaposi sarcoma-associated herpesvirus replication in primary effusion lymphoma cells.
Kanno, Takayuki; Uehara, Taeko; Osawa, Madori; Fukumoto, Hitomi; Mine, Sohtaro; Ueda, Keiji; Hasegawa, Hideki; Katano, Harutaka
Kaposi sarcoma and primary effusion lymphoma cells are infected with Kaposi sarcoma-associated herpesvirus (KSHV), predominantly in the latent form, and KSHV replication is observed rarely. Angiogenesis plays a crucial role in the pathogenesis of both Kaposi sarcoma and primary effusion lymphoma. In this study, we found that fumagillin, a potent angiogenesis inhibitor, induced replication of KSHV in primary effusion lymphoma cell lines. The transcript and protein product of replication transcriptional activator (RTA) were induced by 1-10Ā?M fumagillin at 24 and 48Āh, respectively. Western blot analysis demonstrated that 10Ā?M fumagillin induced not only RTA expression but also other KSHV-encoded lytic proteins. A real-time PCR array detecting KSHV gene expression demonstrated that the expression profiles of KSHV induced by fumagillin were similar to those induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), but the amounts of each transcript were lower than those induced by TPA. Finally, real-time PCR demonstrated an increase in that viral DNA copy number per cell in fumagillin-stimulated primary effusion lymphoma cell lines, indicating replication of KSHV. In addition to TPA, 10Ā?M fumagillin resulted in growth inhibition of primary effusion lymphoma cell lines. These observations suggest that an angiogenesis inhibitor is an agent with potent effects on cell growth and KSHV reactivation in primary effusion lymphoma cells. PMID:26093300
A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation
Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of)] [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of); Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon [Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736 (Korea, Republic of)] [Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Cho, Dong-Hyung, E-mail: email@example.com [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of)] [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of)
Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.
An MMP-2\\/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells
O Nyormoi; L Mills; M Bar-Eli
Several studies have shown that matrix metalloproteases (MMPs) promote tumor growth, invasion, and metastasis. Consequently, MMP inhibitors have been developed as a new class of anticancer drugs, many of which are in clinical trials. The exact mechanism of the antineoplastic activity of MMP antagonists is unknown. To investigate the mechanism, we hypothesized that MMP inhibitors enhance the actions of apoptosis-inducing
Effects of the Oral, Direct Factor Xa Inhibitor Rivaroxaban on Platelet-Induced Thrombin Generation and Prothrombinase Activity
Jochen Graff; Nils von Hentig; Frank Misselwitz; Dagmar Kubitza; Michael Becka; Hans-Klaus Breddin; Sebastian Harder
Rivaroxaban (BAY 59-7939) is an oral, direct factor Xa inhibitor in advanced development. This study was undertaken to investigate its effects on thrombin generation. In this placebo-controlled, randomized, crossover study, 12 healthy subjects received rivaroxaban (single 5- or 30-mg dose) or placebo. Thrombin generation was investigated by measuring the endogenous thrombin potential and prothrombinase-induced clotting time. Maximal effect of rivaroxaban
Mechanism by which mammalian target of rapamycin inhibitors sensitize multiple myeloma cells to dexamethasone-induced apoptosis.
Yan, Huajun; Frost, Patrick; Shi, Yijiang; Hoang, Bao; Sharma, Sanjai; Fisher, Myrna; Gera, Joseph; Lichtenstein, Alan
Mammalian target of rapamycin (mTOR) inhibitors curtail cap-dependent translation. However, they can also induce post-translational modifications of proteins. We assessed both effects to understand the mechanism by which mTOR inhibitors like rapamycin sensitize multiple myeloma cells to dexamethasone-induced apoptosis. Sensitization was achieved in multiple myeloma cells irrespective of their PTEN or p53 status, enhanced by activation of AKT, and associated with stimulation of both intrinsic and extrinsic pathways of apoptosis. The sensitizing effect was not due to post-translational modifications of the RAFTK kinase, Jun kinase, p38 mitogen-activated protein kinase, or BAD. Sensitization was also not associated with a rapamycin-mediated increase in glucocorticoid receptor reporter expression. However, when cap-dependent translation was prevented by transfection with a mutant 4E-BP1 construct, which is resistant to mTOR-induced phosphorylation, cells responded to dexamethasone with enhanced apoptosis, mirroring the effect of coexposure to rapamycin. Thus, sensitization is mediated by inhibition of cap-dependent translation. A high-throughput screening for translational efficiency identified several antiapoptotic proteins whose translation was inhibited by rapamycin. Immunoblot assay confirmed rapamycin-induced down-regulated expressions of XIAP, CIAP1, HSP-27, and BAG-3, which may play a role in the sensitization to apoptosis. Studies in a xenograft model showed synergistic in vivo antimyeloma effects when dexamethasone was combined with the mTOR inhibitor CCI-779. Synergistic effects were associated with an enhanced multiple myeloma cell apoptosis in vivo. This study supports the strategy of combining dexamethasone with mTOR inhibitors in multiple myeloma and identifies a mechanism by which the synergistic effect is achieved. PMID:16489035
The Combined Inducible Nitric Oxide Synthase Inhibitor and Free Radical Scavenger Guanidinoethyldisulfide Prevents Multiple Low-Dose StreptozotocinInduced Diabetes In Vivo and Interleukin1??Induced Suppression of Islet Insulin Secretion In Vitro
Jon G. Mabley; Gary J. Southan; Andrew L. Salzman
Inhibition of inducible nitric oxide synthase has been shown to be antiinflammatory in a variety of disease states. Type I diabetes is an autoimmune disease resulting from the specific destruc- tion of the insulin-producing pancreatic cells. Here we demonstrate that guanidinoethyldisulfide (GED), a combined inducible nitric ox- ide synthase inhibitor and peroxynitrite\\/reactive oxygen species scav- enger reduces the hyperglycemia and
Blockade of nicotine reward and reinstatement by activation of alpha-type peroxisome proliferator-activated receptors
Mascia, Paola; Pistis, Marco; Justinova, Zuzana; Panlilio, Leigh V.; Luchicchi, Antonio; Lecca, Salvatore; Scherma, Maria; Fratta, Walter; Fadda, Paola; Barnes, Chanel; Redhi, Godfrey H.; Yasar, Sevil; Le Foll, Bernard; Tanda, Gianluigi; Piomelli, Daniele; Goldberg, Steven R.
Background Recent findings indicate that inhibitors of fatty acid amide hydrolase (FAAH) counteract the rewarding effects of nicotine in rats. FAAH inhibition increases levels of several endogenous substances in the brain, including the endocannabinoid anandamide and the non-cannabinoid fatty-acid ethanolamides oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), which are ligands for alpha-type peroxisome proliferator-activated nuclear receptors (PPAR-?). Here, we evaluated whether directly-acting PPAR-? agonists can modulate reward-related effects of nicotine. Methods We combined behavioral, neurochemical and electrophysiological approaches to evaluate effects of the PPAR-? agonists WY14643 and methOEA (a long-lasting form of OEA) on: (1) nicotine self-administration in rats and squirrel monkeys; (2) reinstatement of nicotine-seeking behavior in rats and monkeys; (3) nicotine discrimination in rats; (4) nicotine-induced electrophysiological activity of VTA dopamine neurons in anesthetized rats; and (5) nicotine-induced elevation of dopamine levels in the nucleus accumbens shell of freely-moving rats. Results PPAR-? agonists dose-dependently decreased nicotine self-administration and nicotine-induced reinstatement in rats and monkeys, but did not alter food- or cocaine-reinforced operant behavior or the interoceptive effects of nicotine. PPAR-? agonists also dose-dependently decreased nicotine-induced excitation of dopamine neurons in the ventral tegmental area (VTA) and nicotine-induced elevations of dopamine levels in the nucleus accumbens shell of rats. The ability of WY14643 and methOEA to counteract the behavioral, electrophysiological, and neurochemical effects of nicotine was reversed by the PPAR-? antagonist MK886. Conclusions These findings indicate that PPAR-? might provide a valuable new target for anti-smoking medications. PMID:20801430
OCTN3 is a mammalian peroxisomal membrane carnitine transporter
Lamhonwah, Anne-Marie [Division of Neurology, Department of Pediatrics, Hospital for Sick Children, Toronto (Canada); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., M5G 1X8 (Canada); Ackerley, Cameron A. [Department of Pathology, Hospital for Sick Children, Toronto (Canada); Tilups, Aina [Department of Pathology, Hospital for Sick Children, Toronto (Canada); Edwards, Vernon D. [Department of Pathology, Hospital for Sick Children, Toronto (Canada); Wanders, Ronald J. [Department of Pediatrics, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Tein, Ingrid [Division of Neurology, Department of Pediatrics, Hospital for Sick Children, Toronto (Canada) and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., M5G 1X8 (Canada)]. E-mail: firstname.lastname@example.com
Carnitine is a zwitterion essential for the {beta}-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (K {sub m} 20 {mu}M), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.
Pex13 Inactivation in the Mouse Disrupts Peroxisome Biogenesis and Leads to a Zellweger Syndrome Phenotype
Megan Maxwell; Jonas Bjorkman; Tam Nguyen; Peter Sharp; John Finnie; Carol Paterson; Ian Tonks; Barbara C. Paton; Graham F. Kay; Denis I. Crane
Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we
Efficacy of Cilostazol a selective phosphodiesterase-3 inhibitor in rat model of Streptozotocin diabetes induced vascular dementia.
Kumar, Ashwani; Kumar, Amit; Jaggi, Amteshwar S; Singh, Nirmal
The present study has been designed to investigate the potential of Cilostazol a phosphodiesterase-3 (PDE-3) inhibitor in diabetes-induced vascular dementia (Vad) employing Wistar rats. A single dose of Streptozotocin (STZ) was used for the induction of diabetes and subsequent Vad in rats. Memory and learning abilities of rats were evaluated with Morris water maze (MWM) test. Serum glucose, body weight, vascular endothelial function, serum nitrite/nitrate levels, brain oxidative stress levels (viz. brain thiobarbituric acid reactive species and reduced glutathione levels), inflammatory markers (viz. brain myeloperoxidase activity and neutrophil infiltration in the brain hippocampal area) and brain acetylcholinesterase activity were also tested. Donepezil was used as positive control. Streptozotocin treated animals showed poor performance on MWM indicating impairment of learning and memory abilities with a significant reduction in body weight, vascular endothelial function, serum nitrite/nitrate levels, along with an increase in serum glucose, brain oxidative stress levels, inflammatory changes and brain acetylcholinesterase activity. Treatment with selective PDE-3 inhibitor, Cilostazol significantly attenuated, diabetes-induced impairment of learning and memory; endothelial dysfunction, and changes in various biochemical parameters. It is concluded that selective PDE-3 inhibitor, Cilostazol may be considered as the potential pharmacological agent for the management of diabetes-induced vascular dementia. PMID:25987325
Organic cadmium complexes as proteasome inhibitors and apoptosis inducers in human breast cancer cells.
Zhang, Zhen; Bi, Caifeng; Buac, Daniela; Fan, Yuhua; Zhang, Xia; Zuo, Jian; Zhang, Pengfei; Zhang, Nan; Dong, Lili; Dou, Q Ping
Although cadmium (Cd) is a widespread environmental contaminant and human carcinogen, our studies indicate an organic Cd complex to be a potent inhibitor of proteasomal chymotrypsin-like (CT-like) activity, further capable of inducing apoptosis in a cancer cell-specific manner. It has been reported that the ligands indole-3-butyric acid (L1) and indole-3-propionic acid (L2) have cancer-fighting effects when tested in a rat carcinoma model. In addition, 3, 5-diaminobenzoic acid o-vanillin Schiff bases (L3) have high antimicrobial activity and a large number of Schiff base complexes have been reported to have proteasome-inhibitory activity. We therefore hypothesized that synthetic forms of Cd in combination with L1, L2 and L3 may have proteasome-inhibitory and apoptosis-inducing activities, which would be cancer cell-specific. To test this hypothesis, we have synthesized three novel Cd-containing complexes: [Cd2(C12H12O2N)4(H2O)2]Ę2H2O (Cd1), [Cd2(C11H10O2N)4(H2O)2]Ę2H2O (Cd2) and [Cd(C7H4N2O2)(C8H6O2)2]Ę2H2O (Cd3), by using these three ligands. We sought out to characterize and assess the proteasome-inhibitory and anti-proliferative properties of these three Cd complexes in human breast cancer cells. Cd1, Cd2 and Cd3 were found to effectively inhibit the chymotrypsin-like activity of purified 20S proteasome with IC50 values of 2.6, 3.0 and 3.3 ??, respectively. Moreover, inhibition of cancer cell proliferation also correlated with this effect. As a result of proteasomal shutdown, the accumulation of ubiquitinated proteins and the proteasome target I?B-? protein as well as induction of apoptosis were observed. To account for the cancer specificity of this effect, immortalized, non-tumorigenic breast MCF10A cells were used under the same experimental conditions. Our results indicate that MCF10A cells are much less sensitive to the Cd1, Cd2 and Cd3 complexes when compared to MDA MB 231 breast cancer cells. Therefore, our study suggests that these Cd organic complexes are capable of inhibiting tumor cellular proteasome activity and consequently induce cancer cell-specific apoptotic death. PMID:23499788
Secretory Leukocyte Protease Inhibitor: A Macrophage Product Induced by and Antagonistic to Bacterial Lipopolysaccharide
Fen-yu Jin; Carl Nathan; Danuta Radzioch; Aihao Ding
To explore regulation of potentially lethal responses to bacterial lipopolysaccharide (LPS), we used differential display under LPS-free conditions to compare macrophage cell lines from two strains of mice congenic for a locus affecting LPS sensitivity. LPS- hyporesponsive cells, primary macrophages, and polymorphonuclear leukocytes transcribed secretory leukocyte protease inhibitor (SLPI), a known epithelial cell-derived inhibitor of leukocyte serine proteases. Transfection of
Influence of a Human Protease Inhibitor on Surgical Stress Induced Immunosuppression
Nobuhiro Sato; Shigeatsu Endo; Yusuke Kimura; Kenichirou Ikeda; Kiichi Aoki; Takeshi Iwaya; Yuji Akiyama; Yoshinori Noda; Kazuyoshi Saito
Background\\/Aim: Postoperative tissue injury and immunosuppression can occur after major surgery. In this study, we explore the potential benefits of administering a protease inhibitor to treat immunosuppression caused by surgical stress. Methods: Sixteen patients with esophageal cancer were preoperatively allocated at random into two equal groups. A urinary trypsin inhibitor, ulinastatin (UTI), was intravenously administered to the treatment (UTI) group
Effects of Di2-Ethylhexyl Phthalate (DEHP) on Gap-Junctional Intercellular Communication (GJIC), DNA Synthesis, and Peroxisomal Beta Oxidation (PBOX) in Rat, Mouse, and Hamster Liver
Jason S. Isenberg; Lisa M. Kamendulis; Jacqueline H. Smith; David C. Ackley; George Pugh; Arthur W. Lington; James E. Klaunig
The present study evaluated the effect of di-2-ethylhexyl phtha- late (DEHP) on gap-junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX) activity, and replica- tive DNA synthesis in several rodent species with differing sus- ceptibilities to peroxisome proliferator-induced hepatic tumori- genesis. A low (non-tumorigenic) and high (tumorigenic) dietary concentration of DEHP was administered to male F344 rats for 1, 2, 4, and
Inhibition instead of enhancement of lipid peroxidation by pretreatment with the carcinogenic peroxisome proliferator nafenopin in rat liver exposed to a high single dose of corn oil
Wolfgang W. Huber; Bettina Grasl-Kraupp; Herbert Stekel; Christina Gschwentner; Harald Lang; Rolf Schulte-Hermann
Oxidative stress is discussed as a possible hepatocarcinogenic mechanism of peroxisome proliferators (PP) in rodents and\\u000a is suggested to result from the induction of peroxisomal ?-oxidation (PBOX) by PP. The induced PBOX is assumed to produce\\u000a excessive H2O2 from the degradation of fatty acids, ultimately leading to oxidative stress and lipid peroxidation. In the present short\\u000a term-study, we attempted to
mTOR inhibitor RAD001 (everolimus) induces apoptotic, not autophagic cell death, in human nasopharyngeal carcinoma cells.
Cai, Yuchen; Xia, Qing; Su, Quanguan; Luo, Rongzhen; Sun, Yueli; Shi, Yanxia; Jiang, Wenqi
Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and a key element in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Moreover, it is a negative regulator of autophagy and acts as a central regulator in cell growth. For the treatment of cancer, mTOR is a novel and validated therapeutic target. Previous studies have shown that Akt is frequently activated in nasopharyngeal carcinoma (NPC) tissues; thus, the inhibition of mTOR may be a treatment strategy for this tumor type. To evaluate the effect of the mTOR inhibitor RAD001 on NPC cell lines, we performed 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assays, lactate dehydrogenase (LDH) assays, western blotting and flow cytometry to evaluate the mechanisms of cell death. The growth of both CNE-1 and HONE-1 cells was inhibited in a time- and dose-dependent manner. CNE-1 was more sensitive, with a 50% growth inhibition (GI50) of 30.0▒1.0 ĄM compared to HONE-1, cells which had a GI50 of 56.9▒13.1 ĄM. RAD001 induced apoptosis and autophagy in both cell lines. RAD001 induced a significant increase in growth inhibition in the two cell lines when used in combination with the autophagy inhibitor, 3-methyladenine; however, the percentages of apoptotic cells decreased when RAD001 was combined with the caspase inhibitor, z-VAD-fmk. In conclusion, the main mechanism of the mTOR inhibitor RAD001 in these two NPC cells was apoptotic, not autophagic cell death. The combination of RAD001 with autophagy inhibitors may be a useful therapeutic strategy for nasopharyngeal carcinoma. PMID:23426850
Ca 2+ATPase inhibitor induces IL4 and MCP-1 production in RBL-2H3 cells
Jun-ichi Onose; Reiko Teshima; Jun-ichi Sawada
The effects of a Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA), and two hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ) and 2,5-di-(tert-amyl)-1,4-hydroquinone (DTAHQ) on the release of IL-4 and MCP-1 from RBL-2H3 cells were investigated. CPA, DTBHQ and DTAHQ, all of which induce intracellular free Ca2+ concentration ([Ca2+]i) increase, induced IL-4 and MCP-1 release in a dose-dependent manner. The release of TNF-? required both a Ca2+-ATPase
New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.
Cam§es, Fßtima; Islinger, Markus; GuimarŃes, Sofia C; Kilaru, Sreedhar; Schuster, Martin; Godinho, Luis F; Steinberg, Gero; Schrader, Michael
Peroxisomes are ubiquitous organelles which participate in a variety of essential biochemical pathways. An intimate interrelationship between peroxisomes and mitochondria is emerging in mammals, where both organelles cooperate in fatty acid ?-oxidation and cellular lipid homeostasis. As mitochondrial fatty acid ?-oxidation is lacking in yeast and plants, suitable genetically accessible model systems to study this interrelationship are scarce. Here, we propose the filamentous fungus Ustilago maydis as a suitable model for those studies. We combined molecular cell biology, bioinformatics and phylogenetic analyses and provide the first comprehensive inventory of U. maydis peroxisomal proteins and pathways. Studies with a peroxisome-deficient ?pex3 mutant revealed the existence of parallel and complex, cooperative ?-oxidation pathways in peroxisomes and mitochondria, mimicking the situation in mammals. Furthermore, we provide evidence that acyl-CoA dehydrogenases (ACADs) are bona fide peroxisomal proteins in fungi and mammals and together with acyl-CoA oxidases (ACOX) belong to the basic enzymatic repertoire of peroxisomes. A genome comparison with baker's yeast and human gained new insights into the basic peroxisomal protein inventory shared by humans and fungi and revealed novel peroxisomal proteins and functions in U. maydis. The importance of our findings for the evolution and function of the complex interrelationship between peroxisomes and mitochondria in fatty acid ?-oxidation is discussed. PMID:25307522
AT101, a small molecule inhibitor of anti-apoptotic Bcl2 family members, activates the SAPK\\/JNK pathway and enhances radiation-induced apoptosis
Shuraila F Zerp; Rianne Stoter; Gitta Kuipers; Dajun Yang; Marc E Lippman; Wim J van Blitterswijk; Harry Bartelink; Rogier Rooswinkel; Vincent Lafleur; Marcel Verheij
BACKGROUND: Gossypol, a naturally occurring polyphenolic compound has been identified as a small molecule inhibitor of anti-apoptotic Bcl-2 family proteins. It induces apoptosis in a wide range of tumor cell lines and enhances chemotherapy- and radiation-induced cytotoxicity both in vitro and in vivo. Bcl-2 and related proteins are important inhibitors of apoptosis and frequently overexpressed in human tumors. Increased levels
Antiviral and immunomodulating inhibitors of experimentally-induced Punta Toro virus infections.
Sidwell, R W; Huffman, J H; Barnard, D L; Smee, D F; Warren, R P; Chirigos, M A; Kende, M; Huggins, J
A major component of a US Army Medical Research and Development Command-supported program to discover and develop new drugs for the treatment of Rift Valley fever, sandfly fever, and Crimean-Congo hemorrhagic fever has been to study candidate test materials against hepatotropic infections of C57BL/6 mice induced by the related but less biohazardous Punta Toro virus (PTV). The effects of 75 compounds, some of which were considered immunomodulators in their primary mechanism of activity, were studied in the PTV infection model. Of these, ribavirin, ribamidine, ribavirin 2',3',5'-triacetate, tiazofurin, tiazofurin-5'-monophosphate, tiazofurin-2',3',5'-triacetate, selenazofurin, pyrazofurin, 3-deazaguanine, and 3-deazaguanosine were considered significantly inhibitory, acting against the infection by a direct antiviral (non-immunomodulatory) fashion. These compounds had therapeutic indices (TI) ranging from > or = 5 to 65, using increased survivors as the evaluation parameter. Immunomodulators considered significantly inhibitory to this infection were poly (ICLC), ampligen, human recombinant interferon-alpha-A/D, MVE-1, MVE-2, AM-3, AM-5, mannozym, bropirimine, CL246,738, phenyleneamine, and 7-thia-8-oxoguanosine. Utilizing increased survivor numbers as measure of activity, these inhibitors had TI ranging from > or = 16 to 1000. Other antiviral effects exerted by the active compounds included reduction of hepatic icterus, lowered serum glutamic oxaloacetic and pyruvic acid transaminases, and inhibition of recoverable serum and liver virus titers. The active immunomodulators were significantly effective when therapy was initiated as late as 48 h after virus inoculation, at a time when clinical signs of the PTV disease were being manifested in the animal. PMID:7847873
Roflumilast, a phosphodiesterase-4 inhibitor, induces phagocytic activity in Greek COPD patients
Porpodis, Konstantinos; Domvri, Kalliopi; Zarogoulidis, Paul; Petridis, Dimitrios; Tsirgogianni, Katerina; Papaioannou, Antonis; Hatzizisi, Olga; Kioumis, Ioannis; Liaka, Alexandra; Kikidaki, Violeta; Lampaki, Sofia; Organtzis, John; Zarogoulidis, Konstantinos
Background A new approach to the treatment of COPD includes controlling inflammation because of its important role in exacerbation of the disease. Recently, roflumilast has been added as a therapeutic option for COPD. Roflumilast is an oral phosphodiesterase-4 inhibitor that targets inflammatory cells involved in triggering exacerbations of COPD. The objective of the current study was to evaluate roflumilast for its contribution to phagocytic activity in COPD patients. Methods Twenty-one patients diagnosed with COPD received roflumilast once daily for 6 months in combination with fluticasone (an inhaled corticosteroid), salmeterol (a long-acting ?2-agonist), and tiotropium (a long-acting muscarinic antagonist) or combinations of these agents. The main inclusion criterion was stable disease for at least the previous 30 days. Neutrophils and spirometric changes, ie, forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC), were measured in the COPD patients at indicated time points. The first sample was taken before receiving roflumilast, the second 3 months later, and the third after 6 months. Examination of defective phagocytosis was done by flow cytometry using a FagoFlowEx« kit. The statistical analysis was performed using Statistica software. Results Our results indicate that phagocytic activity was increased after 3 and 6 months of treatment when compared with baseline (P<0.001). Similarly, FVC and FEV1 were also increased during the 6-month period, but only FVC differed significantly from baseline (P<0.001). Conclusion Although the number of patients in this study was limited, our results indicate that roflumilast induces phagocytic activity, which improves lung function. PMID:26109853
Inter-?-inhibitor Impairs TSG-6-induced Hyaluronan Cross-linking*
Baranova, Natalia S.; Foulcer, Simon J.; Briggs, David C.; Tilakaratna, Viranga; Enghild, Jan J.; Milner, Caroline M.; Day, Anthony J.; Richter, Ralf P.
Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-?-inhibitor (I?I). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from I?I to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of I?I and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of I?I and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and I?I in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HCĘTSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and I?I leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of I?I will dictate its function. PMID:24005673
Hypomagnesemia Induced by Long-Term Treatment with Proton-Pump Inhibitors
Janett, Simone; Camozzi, Pietro; Peeters, Gabriļlla G. A. M.; Lava, Sebastiano A. G.; Simonetti, Giacomo D.; Goeggel Simonetti, Barbara; Bianchetti, Mario G.; Milani, Gregorio P.
In 2006, hypomagnesemia was first described as a complication of proton-pump inhibitors. To address this issue, we systematically reviewed the literature. Hypomagnesemia, mostly associated with hypocalcemic hypoparathyroidism and hypokalemia, was reported in 64 individuals on long-term proton-pump inhibitors. Hypomagnesemia recurred following replacement of one proton-pump inhibitor with another but not with a histamine type-2 receptor antagonist. The association between proton-pump inhibitors and magnesium metabolism was addressed in 14 case-control, cross-sectional studies. An association was found in 11 of them: 6 reports found that the use of proton-pump inhibitors is associated per se with a tendency towards hypomagnesemia, 2 found that this tendency is more pronounced in patients concurrently treated with diuretics, carboplatin, or cisplatin, and 2 found a relevant tendency to hypomagnesemia in patients with poor renal function. Finally, findings likely reflecting decreased intestinal magnesium uptake were observed on treatment with proton-pump inhibitors. Three studies did not disclose any relationship between magnesium metabolism and treatment with histamine type-2 receptor antagonists. In conclusion, proton-pump inhibitors may cause hypomagnesemia. In these cases, switching to a histamine type-2 receptor antagonist is advised.
Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation.
O'Rourke, J.; Wang, W. P.; Donnelly, L.; Wang, E.; Kreutzer, D. L.
Levels of extravascular tissue plasminogen activator activity (PA) and those of inhibitors of PA and of urokinase (UK) present within the anterior chamber of normal and inflamed feline eyes were assessed with the use of a direct PA assay of microsamples of aqueous humor. Purposes of the study were, first, to confirm prior indirect evidence that this extravascular space normally contains higher levels of uninhibited PA, but lower levels of inhibitor activity, than does plasma and, second, to determine patterns of change in these activities under in vivo conditions imposed by a chronic mycobacterial-induced uveitis (CMIU) disease model. The PA assay utilized a 125I-plasminogen substrate whose cleavage by PA contained in samples was both visualized during gel electrophoreis, and quantified by gamma counting. The results provided the first direct evidence that the higher fibrinolytic activity previously observed in normal aqueous in comparison with plasma is in fact associated with higher levels of available (uninhibited) PA (P less than 0.01) The data also indicated that normal aqueous contains a much higher level of PA inhibitor activity than previously suspected--roughly 40 times more than available PA levels. These normal values for PA and inhibitors occupied a relatively narrow, threefold range, in contrast to the wide scattering of individual values that appeared during 18-20 weeks of the chronic inflammation disease model. Despite this, however, the general pattern of observation for all individual eyes during CMIU was a significant increase in levels of both PA and inhibitors. The net effect of CMIU was thus to cause the 1:40 ratio noted above to be tilted more strongly in favor of inhibitor activity, ie, up to 1:80. Increases in local vasopermeability in this disease model were believed contributory to this change. However, local generations of PA and APA in vivo by inflammatory cells, especially monocyte-macrophages, must also be considered. Assays for UK inhibitor showed levels of activity and directions of change that closely followed those of PA inhibitor, which suggests the possibility that they may be identical. It is surmised that the above patterns, along with results of our prior studies, indicate an apparent need for a multistep, strict inhibitory control of plasmin generation and proteolysis in vivo within normal extravascular spaces such as the anterior chamber.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 2 PMID:3493701
The peroxisomal lumen in Saccharomyces cerevisiae is alkaline.
van Roermund, Carlo W T; de Jong, Mark; IJlst, Lodewijk; van Marle, Jan; Dansen, Tobias B; Wanders, Ronald J A; Waterham, Hans R
Peroxisomes have a central function in lipid metabolism, including the beta-oxidation of various fatty acids. The products and substrates involved in the beta-oxidation have to cross the peroxisomal membrane, which previously has been demonstrated to constitute a closed barrier, implying the existence of specific transport mechanisms. Fatty acid transport across the yeast peroxisomal membrane may follow two routes: one for activated fatty acids, dependent on the peroxisomal ABC half transporter proteins Pxa1p and Pxa2p, and one for free fatty acids, which depends on the peroxisomal acyl-CoA synthetase Faa2p and the ATP transporter Ant1p. A proton gradient across the peroxisomal membrane as part of a proton motive force has been proposed to be required for proper peroxisomal function, but the nature of the peroxisomal pH has remained inconclusive and little is known about its generation. To determine the pH of Sacharomyces cerevisiae peroxisomes in vivo, we have used two different pH-sensitive yellow fluorescent proteins targeted to the peroxisome by virtue of a C-terminal SKL and found the peroxisomal matrix in wild-type cells to be alkaline (pH(per) 8.2), while the cytosolic pH was neutral (pH(cyt) 7.0). No Delta pH was present in ant1 Delta cells, indicating that the peroxisomal pH is regulated in an ATP-dependent way and suggesting that Ant1p activity is directly involved in maintenance of the peroxisomal pH. Moreover, we found a high peroxisomal pH of >8.6 in faa2 Delta cells, while the peroxisomal pH remained 8.1+/-0.2 in pxa2 Delta cells. Our combined results suggest that the proton gradient across the peroxisomal membrane is dependent on Ant1p activity and required for the beta-oxidation of medium chain fatty acids. PMID:15316083
Dynamics of the Light-Dependent Transition of Plant Peroxisomes.
Goto-Yamada, Shino; Mano, Shoji; Yamada, Kenji; Oikawa, Kazusato; Hosokawa, Yoichiroh; Hara-Nishimura, Ikuko; Nishimura, Mikio
Peroxisomes are present in almost all plant cells. These organelles are involved in various metabolic processes, such as lipid catabolism and photorespiration. A notable feature of plant peroxisomes is their flexible adaptive responses to environmental conditions such as light. When plants shift from heterotrophic to autotrophic growth during the post-germinative stage, peroxisomes undergo a dynamic response, i.e. enzymes involved in lipid catabolism are replaced with photorespiratory enzymes. Although the detailed molecular mechanisms underlying the functional transition of peroxisomes have previously been unclear, recent analyses at the cellular level have enabled this detailed machinery to be characterized. During the functional transition, obsolete enzymes are degraded inside peroxisomes by Lon protease, while newly synthesized enzymes are transported into peroxisomes. In parallel, mature and oxidized peroxisomes are eliminated via autophagy; this functional transition occurs in an efficient manner. Moreover, it has become clear that quality control mechanisms are important for the peroxisomal response to environmental stimuli. In this review, we highlight recent advances in elucidating the molecular mechanisms required for the regulation of peroxisomal roles in response to changes in environmental conditions. PMID:26063394
Intracellular Injection of Protein Kinase Inhibitor Blocks the Serotonin-Induced Increase in K+ Conductance in Aplysia Neuron R15
NASA Astrophysics Data System (ADS)
Adams, William B.; Levitan, Irwin B.
Previous work has shown that serotonin induces an increase in membrane K+ conductance in Aplysia neuron R15 and that this response is mediated by cAMP. The present study examines the role of protein phosphorylation in the response to serotonin. A specific inhibitor of cAMP-dependent protein kinase was injected intracellularly into neuron R15. The injection blocked the serotonin-induced increase in K+ conductance completely for at least 4 hours. The blockage was selective because the cell's response to dopamine was not inhibited. Furthermore, the blockage was specifically produced by protein kinase inhibitor because injection of other proteins (? -bungarotoxin and bovine serum albumin) did not affect the serotonin response. The serotonin response recovered fully 5-13 hours after the injection, presumably as a result of intracellular proteolysis of the protein kinase inhibitor. The results indicate that protein phosphorylation is a necessary step in the process that leads to activation of K+ channels by serotonin in neuron R15.
Antioxidants and NOS inhibitors selectively targets manganese-induced cell volume via Na-K-Cl cotransporter-1 in astrocytes.
Alahmari, Khalid A; Prabhakaran, Harini; Prabhakaran, Krishnan; Chandramoorthy, Harish C; Ramugounder, Ramakrishnan
Manganese has shown to be involved in astrocyte swelling. Several factors such as transporters, exchangers and ion channels are attributed to astrocyte swelling as a result in the deregulation of cell volume. Products of oxidation and nitration have been implied to be involved in the pathophysiology of swelling; however, the direct link and mechanism of manganese induced astrocyte swelling has not been fully elucidated. In the current study, we used rat primary astrocyte cultures to investigate the activation of Na-K-Cl cotransporter-1 (NKCC1) a downstream mechanism for free radical induced astrocyte swelling as a result of manganese toxicity. Our results showed manganese, oxidants and NO donors as potent inducer of oxidation and nitration of NKCC1. Our results further confirmed that manganese (50?M) increased the total protein, phosphorylation and activity of NKCC1 as well as cell volume (p<0.05 vs. control). NKCC1 inhibitor (bumetanide), NKCC1-siRNA, antioxidants; DMTU, MnTBAP, tempol, catalase and Vit-E, NOS inhibitor; l-NAME, peroxinitrite scavenger; uric acid all significantly reversed the effects of NKCC1 activation (p<0.05). From the current investigation we infer that manganese or oxidants and NO induced activation, oxidation/nitration of NKCC1 play an important role in the astrocyte swelling. PMID:25817889
Hyperphosphate-Induced Myocardial Hypertrophy through the GATA-4/NFAT-3 Signaling Pathway Is Attenuated by ERK Inhibitor Treatment
Liu, Yao-Lung; Huang, Chiu-Ching; Chang, Chiz-Chung; Chou, Che-Yi; Lin, Shih-Yi; Wang, I-Kuan; Hsieh, Dennis Jine-Yuan; Jong, Gwo-Ping; Huang, Chih-Yang; Wang, Chao-Min
Background/Aims Numerous epidemiological studies have associated elevated serum phosphorus levels with cardiovascular disease and the risk of death in the general population as well as in chronic kidney disease (CKD) and dialysis patients. In this study, we explored whether elevated phosphate conditions induce cardiac hypertrophy and attempted to identify the molecular and cellular mechanisms in the hypertrophic response. Methods H9c2 myocardial cells were incubated in high-phosphate conditions to induce hypertrophy. Pathological hypertrophic responses were measured in terms of cell size, arrangement of actin filaments, and hypertrophy markers such as atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in myocardial cells. Several transcriptional factors involved in cardiac hypertrophy development were measured to investigate the molecular pathways involved in elevated phosphate-induced cardiac hypertrophy. Results High-phosphate conditions induced cellular hypertrophy, marked by increased cell size, reorganization of actin filaments, and upregulation of both ANP and BNP in H9c2 cells. Both upstream calcineurin and downstream transcription factors, including GATA-4 and NFAT-3, were significantly increased under hyperphosphate conditions. Moreover, both MEK1/2 and ERK1/2 expression increased significantly, and cellular hypertrophy was markedly attenuated by U0126, an ERK1/2 inhibitor. Conclusions These results suggest that hyperphosphate conditions induce myocardial hypertrophy through the ERK signaling pathway in H9c2 cells. Our findings provide a link between the hyperphosphate-induced response and the ERK/NFAT-3 signaling pathway that mediates the development of cardiac hypertrophy. In view of the potent and selective activity of the ERK inhibitor U0126, this agent warrants further investigation as a candidate for preventing hyperphosphate-induced cardiac hypertrophy in CKD and dialysis patients. PMID:25999956
Glucosidase trimming inhibitors preferentially perturb I cell activation induced by CD2 mAb
F. J. van Kemenade; F. T. M. Rotteveel; R. A. W. van Lier; F. Miedema
Glycosidase trimming inhibitors may be used to study contribution of N-linked glycan moieties in T cell function. We have studied the effects of castano- spermine (Cas), swainsonine (Swain), 1-deoxynojirimycin (dNM), and 1-deoxymannojirimycin (dMM) on T cell activation and differentiation. Our analysis included a new dNM derivative, N-pentyl-1-deoxynojirimycin (pentyldNM). Previous reports showed inhibitory action of trimming inhibitors, such as Swain and
Effects of tumour necrosis factor-? synthesis inhibitors on rat trinitrobenzene sulphonic acid-induced chronic colitis
Christine Bobin-Dubigeon; Xavier Collin; Nicole Grimaud; Jean-Michel Robert; Guillaume Le Baut; Jean-Yves Petit
The fact that tumour necrosis factor-? (TNF-?) is clearly involved in the pathogenesis of intestinal bowel disease, especially Crohn's disease, suggests that TNF-? synthesis inhibitors could be beneficial for treatment. The present study assessed the effect of chronic oral gavage of two in vitro TNF-? synthesis inhibitors, JM 34 maleate or [N-(4,6-dimethylpyridin-2-yl)-furane-2-carboxamide)] maleate and XC 21 or (N-?picolyl-tetrafluorophtalimide), on colonic
Mouse liver selenium-binding protein decreased in aboundance by peroxisome proliferators.
Giometti, C. S.; Liang, X.; Tollaksen, S. L.; Wall, D. B.; Lubman, D. M.; Subbarao, V.; Sambasiva Rao, M.
Several studies with two-dimensional gel electrophoresis (2-DE) have shown that the abundance of numerous mouse liver proteins is altered in response to treatment with chemicals known to cause peroxisome proliferation. The peptide masses from tryptic digests of two liver proteins showing dramatic decreases in abundance in response to numerous peroxisome proliferators were used to search sequence databases. The selenium-binding protein 2 (SBP2 formerly 56 kDa acetaminophen-binding protein, AP 56) and selenium-binding protein 1 (SBP1 formerly 56 kDa selenium-binding protein, SP 56) in mouse liver, proteins with a high degree of sequence similarity, were the highest ranked identities obtained. Identity with SBP2 was subsequently confirmed by immunodetection with specific antiserum. Treatment of mice with 0.025% ciprofibrate resulted in the more basic of this pair of proteins being decreased to 30% of control abundance while the acidic protein was decreased to 7% of the control amount. Dexamethasone treatment, in contrast, caused increases of 80% and 20% in the abundance of the acidic and basic forms, respectively. Administration of dexamethasone to mice in combination with ciprofibrate produced expression of the acidic SBP2 at 23% of the control level and the basic SBP2 at 36%, a slightly moderated reduction compared with the decrease that occurred with ciprofibrate alone. These data suggest that peroxisome proliferators such as ciprofibrate cause a decrease in the abundance of the SBP2, which leads to increased cell proliferation, even in the presence of an inhibitor such as dexamethasone. Such a decrease in SBP, thought to serve as cell growth regulation factors, could be central to the nongenotoxic carcinogenicity of the peroxisome proliferators observed in rodents.
NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells
Sobhakumari, Arya [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Schickling, Brandon M. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Love-Homan, Laurie; Raeburn, Ayanna [Department of Pathology, The University of Iowa, Iowa City, IA (United States); Fletcher, Elise V.M. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Case, Adam J. [Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Domann, Frederick E. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); Miller, Francis J. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); and others
Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect. - Highlights: Ģ Erlotinib increased LC3B-II and autophagosome formation in HNSCC cells. Ģ Inhibition of autophagy sensitized HNSCC cells to erlotinib. Ģ Erlotinib increased NOX4 promoter and 3?UTR luciferase activity. Ģ Manipulating NOX4 decreases or increases autophagy.
Effects of nimesulide, a preferential cyclooxygenase-2 inhibitor, on carrageenan-induced pleurisy and stress-induced gastric lesions in rats.
Nakatsugi, S; Terada, N; Yoshimura, T; Horie, Y; Furukawa, M
Intrapleural injection of carrageenan in rats increased prostaglandin E2 (PGE2) production and induced newly synthesized cyclooxygenase-2 (COX-2) in pleural exudate cells without affecting COX-1 levels. Nimesulide, a preferential inhibitor of COX-2, reduced pleural PGE2 production and was almost as active as indomethacin and 10 times more active than ibuprofen. Only COX-1, and nc COX-2, was detected in gastric mucosal cells, and PGE2 concentration of gastric mucosa was significantly decreased by indomethacin and ibuprofen. The decrease in gastric PGE2 production induced by indomethacin and ibuprofen was enhanced in stressed rats, resulting in aggravation of stress-induced gastric lesions at anti-inflammatory doses. However, nimesulide did not produce stress-induced gastric lesions even at 30 times the anti-inflammatory dose. This supports the hypothesis that inhibition of COX-1 causes unwanted side effects and inhibition of COX-2 produces anti-inflammatory effects. PMID:9014217
Chloroquine or Chloroquine-PI3K/Akt Pathway Inhibitor Combinations Strongly Promote ?-Irradiation-Induced Cell Death in Primary Stem-Like Glioma Cells
Firat, Elke; Weyerbrock, Astrid; Gaedicke, Simone; Grosu, Anca-Ligia; Niedermann, Gabriele
We asked whether inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is highly active in cancer stem cells (CSCs) and upregulated in response to genotoxic treatments, promote ?-irradiation?IR)-induced cell death in highly radioresistant, patient-derived stem-like glioma cells (SLGCs). Surprisingly, in most cases the inhibitors did not promote ?IR-induced cell death. In contrast, the strongly cytostatic Ly294002 and PI-103 even tended to reduce it. Since autophagy was induced we examined whether addition of the clinically applicable autophagy inhibitor chloroquine (CQ) would trigger cell death in SLGCs. Triple therapy with CQ at doses as low as 5 to 10 ĄM indeed caused strong apoptosis. At slightly higher doses, CQ alone strongly promoted ?IR-induced apoptosis in all SLGC lines examined. The strong apoptosis in combinations with CQ was invariably associated with strong accumulation of the autophagosomal marker LC3-II, indicating inhibition of late autophagy. Thus, autophagy-promoting effects of PI3K/Akt pathway inhibitors apparently hinder cell death induction in ?-irradiated SLGCs. However, as we show here for the first time, the late autophagy inhibitor CQ strongly promotes ?IR-induced cell death in highly radioresistant CSCs, and triple combinations of CQ, ?IR and a PI3K/Akt pathway inhibitor permit reduction of the CQ dose required to trigger cell death. PMID:23091617
Spinal Glia Division Contributes to Conditioning Lesion-Induced Axon Regeneration Into the Injured Spinal Cord: Potential Role of Cyclic AMP-Induced Tissue Inhibitor of Metalloproteinase-1.
Liu, Huaqing; Angert, Mila; Nishihara, Tasuku; Shubayev, Igor; Dolkas, Jennifer; Shubayev, Veronica I
Regeneration of sensory neurons after spinal cord injury depends on the function of dividing neuronal-glial antigen 2 (NG2)-expressing cells. We have shown that increases in the number of dividing NG2-positive cells through short-term pharmacologic inhibition of matrix metalloproteinases contributes to recovery after spinal cord injury. A conditioning sciatic nerve crush (SNC) preceding spinal cord injury stimulates central sensory axon regeneration via the intraganglionic action of cyclic adenosine monophosphate. Here, using bromodeoxyuridine, mitomycin (mitosis inhibitor), and cholera toxin B tracer, we demonstrate that SNC-induced division of spinal glia is related to the spinal induction of tissue inhibitor of metalloproteinase-1 and contributes to central sensory axon growth into the damaged spinal cord. Dividing cells were mainly NG2-positive and Iba1-positive and included myeloid NG2-positive populations. The cells dividing in response to SNC mainly matured into oligodendrocytes and microglia within the injured spinal cord. Some postmitotic cells remained NG2-reactive and were associated with regenerating fibers. Moreover, intraganglionic tissue inhibitor of metalloproteinase-1 expression was induced after administration of SNC or cyclic adenosine monophosphate analog (dbcAMP) to dorsal root ganglia in vivo and in primary adult dorsal root ganglia cultures. Collectively, these findings support a novel model whereby a cyclic adenosine monophosphate-activated regeneration program induced in sensory neurons by a conditioning peripheral nerve lesion uses tissue inhibitor of metalloproteinase-1 to protect against short-term proteolysis, enabling glial cell division and promoting axon growth into the damaged CNS. PMID:25933384
Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 Is a Peroxin That Recruits the PEX1-PEX6 Complex to Peroxisomes[W
Goto, Shino; Mano, Shoji; Nakamori, Chihiro; Nishimura, Mikio
Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H2O2 and ?-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein¢protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants. PMID:21487094
Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.
Quilis, Jordi; L¾pez-GarcĒa, Belķn; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca
Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C.Āsuppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants. PMID:24237606
Effects of Respiration Inhibitors and Uncouplers on Dark- and Light-Induced Leaflet Movements of Cassia fasciculata1
Saeedi, Saed; Roblin, Gabriel
Respiration inhibitors, in particular KCN and NaN3, inhibited slightly the dark-induced (scotonasty) as well as the light-induced (photonasty) leaflet movements of Cassia fasciculata: they act only at concentrations higher than 1 millimolar and 0.1 millimolar, respectively. Amytal induced a stronger inhibitory effect on scotonasty. Salicylhydroxamic acid, which inhibits the cyanide-insensitive respiration pathway, was also poorly effective when applied alone. KCN and salicylhydroxamic acid applied together increased the inhibition. Uncouplers of oxidative phosphorylation were very effective: 2,4-dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone inhibited the scotonastic movements at concentrations higher than 10 ?m and 1 ?m, respectively. Although uncouplers reduced the photonastic movements at higher concentrations, they promoted leaflet opening at other concentrations in an unexpected way. PMID:16665004
HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease.
d'Ydewalle, Constantin; Krishnan, Jyothsna; Chiheb, Driss M; Van Damme, Philip; Irobi, Joy; Kozikowski, Alan P; Vanden Berghe, Pieter; Timmerman, Vincent; Robberecht, Wim; Van Den Bosch, Ludo
Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system. Mutations in the 27-kDa small heat-shock protein gene (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). We developed and characterized transgenic mice expressing two different HSPB1 mutations (S135F and P182L) in neurons only. These mice showed all features of CMT or distal HMN dependent on the mutation. Expression of mutant HSPB1 decreased acetylated ?-tubulin abundance and induced severe axonal transport deficits. An increase of ?-tubulin acetylation induced by pharmacological inhibition of histone deacetylase 6 (HDAC6) corrected the axonal transport defects caused by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of ?-tubulin deacetylation in mutant HSPB1-induced neuropathies and offer perspectives for using HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies. PMID:21785432
The SUV39H1 inhibitor chaetocin induces differentiation and shows synergistic cytotoxicity with other epigenetic drugs in acute myeloid leukemia cells
Lai, Y-S; Chen, J-Y; Tsai, H-J; Chen, T-Y; Hung, W-C
Epigenetic modifying enzymes have a crucial role in the pathogenesis of acute myeloid leukemia (AML). Methylation of lysine 9 on histone H3 by the methyltransferase G9a and SUV39H1 is associated with inhibition of tumor suppressor genes. We studied the effect of G9a and SUV39H1 inhibitors on viability and differentiation of AML cells and tested the cytotoxicity induced by combination of G9a and SUV39H1 inhibitors and various epigenetic drugs. The SUV39H1 inhibitor (chaetocin) and the G9a inhibitor (UNC0638) caused cell death in AML cells at high concentrations. However, only chaetocin-induced CD11b expression and differentiation of AML cells at non-cytotoxic concentration. HL-60 and KG-1a cells were more sensitive to chaetocin than U937 cells. Long-term incubation of chaetocin led to downregulation of SUV39H1 and reduction of H3K9 tri-methylation in HL-60 and KG-1a cells. Combination of chaetocin with suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor) or JQ (a BET (bromodomain extra terminal) bromodomain inhibitor) showed synergistic cytotoxicity. Conversely, no synergism was found by combining chaetocin and UNC0638. More importantly, chaetocin-induced differentiation and combined cytotoxicity were also found in the primary cells of AML patients. Collectively, the SUV39H1 inhibitor chaetocin alone or in combination with other epigenetic drugs may be effective for the treatment of AML. PMID:25978433
Cytokine-induced apoptosis inhibitorĀ1 inhibits the growth and proliferation of multiple myeloma.
Wang, Xiaobo; Pan, Jingxuan; Li, Juan
The present study investigated the differential expression of cytokine-induced apoptosis inhibitorĀ1 (CIAPIN1) in human multiple myeloma (MM) bone marrow tissue and adjacent healthy bone marrow tissue. In addition, the effect of a transduced CIAPIN1 gene on the growth of the RPMI?8226 human MM cell line was investigated. CIAPIN1 protein expression was detected inĀ32Āsamples of paraffin?embedded MM and adjacent healthy bone marrow tissue using immunohistochemistry. The CIAPIN1 gene (Ad?CIAPIN1, small interfering RNA) was inserted into a lentiviral vector and transfected into the RPMI?8226 human MM cell line. The expression of target proteins CIAPIN1 and insulin?like growth factor 1UĀ(IGF?1), cell cycle?regulatory proteins and functional proteins was detected using western blotting. MTT and soft agar colony formation assays were conducted, and cellular tumorigenicity in nude mice was assessed, in order to investigate the proliferative capacity of cells inĀvitro and inĀvivo. Flow cytometry was applied in order to analyze changes in the cell cycle and cell apoptosis. CIAPIN1 expression was significantly reduced in cells from the 32ĀMM samples compared with those from healthy bone marrowĀ(P<0.05). Upregulation of CIAPIN1, following transduction by lentiviral vectors, caused cells to arrest in G1/S phase of the cell cycle and significantly inhibited the growth of the RPMI?8226 MM cell line inĀvitro and inĀvivo. CIAPIN1 was shown to inhibit cell growth. Specifically, it inhibited cyclin?dependent kinaseĀ2, cyclin?dependent kinaseĀ4 and insulin?like growth factor?1. Increased expression of CIAPIN1 also led to an increase in the levels of p27 and Rb, an effect that may have been achieved via regulation of cell cycle proteins and functional proteins. The results of the present study suggest that downregulation of the CIAPIN1 gene in MM cells may be associated with the development of this disease. CIAPIN1 transfection in RPMI?8226 cells significantly inhibited the growth of tumor cells, suggesting that the CIAPIN1 gene is a potential tumor suppressor. PMID:25901506
Tempol protects cardiomyocytes from nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity.
Liu, Yongmin; Shim, Eunwoo; Nguyen, Phuonggiang; Gibbons, Alexander T; Mitchell, James B; Poirier, Miriam C
Nucleoside reverse transcriptase inhibitors (NRTIs), essential components of combinational therapies used for treatment of human immunodeficiency virus-1, damage heart mitochondria. Here, we have shown mitochondrial compromise in H9c2 rat cardiomyocytes exposed for 16 passages (P) to the NRTIs zidovudine (AZT, 50?M) and didanosine (ddI, 50?M), and we have demonstrated protection from mitochondrial compromise in cells treated with 200?M 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (Tempol) or 200?M 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine (Tempol-H), along with AZT/ddI, for 16P. Exposure to AZT/ddI caused a moderate growth inhibition at P3, P5, P7, and P13, which was not altered by addition of Tempol or Tempol-H. Mitochondrial oxidative phosphorylation capacity was determined as uncoupled oxygen consumption rate (OCR) by Seahorse XF24 Analyzer. At P5, P7, and P13, AZT/ddI-exposed cells showed an OCR reduction of 8.8-57.2% in AZT/ddI-exposed cells, compared with unexposed cells. Addition of Tempol or Tempol-H, along with AZT/ddI, resulted in OCR levels increased by about 300% above the values seen with AZT/ddI alone. The Seahorse data were further supported by electron microscopy (EM) studies in which P16 cells exposed to AZT/ddI/Tempol had less mitochondrial pathology than P16 cells exposed to AZT/ddI. Western blots of P5 cells showed that Tempol and Tempol-H upregulated expression of mitochondrial uncoupling protein-2 (UCP-2). However, Complex I activity that was reduced by AZT/ddI, was not restored in the presence of AZT/ddI/Tempol. Superoxide levels were increased in the presence of AZT/ddI and significantly decreased in cells exposed to AZT/3TC/Tempol at P3, P7, and P10. In conclusion, Tempol protects against NRTI-induced mitochondrial compromise, and UCP-2 plays a role through mild uncoupling. PMID:24591154
JNK inhibitor SP600125 protects against lipopolysaccharide-induced acute lung injury via upregulation of claudin-4
Although in vitro studies have previously demonstrated that mitogen-activated protein kinases are important for the activation of transcription factors and the regulation of proinflammatory mediators, the function of c-Jun NH2-terminal kinase (JNK) in acute lung injury (ALI) remains to be fully elucidated. The present study aimed to investigate the effect of the JNK selective inhibitor SP600125 on lipopolysaccharide (LPS)-induced ALI. Pulmonary edema, the expression of inflammatory cytokines and pathological alterations were found to be significantly attenuated in LPS-induced ALI following treatment with SP600125 in vivo. In vitro, it was demonstrated that SP600125 administration significantly improved A549 cell viability in a dose-dependent manner using the Cell Counting kit-8 and the 5-ethynyl-2?-deoxyuridine incorporation assay. Furthermore, flow cytometric analysis demonstrated that the apoptotic rate was significantly reduced in a concentration-dependent manner following SP600125 injection. At the molecular level, SP600125 treatment dose-dependently inhibited JNK activation and upregulated claudin-4 expression in vivo and in vitro. In combination, the results from the present study indicated that the JNK inhibitor SP600125 protected against LPS-induced ALI in vivo and in vitro, possibly by upregulating the expression of claudin-4. PMID:24944614
The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo
Tan, Wei, E-mail: email@example.com [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan (China)] [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan (China); Zhou, Wei; Yu, Hong-gang; Luo, He-Sheng; Shen, Lei [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan (China)] [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan (China)
Highlights: Black-Right-Pointing-Pointer Zebularine inhibited cell growth of gastric cancer in a time- and dose-dependent manner. Black-Right-Pointing-Pointer Chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Zebularine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by zebularine. -- Abstract: DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 {mu}M accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.
Myelin associated inhibitors: a link between injury-induced and experience-dependent plasticity.
Akbik, Feras; Cafferty, William B J; Strittmatter, Stephen M
In the adult, both neurologic recovery and anatomical growth after a CNS injury are limited. Two classes of growth inhibitors, myelin associated inhibitors (MAIs) and extracellular matrix associated inhibitors, limit both functional recovery and anatomical rearrangements in animal models of spinal cord injury. Here we focus on how MAIs limit a wide spectrum of growth that includes regeneration, sprouting, and plasticity in both the intact and lesioned CNS. Three classic myelin associated inhibitors, Nogo-A, MAG, and OMgp, signal through their common receptors, Nogo-66 Receptor-1 (NgR1) and Paired-Immunoglobulin-like-Receptor-B (PirB), to regulate cytoskeletal dynamics and inhibit growth. Initially described as inhibitors of axonal regeneration, subsequent work has demonstrated that MAIs also limit activity and experience-dependent plasticity in the intact, adult CNS. MAIs therefore represent a point of convergence for plasticity that limits anatomical rearrangements regardless of the inciting stimulus, blurring the distinction between injury studies and more "basic" plasticity studies. PMID:21699896
Corticosteroids Augment BRAF Inhibitor Vemurafenib Induced Lymphopenia and Risk of Infection
Sondermann, Wiebke; Griewank, Klaus G.; Schilling, Bastian; Livingstone, Elisabeth; Leyh, Julia C.; Rompoti, Natalia; Cosgarea, Ioana; Schimming, Tobias; Schadendorf, Dirk; Zimmer, Lisa; Hillen, Uwe
We have previously demonstrated an impact of the BRAF inhibitor vemurafenib on patient lymphocyte counts. In the current study, the extent to which concomitant use of corticosteroids in BRAF inhibitor treated patients affects lymphocyte counts and predisposes to infection was investigated. A cohort of 102 patients receiving either the selective BRAF inhibitor vemurafenib or dabrafenib was analyzed. The amount of patients receiving either medication with or without systemic corticosteroids (dexamethasone) was determined and lymphocyte counts before and under therapy assessed. Additionally, the number and severity of infections occurring in these groups was analyzed. Vemurafenib treatment led to a considerable decrease in lymphocyte cell counts, with 62.3% of patients having lymphopenia. Dabrafenib treated patients only rarely demonstrated lymphopenia (12.5%). Dexamethasone co-administration further diminished lymphocyte counts. Lymphopenias were observed in 84.6% of patients receiving vemurafenib and dexamethasone. In our cohort, infections were noted in 9 patients, 4 of these were severe and 2 eventually fatal. All 9 cases with infections demonstrated lymphopenia, 8 of these had received dexamethasone and 7 of these a therapy with vemurafenib. Our findings demonstrate a significant lymphopenia in patients treated with the BRAF inhibitor vemurafenib, which is further augmented by dexamethasone and predisposes to infection. If validated in other studies, risk of infection should be considered when applying corticosteroids in combination with BRAF inhibitors, in particular vemurafenib. PMID:25897843
Myelin Associated Inhibitors: A Link Between Injury-Induced and Experience-Dependent Plasticity
Akbik, Feras; Cafferty, William B. J.; Strittmatter, Stephen M.
SUMMARY In the adult, both neurologic recovery and anatomical growth after a CNS injury are limited. Two classes of growth inhibitors, myelin associated inhibitors (MAIs) and extracellular matrix associated inhibitors, limit both functional recovery and anatomical rearrangements in animal models of spinal cord injury. Here we focus on how MAIs limit a wide spectrum of growth that includes regeneration, sprouting, and plasticity in both the intact and lesioned CNS. Three classic myelin associated inhibitors, Nogo-A, MAG, and OMgp, signal through their common receptors, Nogo-66 Receptor-1 (NgR1) and Paired-Immunoglobulin-like-Receptor-1 (PirB), to regulate cytoskeletal dynamics and inhibit growth. Initially described as inhibitors of axonal regeneration, subsequent work has demonstrated that MAIs also limit activity and experience-dependent plasticity in the intact, adult CNS. MAIs therefore represent a point of convergence for plasticity that limits anatomical rearrangements regardless of the inciting stimulus, blurring the distinction between injury studies and more ōbasicö plasticity studies. PMID:21699896
The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes
Liow, K.Y.; Chow, S.C., E-mail: firstname.lastname@example.com
The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose¢response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: Ģ z-FA-CMK is toxic and induce cell death in the human T cells. Ģ z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. Ģ z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.
Bacillus Calmette-Guķrin vaccine induces a selective serotonin reuptake inhibitor (SSRI)-resistant depression like phenotype in mice.
Vijaya Kumar, K; Rudra, Anjuman; Sreedhara, M V; Siva Subramani, T; Prasad, Durga Shiva; Das, Manish Lal; Murugesan, Senthil; Yadav, Rajbharan; Trivedi, Ravi Kumar; Louis, Justin V; Li, Yu-Wen; Bristow, Linda J; Naidu, Pattipati S; Vikramadithyan, Reeba Kannimel
Preclinical studies have shown that administration of Bacillus Calmette-Guķrin (BCG) vaccine induces depression-like behaviors in mice; however, the effect of antidepressant drug treatment has not been reported earlier. In the present study, we induced depression-like behavior by administering BCG vaccine to BALB/c mice. BCG treatment produced robust serum sickness as shown by a decrease in body weight, reduced spontaneous locomotor activity and reduced voluntary wheel running activity. BCG treatment also elevated plasma IL6 and IFN? levels and produced a marked activation of lung IDO activity. At a time point when serum sickness-related behaviors had fully recovered (i.e., day 14) BCG-treated mice showed a significant increase in immobility in the forced swim test (FST) and tail suspension test (TST) indicative of a pro-depressant phenotype. We observed significant increase in [(3)H]PK11195 binding in cortex and hippocampus regions of BGC-treated mice in comparison to saline-treated mice indicating prominent neuroinflammation. Pharmacological evaluation of FST behavior in BCG-treated mice demonstrated selective resistance to the selective serotonin reuptake inhibitors (SSRIs) fluoxetine and escitalopram. In contrast the tricyclic antidepressant imipramine, the dual serotonin/norepinephrine reuptake inhibitor (SNRI) duloxetine, and the dual dopamine/norepinephrine reuptake inhibitor (DNRI) nomifensine retained antidepressant efficacy in these mice. The lack of efficacy with acute treatment with SSRIs could not be explained either by differences in drug exposure or serotonin transporter (SERT) occupancy. Our results demonstrate that BCG-vaccine induced depression like behavior is selectively resistant to SSRIs and could potentially be employed to evaluate novel therapeutic agents being developed to treat SSRI-resistance in humans. PMID:25016199
The role of poly(ADP-ribose) polymerase-1 inhibitor in carrageenan-induced lung inflammation in mice.
Ahmad, Sheikh Fayaz; Zoheir, Khairy M A; Ansari, Mushtaq Ahmad; Korashy, Hesham M; Bakheet, Saleh A; Ashour, Abdelkader E; Al-Shabanah, Othman A; Al-harbi, Mohammed M; Attia, Sabry M
Increasing indication is unveiling a role for poly(ADP-ribose) polymerase (PARP)-1 in the regulation of inflammatory/immune responses. The aim of the present study was to determine the potential anti-inflammatory effects of PARP-1 inhibitor 5-aminoisoquinolinone (5-AIQ) to explore the role of PARP-1 inhibitor in a mouse model of carrageenan-induced lung inflammation. A single dose of 5-AIQ (1.5mg/kg) was administered intraperitoneally (i.p.) 1h before ?-carrageenan (Cg) administration. We assessed the effects of 5-AIQ treatment on CD25(+), GITR(+), CD25(+)GITR(+), IL-17(+) and Foxp3(+) cells which were investigated using flowcytometry in pleural exudates and heparinized blood. We also evaluated mRNA expressions of IL-6, TNF-?, IL-1?, IL-10, CD11a, l-selectin (CD62L), ICAM-1, MCP-1, iNOS and COX-2 in the lung tissue. We further examined the effects of 5-AIQ on the key mediators of inflammation, namely COX-2, STAT-3, NF-kB p65, PARP-1, IkB-? and IL-4 protein expression in the lung tissue using western blotting. The results illustrated that the numbers of T cell subsets, IL-17(+) cytokine levels were markedly increased and Foxp3(+) production decreased in the Cg group. Furthermore, Cg-induced up-regulation of adhesion molecules, pro-inflammatory mediators and chemokine expressions. Western blot analysis revealed an increased protein expressions of COX-2, STAT-3 NF-kB p65 and PARP-1 and decreased IkB-? and IL-4 in the Cg group. PARP-1 inhibitor via 5-AIQ treatment reverses the action significantly of all the previously mentioned effects. Moreover, histological examinations revealed anti-inflammatory effects of 5-AIQ, whereas Cg-group aggravated Cg-induced inflammation. Present findings demonstrate the potent anti-inflammatory action of the PARP-1 inhibitor in acute lung injury induced by carrageenan. PMID:25304310
Beyond gastric acid reduction: Proton pump inhibitors induce heme oxygenase-1 in gastric and endothelial cells
Becker, Jan C. [Department of Medicine B, University of Muenster, 48149 Muenster (Germany)]. E-mail: email@example.com; Grosser, Nina [Department of Pharmacology and Toxicology, School of Pharmacy, Martin Luther University, Halle-Wittenberg, 06099 Halle (Saale) (Germany); Waltke, Christian [Department of Medicine B, University of Muenster, 48149 Muenster (Germany); Schulz, Stephanie [Department of Pharmacology and Toxicology, School of Pharmacy, Martin Luther University, Halle-Wittenberg, 06099 Halle (Saale) (Germany); Erdmann, Kati [Department of Pharmacology and Toxicology, School of Pharmacy, Martin Luther University, Halle-Wittenberg, 06099 Halle (Saale) (Germany); Domschke, Wolfram [Department of Medicine B, University of Muenster, 48149 Muenster (Germany); Schroeder, Henning [Department of Pharmacology and Toxicology, School of Pharmacy, Martin Luther University, Halle-Wittenberg, 06099 Halle (Saale) (Germany); Pohle, Thorsten [Department of Medicine B, University of Muenster, 48149 Muenster (Germany)
Proton pump inhibitors (PPIs) have been demonstrated to prevent gastric mucosal injury by mechanisms independent of acid inhibition. Here we demonstrate that both omeprazole and lansoprazole protect human gastric epithelial and endothelial cells against oxidative stress. This effect was abrogated in the presence of the heme oxygenase-1 (HO-1) inhibitor ZnBG. Exposure to either PPI resulted in a strong induction of HO-1 expression on mRNA and protein level, and led to an increased activity of this enzyme. Expression of cyclooxygenase isoforms 1 and 2 remained unaffected, and COX-inhibitors did not antagonize HO-1 induction by PPIs. Our results suggest that the antioxidant defense protein HO-1 is a target of PPIs in both endothelial and gastric epithelial cells. HO-1 induction might account for the gastroprotective effects of PPIs independently of acid inhibition, especially in NSAID gastropathy. Moreover, our findings provide additional perspectives for a possible but yet unexplored use of PPIs in vasoprotection.
An Aminopyridazine Inhibitor of Death Associated Protein Kinase Attenuates Hypoxia-Ischemia Induced Brain Damage
Velentza, A.V.; Wainwright, M.S.; Zasadzki, M.; Mirzoeva, S.; Haiech, J.; Focia, P.J.; Egli, M.; Watterson, D.M.
Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.
High content screening for non-classical peroxisome proliferators
Sexton, Jonathan Z; He, Qingping; Forsberg, Lawrence J; Brenman, Jay E
Peroxisomes are ubiquitous cellular organelles that perform vital functions including fatty acid beta-oxidation, plasmalogen synthesis, and detoxification of harmful oxidative species. In rodents numerous compounds that increase peroxisome biogenesis also alleviate metabolic syndrome (MetS)/type 2 diabetes (T2D) symptoms. However, compounds that increase peroxisome biogenesis in rodents largely do not increase peroxisome biogenesis in humans. We designed a novel genetically encoded high throughput screening (HTS) high content assay to identify small molecule compounds that function as peroxisome proliferators in human cells. From this assay we have confirmed that 4-phenylbutyrate (PBA), a PPAR independent peroxisome proliferator and chemical chaperone, increases peroxisome proliferation in human cells and serves as a positive control for our screen. We performed a small pilot and larger 15,000 compound production screen with an overall Z? factor of 0.74 for 384-well plate format, providing a valuable screening tool for identifying peroxisome modulator compounds. From this screen we have identified 4 existing drugs and 10 novel compounds, some with common scaffolds 1000X more potent than PBA. It is hoped that these novel compounds may serve as scaffolds for testing for efficacy in alleviating MetS/T2D symptoms both in mouse models and ultimately human disease. PMID:21132080
Peroxisome Biogenesis: Something Old, Something New, Something Borrowed
NSDL National Science Digital Library
Fred Mast (University of Alberta)
Eukaryotic cells are characterized by their varied complement of organelles. One set of membrane-bound, usually spherical compartments are commonly grouped together under the term peroxisomes. Peroxisomes function in regulating the synthesis and availability of many diverse lipids by harnessing the power of oxidative reactions and contribute to a number of metabolic processes essential for cellular differentiation and organismal development.
Immunohistochemistry for a bifunctional protein in patients with peroxisomal disorders
Atsushi Imamura; Atsushi Kamei; Yasuyuki Suzuki; Naomi Kondo; Tadao Orii; Sachio Takashima
Immunohistochemical studies using antisera against bifunctional protein, a ?-oxidation enzyme, were performed on liver, kidney, and brain tissue specimens from patients with peroxisomal disorders and from controls to investigate the distribution and development of peroxisomes. Bifunctional protein-positive granules were not found in patients with Zellweger syndrome or neonatal adrenoleukodystrophy, whereas positive immunoreactivity was observed from 8 and 6 weeks gestation
Molecular mechanisms of organelle inheritance: lessons from peroxisomes in yeast
Andrei Fagarasanu; Fred D. Mast; Barbara Knoblach; Richard A. Rachubinski
Preserving a functional set of cytoplasmic organelles in a eukaryotic cell requires a process of accurate organelle inheritance at cell division. Studies of peroxisome inheritance in yeast have revealed that polarized transport of a subset of peroxisomes to the emergent daughter cell is balanced by retention mechanisms operating in both mother cell and bud to achieve an equitable distribution of
Survivin selective inhibitor YM155 induce apoptosis in SK-NEP-1 Wilms tumor cells
Background Survivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of mitosis and programmed cell death. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. The aim of this study was to determine the antitumor activity of YM155 in SK-NEP-1 cells. Methods SK-NEP-1 cell growth in vitro and in vivo was assessed by MTT and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis in cell culture. Then gene expression profile of tumor cells treated with YM155 was analyzed with real-time PCR arrays. We then analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis tool. Results YM155 treatment resulted in inhibition of cell proliferation of SK-NEP-1cells in a dose-dependent manner. Annexin V assay, cell cycle, and activation of caspase-3 demonstrates that YM155 induced apoptosis in SK-NEP-1 cells. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ▒ 0.77 cm3; YM155 10 mg/kg: 0.95 ▒ 0.55 cm3) compared to DMSO group (DMSO: 3.70 ▒ 2.4 cm3) or PBS group cells (PBS: 3.78 ▒ 2.20 cm3, ANOVA P < 0.01). YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ▒ 0.24 g; YM155 10 mg/kg: 0.72 ▒ 0.17 g) compared to DMSO group (DMSO: 2.06 ▒ 0.38 g) or PBS group cells (PBS: 2.36 ▒ 0.43 g, ANOVA P < 0.01). Real-time PCR array analysis showed between Test group and control group there are 32 genes significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment. Ingenuity pathway analysis (IPA) showed cell death was the highest rated network with 65 focus molecules and the significance score of 44. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to cell death, cellular function maintenance, cell morphology, carbohydrate metabolism and cellular growth and proliferation. Death receptor signaling (3.87E-19), TNFR1 signaling, induction of apoptosis by HIV1, apoptosis signaling and molecular mechanisms of cancer came out to be the top four most significant pathways. IPA analysis also showed top molecules up-regulated were BBC3, BIRC3, BIRC8, BNIP1, CASP7, CASP9, CD5, CDKN1A, CEBPG and COL4A3, top molecules down-regulated were ZNF443, UTP11L, TP73, TNFSF10, TNFRSF1B, TNFRSF25, TIAF1, STK17A, SST and SPP1, upstream regulator were NR3C1, TP53, dexamethasone , TNF and Akt. Conclusions The present study demonstrates that YM155 treatment resulted in apoptosis and inhibition of cell proliferation of SK-NEP-1cells. YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors. Real-time PCR array analysis firstly showed expression profile of genes dyes-regulated after YM155 treatment. IPA analysis also represents new molecule mechanism of YM155 treatment, such as NR3C1 and dexamethasone may be new target of YM155. And our results may provide new clues of molecular mechanism of apoptosis induced by YM155. PMID:23267699
Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation.
Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid; Gjerl°v, Simon; Birk, Jesper; R÷pke, Carsten; Norrild, Bodil
Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induction of cell death. We have used the osteosarcoma cell line U2OS cells provided with E7 and the cdk2 inhibitor p21 (cip1/waf1) under inducible control, as a model system for the analysis of E7-mediated apoptosis. Our data shows that simultaneous expression of E7 and p21 proteins induces cell death, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge, this is the first time a role for cathepsin B is reported in HPV-induced apoptotic signalling. PMID:15016552
Dexamethasone induces the expression of metalloproteinase inhibitor TIMP-1 in the murine cerebral vascular endothelial cell line cEND
F÷rster, Carola; Kahles, Timo; Kietz, Silke; Drenckhahn, Detlev
In many neuroinflammatory conditions, including multiple sclerosis (MS), encephalitis, meningitis, brain tumours and cerebral ischaemia, the matrix metalloproteinases (MMPs) play an important role in disrupting the blood¢brain barrier (BBB). Normally under tight regulation, increased MMP-9 cerebrospinal fluid levels and excessive proteolytic activity is detected in the blood and cerebrospinal fluid in patients with acute MS. MMP-9 is a member of the type IV collagenases, which attack components of the endothelial basal lamina, including type IV collagen. The disruption of the BBB and clinical symptoms can be reduced with different inhibitors to MMPs including activators of tissue inhibitor of metalloproteinases-1 (TIMP-1), the cognate tissue inhibitor of MMP-9. Since intravenous glucocorticoid (GC) treatment reduces the levels of MMP-9 markedly in patients, we hypothesized that GC effects might be mediated by transcriptional activation of the TIMP-1 gene in addition to reported repressive effects on MMP-9 transcription. Our results provide direct evidence that GCs increase TIMP-1 in the brain endothelial cell line cEND, prevent alterations in microvascular integrin ?1 subunit expression and help maintain endothelial barrier function in response to pro-inflammatory stimuli (TNF? administration). GC-induced up-regulation of TIMP-1 expression by the CNS vascular endo-thelium may thus play a role in preservation of the endothelial basal lamina and maintain integrin ?1 and tight junction protein expression important for vessel wall integrity. PMID:17317742
A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties.
Fang, Evandro Fei; Ng, Tzi Bun
Nephelium lappaceum L., commonly known as "rambutan," is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and ?-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 ?M. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy. PMID:25820360
The Effect of Serine Protease Inhibitors on Airway Inflammation in a Chronic Allergen-Induced Asthma Mouse Model
Lin, Li-Jen; Wang, Shulhn-Der; Chiang, Chung-Jen; Kao, Shung-Te
Serine protease inhibitors reportedly attenuated airway inflammation and had antioxidant in multiorgan. However, the effects of the serine protease inhibitors nafamostat mesilate (FUT), gabexate mesilate (FOY), and ulinastatin (UTI) on a long-term challenged mouse model of chronic asthma are unclear. BALB/c mice (6 mice/group) were intratracheally inoculated with five doses of Dermatophagoides pteronyssinus (Der p; 50??L, 1?mg/mL) at one-week intervals. Therapeutic doses of FUT (0.0625?mg/kg), FOY (20?mg/kg), or UTI (10,000?U/kg) were, respectively, injected intraperitoneally into these mice. Control mice received sterile PBS. At 3 days after the last challenge, mice were sacrificed to assess airway hyperresponsiveness (AHR), remodeling, and inflammation; lung histological features; and cytokine expression profiles. Compared with untreated controls, mice treated with FUT, FOY, and UTI had decreased AHR and goblet cell hyperplasia, decreased eosinophil and neutrophil infiltration, decreased Der p-induced IL-4 levels in serum and IL-5, IL-6, IL-13, and IL-17 levels in bronchoalveolar lavage fluid, and inhibited nuclear factor (NF)-?B activity in lung tissues. The serine protease inhibitors FUT, FOY, and UTI have potential therapeutic benefits for treating asthma by downregulating Th2 cytokines and Th17 cell function and inhibiting NF-?B activation in lung tissue. PMID:25180025
The effect of serine protease inhibitors on airway inflammation in a chronic allergen-induced asthma mouse model.
Lin, Chih-Che; Lin, Li-Jen; Wang, Shulhn-Der; Chiang, Chung-Jen; Chao, Yun-Peng; Lin, Joseph; Kao, Shung-Te
Serine protease inhibitors reportedly attenuated airway inflammation and had antioxidant in multiorgan. However, the effects of the serine protease inhibitors nafamostat mesilate (FUT), gabexate mesilate (FOY), and ulinastatin (UTI) on a long-term challenged mouse model of chronic asthma are unclear. BALB/c mice (6 mice/group) were intratracheally inoculated with five doses of Dermatophagoides pteronyssinus (Der p; 50??L, 1?mg/mL) at one-week intervals. Therapeutic doses of FUT (0.0625?mg/kg), FOY (20?mg/kg), or UTI (10,000?U/kg) were, respectively, injected intraperitoneally into these mice. Control mice received sterile PBS. At 3 days after the last challenge, mice were sacrificed to assess airway hyperresponsiveness (AHR), remodeling, and inflammation; lung histological features; and cytokine expression profiles. Compared with untreated controls, mice treated with FUT, FOY, and UTI had decreased AHR and goblet cell hyperplasia, decreased eosinophil and neutrophil infiltration, decreased Der p-induced IL-4 levels in serum and IL-5, IL-6, IL-13, and IL-17 levels in bronchoalveolar lavage fluid, and inhibited nuclear factor (NF)-?B activity in lung tissues. The serine protease inhibitors FUT, FOY, and UTI have potential therapeutic benefits for treating asthma by downregulating Th2 cytokines and Th17 cell function and inhibiting NF-?B activation in lung tissue. PMID:25180025
HDAC inhibitors mitigate ischemia-induced oligodendrocyte damage: potential roles of oligodendrogenesis, VEGF, and anti-inflammation
Kim, Hyeon Ju; Chuang, De-Maw
White matter injury is an important component of stroke pathology, but its pathophysiology and potential treatment remain relatively elusive and underexplored. We previously reported that after permanent middle cerebral artery occlusion (pMCAO), sodium butyrate (SB) and trichostatin A (TSA) induced neurogenesis via histone deacetylase (HDAC) inhibition in multiple ischemic brain regions in rats; these effects-which depended on activation of brain-derived neurotrophic factor (BDNF)-TrkB signaling-contributed to behavioral improvement. The present study found that SB or TSA robustly protected against ischemia-induced loss of oligodendrocytes detected by confocal microscopy of myelin basic protein (MBP) immunostaining in the ipsilateral subventricular zone (SVZ), striatum, corpus callosum, and frontal cortex seven days post-pMCAO. Co-localization of 5-bromo-2Æ-deoxyuridine (BrdU)+ and MBP+ cells after SB treatment suggested the occurrence of oligodendrogenesis. SB also strongly upregulated vascular endothelial growth factor (VEGF), which plays a major role in neurogenesis, angiogenesis, and functional recovery after stroke. These SB-induced effects were markedly suppressed by blocking the TrkB signaling pathway with K252a. pMCAO-induced activation of microglia (OX42+) and macrophages/monocytes (ED1+)-which has been linked to white matter injury-was robustly suppressed by SB in a K252a-sensitive manner. In addition, SB treatment largely blocked caspase-3+ and OX42+ cells in ipsilateral brain regions. Our results suggest that HDAC inhibitor-mediated protection against ischemia-induced oligodendrocyte loss may involve multiple mechanisms including oligodendrogenesis, VEGF upregulation, anti-inflammation, and caspase-3 downregulation. Taken together, the results suggest that post-insult treatment with HDAC inhibitors is a rational strategy to mitigate white matter injury following ischemic stroke. PMID:24936215
Brainstem brain-derived neurotrophic factor signaling is required for histone deacetylase inhibitor-induced pain relief.
Tao, Wenjuan; Chen, Quan; Wang, Lu; Zhou, Wenjie; Wang, Yunping; Zhang, Zhi
Our previous study demonstrated that persistent pain can epigenetically suppress the transcription of Gad2 [encoding glutamic acid decarboxylase 65 (GAD65)] and consequently impair the inhibitory function of GABAergic synapses in central pain-modulating neurons. This contributes to the development of persistent pain sensitization. Histone deacetylase (HDAC) inhibitors increased GAD65 activity considerably, restored GABA synaptic function, and rendered sensitized pain behavior less pronounced. However, the molecular mechanisms by which HDAC regulates GABAergic transmission through GAD65 under pain conditions are unknown. This work showed that HDAC inhibitor-induced increases in colocalization of GAD65 and synaptic protein synapsin I on the presynaptic axon terminals of the nucleus raphe magnus (NRM) were blocked by a TrkB receptor antagonist K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], indicating that BDNF-TrkB signaling may be required in GAD65 modulation of GABA synaptic function. At the brain-derived neurotrophic factor (BDNF) promoter, HDAC inhibitors induced significant increases in H3 hyperacetylation, consistent with the increase in BDNF mRNA and total proteins. Although exogenous BDNF facilitated GABA miniature inhibitory postsynaptic currents and GAD65 accumulation in NRM neuronal synapses in normal rats, it failed to do so in animals subjected to persistent inflammation. In addition, blockade of the TrkB receptor with K252a has no effect on miniature inhibitory postsynaptic currents and synaptic GAD65 accumulation under normal conditions. In addition, the analgesic effects of HDAC inhibitors on behavior were blocked by NRM infusion of K252a. These findings suggest that BDNF-TrkB signaling is required for drugs that reverse the epigenetic effects of chronic pain at the gene level, such as HDAC inhibitors. PMID:25852071
Computational design, chemical synthesis, and biological evaluation of a novel ERK inhibitor (BL-EI001) with apoptosis-inducing mechanisms in breast cancer
Liu, Bo; Fu, Leilei; Zhang, Cui; Zhang, Lan; Zhang, Yonghui; Ouyang, Liang; He, Gu; Huang, Jian
Extracellular signal-regulated kinase1/2 (ERK1/2) plays a crucial role in the resistance of apoptosis in carcinogenesis; however, its targeted small-molecule inhibitors still remain to be discovered. Thus, in this study, we computationally and experimentally screened a series of small-molecule inhibitors targeting ERK toward different types of human breast cancer cells. Subsequently, we synthesized some candidate ERK inhibitors, identified a novel ERK inhibitor (BL-EI001) with anti-proliferative activities, and analyzed the BL-EI001/ERK complex. Moreover, we found that BL-EI001 induced breast cancer cell apoptosis via mitochondrial pathway but independent on Ras/Raf/MEK pathway. In addition, we carried out proteomics analyses for exploring some possible BL-EI001-induced apoptotic pathways, and further found that BL-EI001-induced apoptosis affected ERK phosphorylation in breast cancer. Further, we found that BL-EI001 bear anti-tumor activities without remarkable toxicities, and also induced mitochondrial apoptosis by targeting ERK in vivo. Taken together, these results demonstrate that in silico design and experimental discovery of a synthesized small-molecule ERK inhibitor (BL-EI001) as a potential novel apoptosis-inducing drug in the treatment of breast cancer. PMID:25742792
Involvement of the strychnine-sensitive glycine receptor in the anxiolytic effects of GlyT1 inhibitors on maternal separation-induced ultrasonic vocalization in rat pups.
Komatsu, Hiroko; Furuya, Yoshiaki; Sawada, Kohei; Asada, Takashi
Several studies have shown that glycine transporter 1 (GlyT1) inhibitors have anxiolytic actions. There are two types of glycine receptor: the strychnine-sensitive glycine receptor (GlyA) and the strychnine-insensitive glycine receptor (GlyB); however, which receptor is the main contributor to the anxiolytic actions of GlyT1 inhibitors is yet to be determined. Here, we clarified which glycine receptor is the main contributor to the anxiolytic effects of GlyT1 inhibitors by using maternal separation-induced ultrasonic vocalization (USV) by rat pups as an index of anxiety. We confirmed that administration of the benzodiazepine diazepam or the selective serotonin reuptake inhibitor escitaloplam, which are both clinically proven anxiolytics, or the GlyT1 inhibitor SSR504734 (2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide), decreases USV in rat pups. In addition, we showed that another GlyT1 inhibitor, ALX5407 ((R)-N-[3-(4'-fluorophenyl)-3(4'-phenylphenoxy)propyl]sarcosine) also decreases USV in rat pups. SSR504734- or ALX5407-induced decreases in USV were dose-dependently reversed by administration of the GlyA antagonist strychnine, whereas the diazepam- or escitalopram-induced decreases in USV were not. Furthermore, GlyT1-induced decreases in USV were not reversed by administration of the GlyB antagonist L-687,414. Together, these results suggest that GlyA activation is the main contributor to the anxiolytic actions of GlyT1 inhibitors and that the anxiolytic actions of diazepam and escitalopram cannot be attributed to GlyA activation. Our findings provide new insights into the importance of the activation of GlyA in the anxiolytic effects of GlyT1 inhibitors. PMID:25435080
Inhibitor of Growth 4 Induces Growth Suppression and Apoptosis in Glioma U87MG
XiaoMei Li; LiMin Cai; Hui Chen; QingYuan Zhang; ShuJun Zhang; YanHua Wang; YanYan Dong; Hui Cheng; JiPing Qi
Objective: Inhibitor of growth (ING) 4 is a member of the ING family proteins. It has been shown to play an important role in cell cycle, transcription and oncogenesis, but the molecular mechanism of ING4 on tumor growth inhibition has not yet been elucidated. The goal of this study is to investigate the inhibitory effects of ING4 on gliomas and
Fugu (Takifugu rubripes) Sexual Differentiation: CYP19 Regulation and Aromatase Inhibitor Induced Testicular Development
H. Rashid; H. Kitano; K. Hoon Lee; S. Nii; T. Shigematsu; K. Kadomura; A. Yamaguchi; M. Matsuyama
In order to assess the involvement of aromatase CYP19 isoforms and endogenous sex steroids in gonadal sex differentiation and development of the Japanese fugu (Takifugu rubripes), an aromatase inhibitor (AI, fadrozole) was administered to developing fishes from the æfirst feedingÆ till the 100th day after hatching. It was observed that ovarian cavity formation was inhibited by fadrozole at doses of
The effect of marimastat, a metalloprotease inhibitor, on allergen-induced asthmatic hyper-reactivity
Bruce, Colleen [Department of Respiratory Medicine, Faculty of Medicine, University of New South Wales, Prince of Wales Hospital, Randwick, NSW 2031 (Australia); Thomas, Paul S. [Department of Respiratory Medicine, Faculty of Medicine, University of New South Wales, Prince of Wales Hospital, Randwick, NSW 2031 (Australia)]. E-mail: email@example.com
This pilot study was designed to assess whether a synthetic matrix metalloproteinase (MMP) inhibitor has anti-inflammatory properties in mild asthma. Tumor necrosis factor alpha (TNF{alpha}) has been shown to be an important cytokine in the pathogenesis of allergic airway inflammatory responses, and its release can be inhibited by MMP inhibitors. Twelve atopic asthmatic subjects received the MMP inhibitor marimastat (5 mg) or placebo, twice daily for 3 weeks, separated by a 6-week washout period in a randomized, double-blind, cross-over manner. All subjects underwent an allergen inhalation provocation test to Dermatophagoides pteronyssinus before and after each study phase. Spirometry, exhaled NO (eNO) levels, differential sputum cell counts, an asthma symptom questionnaire, peak flow, and {beta}{sub 2}-agonist usage were measured. Nine subjects completed the study, and, when compared with placebo, marimastat reduced bronchial hyper-responsiveness to inhaled allergen in these subjects from an allergen PC{sub 20} of 22.2 AU/ml (95%CI 11.7-32.6) to 17.0 AU/ml (95%CI 7.6-26.4, P = 0.02). The marimastat phase showed a nonsignificant fall in sputum inflammatory cells. Marimastat did not modify eNO, FEV{sub 1}, asthma symptoms, or albuterol usage. In conclusion, airway responsiveness to allergen may be modified by a MMP inhibitor, perhaps via TNF{alpha} playing a role in airway inflammation and remodeling.
Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors
Granville, David J.; Tashakkor, Babak; Takeuchi, Cindy; Gustafsson, ┼sa B.; Huang, Chengqun; Sayen, M. Richard; Wentworth, Paul; Yeager, Mark; Gottlieb, Roberta A.
Ischemia and reperfusion both contribute to tissue damage after myocardial infarction. Although many drugs have been shown to reduce infarct size when administered before ischemia, few have been shown to be effective when administered at reperfusion. Moreover, although it is generally accepted that a burst of reactive oxygen species (ROS) occurs at the onset of reperfusion and contributes to tissue damage, the source of ROS and the mechanism of injury is unclear. We now report the finding that chloramphenicol administered at reperfusion reduced infarct size by 60% in a Langendorff isolated perfused rat heart model, and that ROS production was also substantially reduced. Chloramphenicol is an inhibitor of mitochondrial protein synthesis and is also an inhibitor of a subset of cytochrome P450 monooxygenases (CYPs). We could not detect any effect on mitochondrial encoded proteins or mitochondrial respiration in chloramphenicol-perfused hearts, and hypothesized that the effect was caused by inhibition of CYPs. We tested additional CYP inhibitors and found that cimetidine and sulfaphenazole, two CYP inhibitors that have no effect on mitochondrial protein synthesis, were also able to reduce creatine kinase release and infarct size in the Langendorff model. We also showed that chloramphenicol reduced infarct size in an open chest rabbit model of regional ischemia. Taken together, these findings implicate CYPs in myocardial ischemia/reperfusion injury. PMID:14734800
Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli
Daniel J Dwyer; Michael A Kohanski; Boris Hayete; James J Collins
Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, double-stranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and
Proteasome inhibitor bortezomib impairs both myelofibrosis and osteosclerosis induced by high thrombopoietin levels in mice
Orianne Wagner-Ballon; Didier F. Pisani; Thomas Gastinne; Micheline Tulliez; Ronan Chaligne; Catherine Lacout; Jean-Luc Villeval; Patrick Gonin; William Vainchenker; Stephane Giraudier
Primary myelofibrosis (PMF) is the most serious myeloproliferative disorder, char- acterized by clonal myeloproliferation as- sociated with cytokine-mediated bone marrow stromal reaction including fibro- sis and osteosclerosis. Current drug therapy remains mainly palliative. Be- cause the NF-B pathway is implicated in the abnormal release of cytokines in PMF, the proteasome inhibitor bortezomib might be a potential therapy. To test its
The role of ATF-2 family transcription factors in adipocyte differentiation: antiobesity effects of p38 inhibitors.
Maekawa, Toshio; Jin, Wanzhu; Ishii, Shunsuke
ATF-2 is a member of the ATF/CREB family of transcription factors and is activated by stress-activated protein kinases, such as p38. To analyze the physiological role of ATF-2 family transcription factors, we have generated mice with mutations in Atf-2 and Cre-bpa, an Atf-2-related gene. The trans-heterozygotes of both mutants were lean and had reduced white adipose tissue (WAT). ATF-2 and CRE-BPa were required for bone morphogenetic protein 2 (BMP-2)-and p38-dependent induction of peroxisome proliferator-activated receptor gamma2 (PPARgamma2), a key transcription factor mediating adipocyte differentiation. Since stored fat supplies have been recognized as a possible target for antiobesity treatments, we tested whether inhibition of the p38-ATF-2 pathway suppresses adipocyte differentiation and leads to reduced WAT by treating mice with a p38 inhibitor for long periods of time. High-fat diet (HFD)-induced obesity was significantly reduced in mice fed the p38 inhibitor. Furthermore, the p38 inhibitor alleviated HFD-induced insulin resistance. In p38 inhibitor-treated mice, macrophage infiltration into WAT was reduced and the tumor necrosis factor alpha (TNF-alpha) levels were lower than control mice. Thus, p38 inhibitors may provide a novel antiobesity treatment. PMID:19948881
The NF?B inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype.
Zhang, Li; Ren, Xingcong; Cheng, Yan; Liu, Xiuping; Allen, Joshua E; Zhang, Yi; Yuan, Yunsheng; Huang, Siu-Yuan; Yang, Weiwei; Berg, Arthur; Webb, Becky S; Connor, James; Liu, Chang-Gong; Lu, Zhimin; El-Deiry, Wafik S; Yang, Jin-Ming
The malignant phenotype of glioblastoma multiforme (GBM) is believed to be largely driven by glioma stem-like cells (GSCs), and targeting GSCs is now considered a promising new approach to treatment of this devastating disease. Here, we show that SN50, a cell-permeable peptide inhibitor of NF?B, induced robust differentiation of human GSCs, causing loss of their oncogenic potential. We observed that following treatment of GSCs with SN50, their differentiated progeny cells showed significant decreases in their capability to form neuro-spheres and to invade in vitro and a reduction in their tumorigenicity in mouse xenograft models, but had increased sensitivity to the chemotherapeutic drug temozolomide and to radiation treatment. These results suggest that blocking the NF?B pathway may be explored as a useful mean to induce differentiation of GSCs, and provide another supportive evidence for the promise of differentiation therapy in treatment of malignant brain tumors. PMID:24557012
The NF?B inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype
Zhang, Li; Ren, Xingcong; Cheng, Yan; Liu, Xiuping; Allen, Joshua E; Zhang, Yi; Yuan, Yunsheng; Huang, Siu-Yuan; Yang, Weiwei; Berg, Arthur; Webb, Becky S; Connor, James; Liu, Chang-gong; Lu, Zhimin; El-Deiry, Wafik S; Yang, Jin-Ming
The malignant phenotype of glioblastoma multiforme (GBM) is believed to be largely driven by glioma stem-like cells (GSCs), and targeting GSCs is now considered a promising new approach to treatment of this devastating disease. Here, we show that SN50, a cell-permeable peptide inhibitor of NF?B, induced robust differentiation of human GSCs, causing loss of their oncogenic potential. We observed that following treatment of GSCs with SN50, their differentiated progeny cells showed significant decreases in their capability to form neuro-spheres and to invade in vitro and a reduction in their tumorigenicity in mouse xenograft models, but had increased sensitivity to the chemotherapeutic drug temozolomide and to radiation treatment. These results suggest that blocking the NF?B pathway may be explored as a useful mean to induce differentiation of GSCs, and provide another supportive evidence for the promise of differentiation therapy in treatment of malignant brain tumors. PMID:24557012
Artesunate-enhanced apoptosis of human high-risk myelodysplastic cells induced by the DNA methyltransferase inhibitor decitabine
The present study aimed to investigate whether artesunate (ART) could enhance the rate of apoptosis induced by decitabine (DAC) in the high-risk myelodysplastic syndrome (MDS) SKM-1 cell line, and examine the potential underlying mechanisms. The cytotoxicity and effect upon the apoptosis of ART and DAC in the SKM-1 cells was detected using the cell counting kit-8 assay and flow cytometry, respectively. The SKM-1 protein expression levels of activated caspase-3, ?9 and ?8, cleaved poly(ADP-ribose) polymerase and apoptosis-inducing factor (AIF) were measured by western blotting. The laser confocal microscope analysis revealed AIF transfer to the nucleus. The growth inhibition and apoptosis rates of the ART- and DAC-treated SKM-1 cells were significantly increased compared with those of the single agent-treated SKM-1 cells (P<0.05). In addition, ART and DAC induced caspase-dependent apoptosis, while ART, but not DAC, induced caspase-independent apoptosis via AIF transfer from the mitochondria to the nucleus. In addition, ART-DAC-induced cell death was not attenuated by the caspase-3/7 inhibitor, Ac-DEVD-CHO. The results of the present study suggested that the ART-DAC combination exhibited increased effectiveness compared with the single-agent therapy, in vitro. The ART-DAC combined therapy not only activated a caspase-dependent apoptotic pathway, but also a caspase-independent mitochondrial pathway. PMID:26137088
The Proteasome Inhibitor Carfilzomib Suppresses Parathyroid Hormone-induced Osteoclastogenesis through a RANKL-mediated Signaling Pathway.
Yang, Yanmei; Blair, Harry C; Shapiro, Irving M; Wang, Bin
Parathyroid hormone (PTH) induces osteoclast formation and activity by increasing the ratio of RANKL/OPG in osteoblasts. The proteasome inhibitor carfilzomib (CFZ) has been used as an effective therapy for multiple myeloma via the inhibition of pathologic bone destruction. However, the effect of combination of PTH and CFZ on osteoclastogenesis is unknown. We now report that CFZ inhibits PTH-induced RANKL expression and secretion without affecting PTH inhibition of OPG expression, and it does so by blocking HDAC4 proteasomal degradation in osteoblasts. Furthermore, we used different types of culture systems, including co-culture, indirect co-culture, and transactivation, to assess the effect of CFZ on PTH action to induce osteoclastogenesis. Our results demonstrated that CFZ blocks PTH-induced osteoclast formation and bone resorption by its additional effect to inhibit RANKL-mediated I?B degradation and NF-?B activation in osteoclasts. This study showed for the first time that CFZ targets both osteoblasts and osteoclasts to suppress PTH-induced osteoclast differentiation and bone resorption. These findings warrant further investigation of this novel combination in animal models of osteoporosis and in patients. PMID:25979341
Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly
Matsuzono, Yuji; Kinoshita, Naohiko; Tamura, Shigehiko; Shimozawa, Nobuyuki; Hamasaki, Maho; Ghaedi, Kamran; Wanders, Ronald J. A.; Suzuki, Yasuyuki; Kondo, Naomi; Fujiki, Yukio
At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein. PMID:10051604
Induced opening of influenza virus neuraminidase N2 150-loop suggests an important role in inhibitor binding
Wu, Yan; Qin, Guangrong; Gao, Feng; Liu, Yue; Vavricka, Christopher J.; Qi, Jianxun; Jiang, Hualiang; Yu, Kunqian; Gao, George F.
The recently discovered 150-cavity (formed by loop residues 147¢152, N2 numbering) adjacent to the enzymatic active site of group 1 influenza A neuraminidase (NA) has introduced a novel target for the design of next-generation NA inhibitors. However, only group 1 NAs, with the exception of the 2009 pandemic H1N1 NA, possess a 150-cavity, and no 150-cavity has been observed in group 2 NAs. The role of the 150-cavity played in enzymatic activity and inhibitor binding is not well understood. Here, we demonstrate for the first time that oseltamivir carboxylate can induce opening of the rigid closed N2 150-loop and provide a novel mechanism for 150-loop movement using molecular dynamics simulations. Our results provide the structural and biophysical basis of the open form of 150-loop and illustrates that the inherent flexibility and the ligand induced flexibility of the 150-loop should be taken into consideration for future drug design. PMID:23531861
A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.
Burkholder, Kristin M; Perry, Jeffrey W; Wobus, Christiane E; Donato, Nicholas J; Showalter, Hollis D; Kapuria, Vaibhav; O'Riordan, Mary X D
Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking. PMID:21911458
A Small Molecule Deubiquitinase Inhibitor Increases Localization of Inducible Nitric Oxide Synthase to the Macrophage Phagosome and Enhances Bacterial Killing?å
Burkholder, Kristin M.; Perry, Jeffrey W.; Wobus, Christiane E.; Donato, Nicholas J.; Showalter, Hollis D.; Kapuria, Vaibhav; O'Riordan, Mary X. D.
Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking. PMID:21911458
The Hsp90 inhibitor SNX-2112 induces apoptosis of human hepatocellular carcinoma cells: the role of ER stress.
Wang, Xiao; Wang, Shaoxiang; Liu, Yuting; Ding, Weichao; Zheng, Kai; Xiang, Yangfei; Liu, Kaisheng; Wang, Dongmei; Zeng, Yaoying; Xia, Min; Yang, Depo; Wang, Yifei
Heat shock protein 90 (Hsp90) has been predicted to be involved in hepatocellular carcinoma (HCC) therapy; however, the mechanisms of action remain elusive. SNX-2112 is an Hsp90 inhibitor showing broad antitumor activity. Here we aim to determine the role of the endoplasmic reticulum (ER) stress in SNX-2112-induced apoptosis in HCC cells. In general, three HCC cells (i.e., HepG2, Huh7, and SK-Hep1) were used in our experiments. The cell viability was determined by the CCK-8 assay. The apoptosis was analyzed using flow cytometry, laser scanning confocal microscopy (LSM) and Western blotting. The efficacy and mechanisms of action of SNX-2112 were also evaluated in a mouse xenograft model. We found that SNX-2112 showed stronger inhibition on cell growth than 17-AAG, a classical Hsp90 inhibitor. SNX-2112 treatment led to the caspase-dependent apoptosis. Interestingly, SNX-2112 decreased the expression levels of the ER chaperone proteins calnexin and immunoglobulin binding protein (BiP). It also inhibited all three ER stress sensors, namely, inositol-requiring gene 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF-6) in vitro and/or in vivo. However, the ER stress inducer tunicamycin strongly enhanced SNX-2112-induced apoptosis, whereas the IRE1 knockdown did not. Taken together, we for the first time indicated the possible apoptotic pathways of SNX-2112 in HCC cells, raising the possibility that the induction of ER stress might be favorable for SNX-2112-induced apoptosis. PMID:24582562
Cancerous inhibitor of protein phosphatase 2A mediates bortezomib-induced autophagy in hepatocellular carcinoma independent of proteasome.
Yu, Hui-Chuan; Hou, Duen-Ren; Liu, Chun-Yu; Lin, Chen-Si; Shiau, Chung-Wai; Cheng, Ann-Lii; Chen, Kuen-Feng
Previously, we reported that cancerous inhibitor of protein phosphatase 2A (CIP2A) mediates the apoptotic effect of bortezomib in hepatocellular carcinoma (HCC). Here, we report a proteasome-independent mechanism by which bortezomib induces autophagy in HCC. Our data indicate that bortezomib activated autophagy in a dose- and time- dependent manner in HCC cell lines including Huh-7, Sk-Hep1, and Hep3B. Bortezomib downregulated CIP2A, phospho-Akt (P-Akt) and phospho-4EBP1 (P-4EBP1) in a dose- and time-dependent manner in all tested HCC cells. Ectopic expression of CIP2A abolished the effect of bortezomib on autophagy. Co-treatment of bortezomib and calyculin A, a PP2A inhibitor, reduced the effect of bortezomib on P-Akt, P-4EBP1, and autophagy. Increased phosphorylation of either Akt or 4EBP1 by ectopic overexpression protected cells from bortezomib-induced autophagy. Furthermore, we examined the effect of ?Btz, a bortezomib derivative that closely resembles bortezomib structurally but has no proteasome activity, in HCC. Interestingly, ?Btz demonstrated similar effects to bortezomib on autophagy, CIP2A, P-Akt and P-4EBP1, suggesting that the effect of bortezomib on autophagy is independent of proteasome inhibition. Moreover, our in vivo data showed that both bortezomib and ?Btz inhibited tumor growth, downregulated CIP2A, P-Akt and induced autophagy in Huh-7 tumors. In conclusion, bortezomib induces autophagy in HCC through a CIP2A-PP2A-Akt-4EBP1 pathway. PMID:23383345
Heat Shock Protein 90 Inhibitors Prevent LPS-Induced Endothelial Barrier Dysfunction by Disrupting RhoA Signaling
Joshi, Atul D.; Dimitropoulou, Christiana; Thangjam, Gagan; Snead, Connie; Feldman, Sara; Barabutis, Nektarios; Fulton, David; Hou, Yali; Kumar, Sanjiv; Patel, Vijay; Gorshkov, Boris; Verin, Alexander D.; Black, Stephen M.
Permeability of the endothelial monolayer is increased when exposed to the bacterial endotoxin LPS. Our previous studies have shown that heat shock protein (Hsp) 90 inhibitors protect and restore LPS-mediated hyperpermeability in bovine pulmonary arterial endothelial cells. In this study, we assessed the effect of Hsp90 inhibition against LPS-mediated hyperpermeability in cultured human lung microvascular endothelial cells (HLMVECs) and delineated the underlying molecular mechanisms. We demonstrate that Hsp90 inhibition is critical in the early phase, to prevent LPS-mediated hyperpermeability, and also in the later phase, to restore LPS-mediated hyperpermeability in HLMVECs. Because RhoA is a well known mediator of endothelial hyperpermeability, we investigated the effect of Hsp90 inhibition on LPS-mediated RhoA signaling. RhoA nitration and activity were increased by LPS in HLMVECs and suppressed when pretreated with the Hsp90 inhibitor, 17-allylamino-17 demethoxy-geldanamycin (17-AAG). In addition, inhibition of Rho kinase, a downstream effector of RhoA, protected HLMVECs from LPS-mediated hyperpermeability and abolished LPS-induced myosin light chain (MLC) phosphorylation, a target of Rho kinase. In agreement with these findings, 17-AAG or dominant-negative RhoA attenuated LPS-induced MLC phosphorylation. MLC phosphorylation induced by constitutively active RhoA was also suppressed by 17-AAG, suggesting a role for Hsp90 downstream of RhoA. Inhibition of Src family kinases also suppressed RhoA activity and MLC phosphorylation. Together, these data indicate that Hsp90 inhibition prevents and repairs LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling. PMID:23972231
Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. Angiogenesis inhibitors block vascular endothelial growth factor-induced down-regulation of caveolin-1.
Liu, J; Razani, B; Tang, S; Terman, B I; Ware, J A; Lisanti, M P
Angiogenesis is the process by which new blood vessels are formed via proliferation of vascular endothelial cells. A variety of angiogenesis inhibitors that antagonize the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects remains largely unknown. Caveolin-1 and -2 are known to be highly expressed in vascular endothelial cells both in vitro and in vivo. Here, we examine the potential role of caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF, bFGF, or hepatocyte growth factor/scatter factor), resulted in a dramatic reduction in the expression of caveolin-1. This down-regulation event was selective for caveolin-1, as caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of caveolin-1 expression also resulted in the morphological loss of cell surface caveolae organelles as seen by transmission electron microscopy. A variety of well characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-beta, and thalidomide) effectively blocked VEGF-induced down-regulation of caveolin-1 as seen by immunoblotting and immunofluorescence microscopy. However, treatment with angiogenesis inhibitors alone did not significantly affect the expression of caveolin-1. PD98059, a specific inhibitor of mitogen-activated protein kinase and a known angiogenesis inhibitor, also blocked the observed VEGF-induced down-regulation of caveolin-1. Furthermore, we show that caveolin-1 can function as a negative regulator of VEGF-R (KDR) signal transduction in vivo. Thus, down-regulation of caveolin-1 may be an important step along the pathway toward endothelial cell proliferation. PMID:10336480
Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus
Amaia Orbea; Konstantin Beier; Alfred V÷lkl; H. Dariush Fahimi; Miren P. Cajaraville
Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity
Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids
Houten, Sander M.; Denis, Simone; Argmann, Carmen A.; Jia, Yuzhi; Ferdinandusse, Sacha; Reddy, Janardan K.; Wanders, Ronald J. A.
L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid ?-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor ? (PPAR?) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPAR?, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ?-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting. PMID:22534643
Thiazolidinedione Class of Peroxisome Proliferator-Activated Receptor Ā Agonists Prevents Neuronal Damage, Motor Dysfunction, Myelin Loss, Neuropathic Pain, and Inflammation after Spinal Cord Injury in Adult Rats
Seung-Won Park; Jae-Hyuk Yi; Guruwattan Miranpuri; Irawan Satriotomo; Kellie Bowen; Daniel K. Resnick; Raghu Vemuganti
Thiazolidinediones (TZDs) are potent synthetic agonists of the ligand-activated transcription factor peroxisome proliferator- activated receptor- (PPAR). TZDs were shown to induce neuroprotection after cerebral ischemia by blocking inflamma- tion. As spinal cord injury (SCI) induces massive inflammation that precipitates secondary neuronal death, we currently ana- lyzed the therapeutic efficacy of TZDs pioglitazone and rosigli- tazone after SCI in adult rats.
Inhibition of autophagy promotes cell apoptosis induced by the proteasome inhibitor MG-132 in human esophageal squamous cell carcinoma EC9706 cells
Lysosome-dependent macroautophagy, also termed autophagy, and the ubiquitin-proteasome system and are the primary intracellular pathways involved in protein degradation. Previous studies have demonstrated that proteasome inhibitors are able to inhibit tumor growth and activate autophagy. The present study investigated the effect of the proteasome inhibitor MG-132 on cellular proliferation using a cell counting kit 8 assay, and the effect of the agent on apoptosis and autophagy was assessed using flow cytometry and monodansylcadaverine, respectively. Western blot analysis was used to investigate protein changes during the course of treatment. It was revealed that MG-132 inhibited cell proliferation, activated autophagy and induced cell death in EC9706 cells. Autophagy was activated through the class III PI3K pathway, and the expression of the Beclin-1 protein was determined to be significantly upregulated. However, the autophagy inhibitor 3-methyladenine (3-MA) inhibited the expression of the autophagy-associated protein Beclin-1 and reduced the accumulation of autophagic vacuoles induced by MG-132. MG-132-induced apoptosis was enhanced by the autophagy inhibitor 3-MA, which may be a result of caspase-3 activation in the EC9706 cells. These findings suggest that inhibition of the proteasome can induce autophagy in human ESCC cells, and also increase cell death. This indicates that proteasome inhibitors may be potential novel anti-cancer agents for the adjuvant treatment of esophageal squamous cell carcinoma.
Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion.
Kovacs, Krisztina; Toth, Ambrus; Deres, Peter; Kalai, Tamas; Hideg, Kalman; Gallyas, Ferenc; Sumegi, Balazs
Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion. PMID:16337154
Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and Osteoclast-Mediated Bone Resorption
Ren, Zhong-Yuan; Machuca-Gayet, Irma; Domenget, Chantal; Buchet, Rene; Wu, Yuqing; Jurdic, Pierre; Mebarek, Saida
Aim The cysteine protease cathepsin K (CatK), abundantly expressed in osteoclasts, is responsible for the degradation of bone matrix proteins, including collagen type 1. Thus, CatK is an attractive target for new anti-resorptive osteoporosis therapies, but the wider effects of CatK inhibitors on bone cells also need to be evaluated to assess their effects on bone. Therefore, we selected, among a series of synthetized isothiosemicarbazides, two molecules which are highly selective CatK inhibitors (CKIs) to test their effects on osteoblasts and osteoclasts. Research Design and Methods Cell viability upon treatment of CKIs were was assayed on human osteoblast-like Saos-2, mouse monocyte cell line RAW 264.7 and mature mouse osteoclasts differentiated from bone marrow. Osteoblast-induced mineralization in Saos-2 cells and in mouse primary osteoblasts from calvaria, with or without CKIs,; were was monitored by Alizarin Red staining and alkaline phosphatase activity, while osteoclast-induced bone resorption was performed on bovine slices. Results Treatments with two CKIs, CKI-8 and CKI-13 in human osteoblast-like Saos-2, murine RAW 264.7 macrophages stimulated with RANKL and mouse osteoclasts differentiated from bone marrow stimulated with RANKL and MCSF were found not to be toxic at doses of up to 100 nM. As probed by Alizarin Red staining, CKI-8 did not inhibit osteoblast-induced mineralization in mouse primary osteoblasts as well as in osteoblast-like Saos-2 cells. However, CKI-13 led to a reduction in mineralization of around 40% at 10¢100 nM concentrations in osteoblast-like Saos-2 cells while it did not in primary cells. After a 48-hour incubation, both CKI-8 and CKI-13 decreased bone resorption on bovine bone slices. CKI-13 was more efficient than the commercial inhibitor E-64 in inhibiting bone resorption induced by osteoclasts on bovine bone slices. Both CKI-8 and CKI-13 created smaller bone resorption pits on bovine bone slices, suggesting that the mobility of osteoclasts was slowed down by the addition of CKI-8 and CKI-13. Conclusion CKI-8 and CKI-13 screened here show promise as antiresorptive osteoporosis therapeutics but some off target effects on osteoblasts were found with CKI-13. PMID:26168340
Fungal siderophore biosynthesis is partially localized in peroxisomes
Gr³ndlinger, Mario; Yasmin, Sabiha; Lechner, Beatrix Elisabeth; Geley, Stephan; Schrettl, Markus; Hynes, Michael; Haas, Hubertus
Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein-tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl-CoA ligase), SidH (mevalonyl-CoA hydratase) and SidF (anhydromevalonyl-CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH-targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen-type siderophore-producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes. PMID:23617799
Identification of proteasome gene regulation in a rat model for HIV protease inhibitor-induced hyperlipidemia
Jeffrey F. Waring; Rita Ciurlionis; Kennan Marsh; Larry L. Klein; David A. DeGoey; John T. Randolph; Brian Spear; Dale J. Kempf
Patients treated with highly active antiretroviral therapy may develop metabolic side effects such as hyperlipidemia, insulin\\u000a resistance, lipoatrophy and lactic acidosis. The pathophysiology of these metabolic abnormalities is unknown, although some,\\u000a e.g., lactic acidosis and lipoatrophy, are more associated with nucleoside use while protease inhibitors (PIs) have been shown\\u000a to contribute to hyperlipidemia and insulin resistance. Identifying new PIs that
Dupuytren's disease and frozen shoulder induced by treatment with a matrix metalloproteinase inhibitor.
Hutchinson, J W; Tierney, G M; Parsons, S L; Davis, T R
In a series of 12 patients with inoperable gastric carcinoma who had treatment with a synthetic matrix metalloproteinase inhibitor (Marimastat) for more than one month, six developed a frozen shoulder or a condition resembling Dupuytren's disease. This suggests that the matrix metalloproteinases, a family of naturally occurring proteinases, may be involved in the pathogenesis of these two conditions. Our observation opens avenues for further research which could lead to local or systemic therapeutic interventions for frozen shoulder and Dupuytren's disease. PMID:9768907
BIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR Mutations
Daniel B Costa; Balßzs Halmos; Amit Kumar; Susan T Schumer; Mark S Huberman; Titus J Boggon; Daniel G Tenen; Susumu Kobayashi
BackgroundEpidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in
Low-dose cholinesterase inhibitors do not induce delayed effects on cerebral blood flow and metabolism
Oscar U. Scremin; Tsung-Ming Shih; Ly Huynh; Margareth Roch; Wei Sun; Dante R. Chialvo; Donald J. Jenden
The acetylcholinesterase (AChE) inhibitors sarin and pyridostigmine bromide (PB) have been proposed as causes of neurobehavioral dysfunction in Persian Gulf War veterans. To test possible delayed effects of these agents, we exposed rats to low (subsymptomatic) levels of sarin (0.5 LD50 s.c. 3 times weekly) and\\/or PB (80 mg\\/L in drinking water) for 3 weeks. Controls received saline s.c. and
Subcutaneous Exposure To Carbamate Acetylcholinesterase Inhibitors Does Not Induce Apoptosis In Mouse Brain
B. S. Mauck; S. J. Paton; J. B. Lucot; R. D. Grubbs
Pyridostigmine bromide (PB), a cholinesterase inhibitor used as a prophylactic against nerve agents, has been reported to produce neuronal apoptosis in rats. The goal of this study was to determine if PB produced similar levels of apoptosis in a C57BL\\/6J mouse model. Since the ability of PB to cross the blood-brain barrier is controversial, we used physostigmine (PHY), a cholinesterase
The HIV protease inhibitor indinavir impairs sterol regulatory element-binding protein-1 intranuclear localization, inhibits preadipocyte differentiation, and induces insulin resistance.
Caron, M; Auclair, M; Vigouroux, C; Glorian, M; Forest, C; Capeau, J
Protease inhibitors used in the treatment of HIV infection have been causally associated with lipodystrophy and insulin resistance and were shown to alter adipocyte differentiation in cultured cells. We aimed to delineate the mechanism by which indinavir impaired adipocyte function. We report that indinavir altered neither the growth nor insulin sensitivity of 3T3-F442A preadipocytes, nor did it alter the initial step of their differentiation, i.e., clonal proliferation. However, adipose conversion was inhibited by indinavir (by 50-60%), as shown by 1) the decrease in the number of newly formed adipocytes; 2) the lower level of the adipogenic protein markers, sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and the insulin receptor (IR); and 3) the lack of SREBP-1 and PPAR-gamma immunoreactivity in the nucleus of most indinavir-treated cells. Partial adipose conversion also correlated with an accumulation of SREBP-1 at the nuclear periphery and an alteration in its electrophoretic mobility. Defective expression and nuclear localization of PPAR-gamma probably resulted from the decreased level of nuclear SREBP-1. Indinavir also rendered 3T3-F442A adipocytes resistant to insulin for mitogen-activated protein kinase activation at a step distal to IR substrate-1 tyrosine phosphorylation. Hence, indinavir impairs differentiation at an early step of adipose conversion probably involving the process controlling SREBP-1 intranuclear localization. PMID:11375339
Sequence selectivity of the cleavage sites induced by topoisomerase I inhibitors: a molecular dynamics study
Siu, Fung-Ming; Pommier, Yves
Topoisomerase IB (Top1) inhibitors, such as camptothecin (CPT), stabilize the Top1-DNA cleavage complex in a DNA sequence-dependent manner. The sequence selectivity of Top1 inhibitors is important for targeting specific genomic sequences of therapeutic value. However, the molecular mechanisms underlying this selectivity remain largely unknown. We performed molecular dynamics simulations to delineate structural, dynamic and energetic features that contribute to the differential sequence selectivity of the Top1 inhibitors. We found the sequence selectivity of CPT to be highly correlated with the drug binding energies, dynamic and structural properties of the linker domain. Chemical insights, gained by per-residue binding energy analysis revealed that the non-polar interaction between CPT and nucleotide at the +1 position of the cleavage site was the major (favorable) contributor to the total binding energy. Mechanistic insights gained by a potential of mean force analysis implicated that the drug dissociation step was associated with the sequence selectivity. Pharmaceutical insights gained by our molecular dynamics analyses explained why LMP-776, an indenoisoquinoline derivative under clinical development at the National Institutes of Health, displays different sequence selectivity when compared with camptothecin and its clinical derivatives. PMID:24021629
Risk factor-induced cardiovascular remodeling and the effects of angiotensin-converting enzyme inhibitors.
Galderisi, Maurizio; de Divitiis, Oreste
Cardiovascular (CV) risk factors, primarily arterial hypertension, modify the structural and functional features of the myocardium and the blood vessels, a process known as CV remodeling. Cardiac remodeling refers to changes in left ventricular (LV) geometry, such as concentric LV geometry and LV hypertrophy (LVH), an independent hallmark of CV risk. Vascular remodeling consists of structural changes of the arterial walls, such as increased intima-media thickness, arterial stiffening, and deteriorating endothelial function. CV remodeling is to a large extent a result of compensatory mechanisms, and its pathophysiology is partially mediated by the activation of the renin-angiotensin-aldosterone system and its primary effector peptide angiotensin II, which together play a key role in the progression of CV disease. Angiotensin-converting enzyme (ACE) inhibitors, a class of drugs used in the treatment of arterial hypertension, appear to be effective in controlling or even reversing CV remodeling, independently of simple blood pressure reduction. Evidence shows that ACE inhibitors counteract CV remodeling by reducing LV mass and regressing LVH, attenuating vascular atherosclerosis and improving vascular compliance. ACE inhibitors possessing a sulfhydryl moiety appear to have additional benefits in increasing nitric-oxide release and improving vascular endothelial function. PMID:18520954
Synthesis, Pharmacological Assessment, and Molecular Modeling of Acetylcholinesterase/Butyrylcholinesterase Inhibitors: Effect against Amyloid-?-Induced Neurotoxicity
The synthesis, molecular modeling, and pharmacological analysis of phenoxyalkylamino-4-phenylnicotinates (2¢7), phenoxyalkoxybenzylidenemalononitriles (12, 13), pyridonepezils (14¢18), and quinolinodonepezils (19¢21) are described. Pyridonepezils 15¢18 were found to be selective and moderately potent regarding the inhibition of hAChE, whereas quinolinodonepezils 19¢21 were found to be poor inhibitors of hAChE. The most potent and selective hAChE inhibitor was ethyl 6-(4-(1-benzylpiperidin-4-yl)butylamino)-5-cyano-2-methyl-4-phenylnicotinate (18) [IC50 (hAChE) = 0.25 ▒ 0.02 ?M]. Pyridonepezils 15¢18 and quinolinodonepezils 20¢21 are more potent selective inhibitors of EeAChE than hAChE. The most potent and selective EeAChE inhibitor was ethyl 6-(2-(1-benzylpiperidin-4-yl)ethylamino)-5-cyano-2-methyl-4-phenylnicotinate (16) [IC50 (EeAChE) = 0.0167 ▒ 0.0002 ?M], which exhibits the same inhibitory potency as donepezil against hAChE. Compounds 2, 7, 13, 17, 18, 35, and 36 significantly prevented the decrease in cell viability caused by A?1¢42. All compounds were effective in preventing the enhancement of AChE activity induced by A?1¢42. Compounds 2¢7 caused a significant reduction whereas pyridonepezils 17 and 18, and compound 16 also showed some activity. The pyrazolo[3,4-b]quinolines 36 and 38 also prevented the upregulation of AChE induced by A?1¢42. Compounds 2, 7, 12, 13, 17, 18, and 36 may act as antagonists of voltage sensitive calcium channels, since they significantly prevented the Ca2+ influx evoked by KCl depolarization. Docking studies show that compounds 16 and 18 adopted different orientations and conformations inside the active-site gorges of hAChE and hBuChE. The structural and energetic features of the 16-AChE and 18-AChE complexes compared to the 16-BuChE and 18-BuChE complexes account for a higher affinity of the ligand toward AChE. The present data indicate that compounds 2, 7, 17, 18, and 36 may represent attractive multipotent molecules for the potential treatment of AlzheimerÆs disease. PMID:23379636
Berberine Inhibits HIV Protease Inhibitor-Induced Inflammatory Response by Modulating ER Stress Signaling Pathways in Murine Macrophages
Zha, Weibin; Liang, Guang; Xiao, Jian; Studer, Elaine J.; Hylemon, Phillip B.; Pandak,, William M.; Wang, Guangji; Li, Xiaokun; Zhou, Huiping
Background HIV protease inhibitor (PI)-induced inflammatory response plays an important role in HIV PI-associated dyslipidemia and cardiovascular complications. This study examined the effect of berberine, a traditional herb medicine, on HIV PI-induced inflammatory response and further investigated the underlying cellular/molecular mechanisms in macrophages. Methodology and Principal Findings Cultured mouse J774A.1 macrophages and primary mouse macrophages were used in this study. The expression of TNF-? and IL-6 were detected by real-time RT-PCR and ELISA. Activations of ER stress and ERK signaling pathways were determined by Western blot analysis. Immunofluorescent staining was used to determine the intracellular localization of RNA binding protein HuR. RNA-pull down assay was used to determine the association of HuR with endogenous TNF-? and IL-6. Berberine significantly inhibited HIV PI-induced TNF-? and IL-6 expression by modulating ER stress signaling pathways and subsequent ERK activation, in turn preventing the accumulation of the RNA binding protein HuR in cytosol and inhibiting the binding of HuR to the 3?-UTRs of TNF-? and IL-6 in macrophages. Conclusions and Significance Inhibition of ER stress represents a key mechanism by which berberine prevents HIV PI-induced inflammatory response. Our findings provide a new insight into the molecular mechanisms of berberine and show the potential application of berberine as a complimentary therapeutic agent for HIV infection. PMID:20161729
Treatment with anti-TNF alpha protects against the neuropathy induced by the proteasome inhibitor bortezomib in a mouse model.
Alķ, Albert; Bruna, Jordi; Morell, Marta; van de Velde, Helgi; Monbaliu, Johan; Navarro, Xavier; Udina, Esther
Bortezomib (BTZ), a proteasome inhibitor, is an effective anti-neoplastic drug used in the treatment of multiple myeloma and mantle cell lymphoma. However, it can induce a reversible peripheral neuropathy that may lead to treatment discontinuation. The mechanism through which BTZ exerts toxic effects in peripheral neurons is not clear. Release of proinflammatory cytokines after nerve damage can induce neurodegeneration, but the effects of BTZ on cytokine expression in neurons are unknown, although BTZ modulates the expression of cytokines, such as TNF-? and IL-6, in tumor cells. The aim of this study was to evaluate the expression and the role of these cytokines on the course of BTZ induced neuropathy in mice. IL-6, TNF-?, TGF-?1 and IL-1? were up-regulated in dorsal root ganglia but TNF-? and IL-6 increased faster and higher. Then, we studied the potential neuroprotective effect of selective antibodies anti-TNF-? and anti-IL-6 on the evolution of the neuropathy. Treatment with anti-TNF-? but not with anti-IL-6 significantly prevented the decrease of sensory nerve action potentials amplitude and the loss of myelinated and unmyelinated fibers. We conclude that monoclonal antibodies directed against TNF-? may be a suitable neuroprotective therapy against the neurotoxicity induced by BTZ. PMID:24406455
Administration of a tropomyosin receptor kinase inhibitor attenuates sarcoma-induced nerve sprouting, neuroma formation and bone cancer pain
Pain often accompanies cancer and most current therapies for treating cancer pain have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomyosin receptor kinase A (TrkA) has become an attractive target for attenuating chronic pain. In the present report, we use a mouse model of bone cancer pain and examine whether oral administration of a selective small molecule Trk inhibitor (ARRY-470, which blocks TrkA, TrkB and TrkC kinase activity at low nm concentrations) has a significant effect on cancer-induced pain behaviors, tumor-induced remodeling of sensory nerve fibers, tumor growth and tumor-induced bone remodeling. Early/sustained (initiated day 6 post cancer cell injection), but not late/acute (initiated day 18 post cancer cell injection) administration of ARRY-470 markedly attenuated bone cancer pain and significantly blocked the ectopic sprouting of sensory nerve fibers and the formation of neuroma-like structures in the tumor bearing bone, but did not have a significant effect on tumor growth or bone remodeling. These data suggest that, like therapies that target the cancer itself, the earlier that the blockade of TrkA occurs, the more effective the control of cancer pain and the tumor-induced remodeling of sensory nerve fibers. Developing targeted therapies that relieve cancer pain without the side effects of current analgesics has the potential to significantly improve the quality of life and functional status of cancer patients. PMID:21138586
Exposure to histone deacetylase inhibitors during Pavlovian conditioning enhances subsequent cue-induced reinstatement of operant behavior
Ploense, Kyle L.; Kerstetter, Kerry A.; Wade, Matthew A.; Woodward, Nicholas C.; Maliniak, Dan; Reyes, Michael; Uchizono, Russell S.; Bredy, Timothy W.; Kippin, Tod E.
Histone deacetylase inhibitors (HDACIs) strengthen memory following fear conditioning and cocaine-induced conditioned place preference. Here, we examined the effects of two non-specific HDACIs, valproic acid (VPA) and sodium butyrate (NaB), on appetitive learning measured via conditioned stimulus (CS)-induced reinstatement of operant responding. Rats were trained to lever press for food reinforcement and then injected with VPA (50¢200 mg/kg, i.p.), NaB (250¢1000 mg/kg, i.p.), or saline vehicle (1.0 ml/kg), 2h before receiving pairings of noncontingent presentation of food pellets preceded by a tone+light cue CS. Rats next underwent extinction of operant responding followed by response-contingent re-exposure to the CS. Rats receiving VPA (100 mg/kg) or NaB (1000 mg/kg) prior to conditioning displayed significantly higher cue-induced reinstatement than did saline controls. Rats that receiving either vehicle or VPA (100 mg/kg) prior to a conditioning session with a randomized relation between presentation of food pellets and the CS failed to show subsequent cue-induced reinstatement with no difference between the two groups. These findings indicate that, under certain contexts, HDACIs strengthen memory formation by specifically increasing the associative strength of the CS, not through an increasing motivation to seek reinforcement. PMID:23604166
1-[4-( N-Benzylamino)phenyl]-3-phenylurea derivatives as a new class of hypoxia-inducible factor-1? inhibitors
Masaharu Uno; Hyun Seung Ban; Hiroyuki Nakamura
A series of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives 2a¢r were synthesized as HIF-1? inhibitors. Among the compounds synthesized, compound 2k was found to be a potent inhibitor against HIF-1? accumulation under hypoxic condition and inhibited the hypoxia-induced HIF-1 transcriptional activity in HEK293 cells (IC50=7.2?M). Furthermore, compound 2k suppressed the hypoxia-induced secretion of VEGF in HeLa cells (IC50=15?M).
Regression of fibrosis and reversal of cirrhosis in rats by galectin inhibitors in thioacetamide-induced liver disease.
Traber, Peter G; Chou, Hsin; Zomer, Eliezer; Hong, Feng; Klyosov, Anatole; Fiel, Maria-Isabel; Friedman, Scott L
Galectin-3 protein is critical to the development of liver fibrosis because galectin-3 null mice have attenuated fibrosis after liver injury. Therefore, we examined the ability of novel complex carbohydrate galectin inhibitors to treat toxin-induced fibrosis and cirrhosis. Fibrosis was induced in rats by intraperitoneal injections with thioacetamide (TAA) and groups were treated with vehicle, GR-MD-02 (galactoarabino-rhamnogalaturonan) or GM-CT-01 (galactomannan). In initial experiments, 4 weeks of treatment with GR-MD-02 following completion of 8 weeks of TAA significantly reduced collagen content by almost 50% based on Sirius red staining. Rats were then exposed to more intense and longer TAA treatment, which included either GR-MD-02 or GM-CT-01 during weeks 8 through 11. TAA rats treated with vehicle developed extensive fibrosis and pathological stage 6 Ishak fibrosis, or cirrhosis. Treatment with either GR-MD-02 (90 mg/kg ip) or GM-CT-01 (180 mg/kg ip) given once weekly during weeks 8-11 led to marked reduction in fibrosis with reduction in portal and septal galectin-3 positive macrophages and reduction in portal pressure. Vehicle-treated animals had cirrhosis whereas in the treated animals the fibrosis stage was significantly reduced, with evidence of resolved or resolving cirrhosis and reduced portal inflammation and ballooning. In this model of toxin-induced liver fibrosis, treatment with two galectin protein inhibitors with different chemical compositions significantly reduced fibrosis, reversed cirrhosis, reduced galectin-3 expressing portal and septal macrophages, and reduced portal pressure. These findings suggest a potential role of these drugs in human liver fibrosis and cirrhosis. PMID:24130706
Regression of Fibrosis and Reversal of Cirrhosis in Rats by Galectin Inhibitors in Thioacetamide-Induced Liver Disease
Traber, Peter G.; Chou, Hsin; Zomer, Eliezer; Hong, Feng; Klyosov, Anatole; Fiel, Maria-Isabel; Friedman, Scott L.
Galectin-3 protein is critical to the development of liver fibrosis because galectin-3 null mice have attenuated fibrosis after liver injury. Therefore, we examined the ability of novel complex carbohydrate galectin inhibitors to treat toxin-induced fibrosis and cirrhosis. Fibrosis was induced in rats by intraperitoneal injections with thioacetamide (TAA) and groups were treated with vehicle, GR-MD-02 (galactoarabino-rhamnogalaturonan) or GM-CT-01 (galactomannan). In initial experiments, 4 weeks of treatment with GR-MD-02 following completion of 8 weeks of TAA significantly reduced collagen content by almost 50% based on Sirius red staining. Rats were then exposed to more intense and longer TAA treatment, which included either GR-MD-02 or GM-CT-01 during weeks 8 through 11. TAA rats treated with vehicle developed extensive fibrosis and pathological stage 6 Ishak fibrosis, or cirrhosis. Treatment with either GR-MD-02 (90 mg/kg ip) or GM-CT-01 (180 mg/kg ip) given once weekly during weeks 8¢11 led to marked reduction in fibrosis with reduction in portal and septal galectin-3 positive macrophages and reduction in portal pressure. Vehicle-treated animals had cirrhosis whereas in the treated animals the fibrosis stage was significantly reduced, with evidence of resolved or resolving cirrhosis and reduced portal inflammation and ballooning. In this model of toxin-induced liver fibrosis, treatment with two galectin protein inhibitors with different chemical compositions significantly reduced fibrosis, reversed cirrhosis, reduced galectin-3 expressing portal and septal macrophages, and reduced portal pressure. These findings suggest a potential role of these drugs in human liver fibrosis and cirrhosis. PMID:24130706
Nanoparticle-Mediated Expression of an Angiogenic Inhibitor Ameliorates Ischemia-Induced Retinal Neovascularization and Diabetes-Induced Retinal Vascular Leakage
Park, Kyoungmin; Chen, Ying; Hu, Yang; Mayo, Aaron S.; Kompella, Uday B.; Longeras, Richard; Ma, Jian-xing
OBJECTIVE The aim of the study is to evaluate the effect of nanoparticle-mediated gene delivery of angiogenic inhibitors on retinal inflammation, vascular leakage, and neovascularization in diabetic retinopathy. RESEARCH DESIGN AND METHODS An expression plasmid of plasminogen kringle 5 (K5), a natural angiogenic inhibitor, was encapsulated with poly(lactide-coglycolide) to form K5 nanoparticles (K5-NP). Expression of K5 was determined by Western blot analysis and immunohistochemistry, and retinal vascular leakage was measured by permeability assay. Retinal neovascularization was evaluated using fluorescein-angiography and counting preretinal vascular cells in rats with oxygen-induced retinopathy. Effects of K5-NP on retinal inflammation were evaluated in streptozotocin-induced diabetic rats by leukostasis assay and Western blot analysis of intracellular adhesion molecule and vascular endothelial growth factor. Possible toxicities of K5-NP were evaluated using histology examination, retinal thickness measurement, and electroretinogram recording. RESULTS K5-NP mediated efficient expression of K5 and specifically inhibited growth of endothelial cells. An intravitreal injection of K5-NP resulted in high-level expression of K5 in the inner retina of rats during the 4 weeks they were analyzed. Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats. K5-NP attenuated vascular endothelial growth factor and intracellular adhesion molecule-1 overexpression and reduced leukostasis and vascular leakage for at least 4 weeks after a single injection in the retina of streptozotocin-induced diabetic rats. No toxicities of K5-NP were detected to retinal structure and function. CONCLUSIONS K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy. PMID:19491211
Studies were conducted to investigate the effect of ozone in prolonging pentobarbital (PEN)-induced sleeping time (S.T.). Since ozone is a common air pollutant, an ozone-induced alteration of mechanisms of drug action could have public health implications. It was shown that a 5-h...
Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells
Daniel, Kenyon G; Chen, Di; Orlu, Shirley; Cui, Qiuzhi Cindy; Miller, Fred R; Dou, Q Ping
Introduction A physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison. Methods Breast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage. Results When in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations similar to those found in patients, when treated with just CQ or PDTC alone, but not TM, undergo proteasome inhibition and apoptosis. Conclusion The feature of breast cancer cells and tissues to accumulate copper can be used as a targeting method for anticancer therapy through treatment with novel compounds such as CQ and PDTC that become active proteasome inhibitors and breast cancer cell killers in the presence of copper. PMID:16280039
Tirucallic acids are novel pleckstrin homology domain-dependent Akt inhibitors inducing apoptosis in prostate cancer cells.
Estrada, Aydee C; Syrovets, Tatiana; Pitterle, Kai; Lunov, Oleg; B³chele, Berthold; Schimana-Pfeifer, Judith; Schmidt, Thomas; Morad, Samy A F; Simmet, Thomas
Activation of the serine/threonine kinase Akt is associated with aggressive clinical behavior of prostate cancer. We found that the human prostate cancer cell lines LNCaP and PC-3 express predominantly Akt1 and Akt2. Selective down-regulation of Akt1, but not Akt2, by short-hairpin RNA reduced the viability of prostate cancer cells. In addition, structurally different Akt inhibitors were cytotoxic for the prostate cancer cells, confirming that the Akt pathway is indispensable for their viability. We have purified the tetracyclic triterpenoids 3-oxo-tirucallic acid, 3-alpha-acetoxy-tirucallic acid, and 3-beta-acetoxy-tirucallic acid from the oleogum resin of Boswellia carterii to chemical homogeneity. The acetoxy-derivatives in particular potently inhibited the activities of human recombinant Akt1 and Akt2 and of constitutively active Akt immunoprecipitated from PC-3 cells, whereas inhibitor of nuclear factor-kappaB kinases remained unaffected. Docking data indicated that these tetracyclic triterpenoids form hydrogen bonds within the phosphatidylinositol binding pocket of the Akt pleckstrin homology domain. Accordingly, 3-beta-acetoxy-tirucallic acid did not inhibit the activity of Akt1 lacking the pleckstrin homology domain. In the prostate cancer cell lines investigated, these compounds inhibited the phosphorylation of cellular Akt and the Akt signaling pathways, including glycogen synthase kinase-3beta and BAD phosphorylation, nuclear accumulation of p65, the androgen receptor, beta-catenin, and c-Myc. These events culminated in the induction of apoptosis in prostate cancer, but not in nontumorigenic cells. The tirucallic acid derivatives inhibited proliferation and induced apoptosis in tumors xenografted onto chick chorioallantoic membranes and decreased the growth of pre-established prostate tumors in nude mice without overt systemic toxicity. Thus, tirucallic acid derivatives represent a new class of Akt inhibitors with antitumor properties. PMID:20018812
Influence of cholinesterase inhibitors, donepezil and rivastigmine on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference in rats.
Gawel, Kinga; Labuz, Krzysztof; Jenda, Malgorzata; Silberring, Jerzy; Kotlinska, Jolanta H
The influence of systemic administration of cholinesterase inhibitors, donepezil and rivastigmine on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference (CPP) was examined in rats. Additionally, this study aimed to compare the effects of donepezil, which selectively inhibits acetylcholinesterase, and rivastigmine, which inhibits both acetylcholinesterase and butyrylcholinesterase on morphine reward. Morphine-induced CPP (unbiased method) was induced by four injections of morphine (5 mg/kg, i.p.). Donepezil (0.5, 1, and 3 mg/kg, i.p.) or rivastigmine (0.03, 0.5, and 1 mg/kg, i.p.) were given 20 min before morphine during conditioning phase and 20 min before the expression or reinstatement of morphine-induced CPP. Our results indicated that both inhibitors of cholinesterase attenuated the acquisition and expression of morphine CPP. The results were more significant after rivastigmine due to a broader inhibitory spectrum of this drug. Moreover, donepezil (1 mg/kg) and rivastigmine (0.5 mg/kg) attenuated the morphine CPP reinstated by priming injection of 5mg/kg morphine. These properties of both cholinesterase inhibitors were reversed by mecamylamine (3 mg/kg, i.p.), a nicotinic acetylcholine receptor antagonist but not scopolamine (0.5 mg/kg, i.p.), a muscarinic acetylcholine receptor antagonist. All effects of cholinesterase inhibitors were observed at the doses that had no effects on locomotor activity of animals. Our results suggest beneficial role of cholinesterase inhibitors in reduction of morphine reward and morphine-induced seeking behavior. Finally, we found that the efficacy of cholinesterase inhibitors in attenuating reinstatement of morphine CPP provoked by priming injection may be due to stimulation of nicotinic acetylcholine receptors. PMID:24755308
Paradoxical role of PKA inhibitor on amyloid?-induced memory deficit.
Amini, Elham; Nassireslami, Ehsan; Payandemehr, Borna; Khodagholi, Fariba; Foolad, Forough; Khalaj, Sara; Hamedani, Mostafa Pirali; Azimi, Leyla; Sharifzadeh, Mohammad
In spite of characterizing the role of protein kinase A (PKA) in activating biochemical mechanisms, few studies have investigated the effects of PKA inhibitors on memory functions. In the present study, we used Pavlovian fear conditioning paradigm to evaluate memory alterations caused by two doses of H89 (as a conditional inhibitor of PKA) alone and in combination with amyloid-? (A?) in rats. Moreover, we used the Western blotting method to investigate the alterations in markers of transcription, oxidative stress, inflammation, and apoptosis pathways involved in memory impairment. Stereotaxic surgery was done to inject A? (30ng/side) directly into the hippocampal CA1 area bilaterally and H89 (5 or 10?M) intracerebroventricular unilaterally. One series of rats were trained 7days after injections, then contextual and tone tests were conducted on days 8 and 9, respectively. Second series of rats were trained 14days after the injections and tests were carried out on days 15 and 16. Our behavioral results showed that H89 (5?M) not only has no destructive effect on memory, but also attenuates memory deficit caused by A? in combination groups. In contrast, H89 (10?M) has a reversible destructive effect on memory. Our molecular findings indicated that low dose of H89 increases CREB phosphorylation, Nrf2 and HO-1 which results in survival resistance to the stress. On the contrary, H89 with higher concentration leads to substantial increase of NF-?B and caspase-3 levels, which impair memory functions. In conclusion, our data suggest that H89 as a PKA inhibitor influences memory process through a dose and time dependent manner. PMID:26037462
Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury.
Xie, Yuchao; Ramachandran, Anup; Breckenridge, David G; Liles, John T; Lebofsky, Margitta; Farhood, Anwar; Jaeschke, Hartmut
Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24h observation period. Pretreatment with 30mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. PMID:25818599
Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage
Bate, Clive; Rumbold, Louis; Williams, Alun
Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902
Abstract Aging is associated with alterations in hepatic peroxisomal metabolism and susceptibility to hepatocarcinogenecity produced by agonists of peroxisome proliferator-activated receptor alpha (PPARa). Mechanisms involved in these effects are not well understood. Howev...
The proteasome inhibitor bortezomib promotes mitochondrial injury and apoptosis induced by the small molecule Bcl2 inhibitor HA14-1 in multiple myeloma cells
X-Y Pei; Y Dai; S Grant
Interactions between the small molecule Bcl-2 inhibitor HA14-1 and proteasome inhibitors, including bortezomib (VelcadeÖ; formerly known as PS-341) and MG-132, have been examined in human multiple myeloma cells. Sequential (but not simultaneous) exposure of MM.1S cells to bortezomib or MG-132 (10 h) followed by HA14-1 (8 h) resulted in a marked increase in mitochondrial injury (loss of ??m, cytochrome c,
Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates.
Siebels, Ilka; Dr÷se, Stefan
The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology. PMID:23800966
Pan-mammalian target of rapamycin (mTOR) inhibitor AZD8055 primes rhabdomyosarcoma cells for ABT-737-induced apoptosis by down-regulating Mcl-1 protein.
Preuss, Ellen; Hugle, Manuela; Reimann, Romy; Schlecht, Marcel; Fulda, Simone
The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS. PMID:24133218
Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein*
Preuss, Ellen; Hugle, Manuela; Reimann, Romy; Schlecht, Marcel; Fulda, Simone
The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS. PMID:24133218
LW6, a hypoxia?inducible factorĀ1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.
Sato, Mariko; Hirose, Katsumi; Kashiwakura, Ikuo; Aoki, Masahiko; Kawaguchi, Hideo; Hatayama, Yoshiomi; Akimoto, Hiroyoshi; Narita, Yuichiro; Takai, Yoshihiro
Hypoxia?inducible factorĀ1 (HIF?1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF?1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF?1?. A clear anti?tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF?1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF?1? induced by hypoxia in A549 cells at 20ĀmM, independently of the vonĀHippel?Lindau protein. In addition, at this concentration, LW6 induced hypoxia?selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6?treated hypoxic A549 cells and LW6 induced a hypoxia?selective increase of mitochondrial O2Ģ?. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells. PMID:26017562
Evaluation of Whether Gemfibrozil is a Peroxisome Proliferator in Fish
Gemfibrozil is a pharmaceutical that indirectly modulates cholesterol biosynthesis through effects on peroxisome proliferator-activated receptors (PPAR), which are transcriptional cofactors that regulate expression of genes related to lipid metabolism. An enzyme found in the pero...
Valentine, Cathleen D.; Zhang, Hua; Phuan, Puay-Wah; Nguyen, Juliane; Verkman, A.S.; Haggie, Peter M.
SUMMARY P. aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-?B transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-?B activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restored NF-?B-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermal C12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy. PMID:23910799
Isolation and characterization of wound-inducible carboxypeptidase inhibitor from tomato leaves.
DĒez-DĒaz, Monica; Conejero, Vicente; Rodrigo, Ismael; Pearce, Gregory; Ryan, Clarence A
In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack. PMID:15279998
The mTOR inhibitor RAD001 potentiates autophagic cell death induced by temozolomide in a glioblastoma cell line.
Josset, Elodie; Burckel, HķlĶne; Noļl, Georges; Bischoff, Pierre
We have studied the consequences of the combination of the mammalian target of rapamycin (mTOR) inhibitor RAD001 and temozolomide on the growth and cell death of the glioblastoma cell line U-87 in vitro. A progressive decrease of cell proliferation was recorded with increasing concentrations of temozolomide, which was markedly reinforced and prolonged by the addition of RAD001. While this combination treatment resulted in only a low level of apoptosis, it led to a pronounced enhancement of autophagic cell death. When combined with ?-ray irradiation, a significant reinforcement of the overall cytotoxicity was obtained, suggesting the efficacy of such a multipronged approach for the treatment of glioblastoma. RAD001 strongly contributes to the reinforcement of temozolomide-induced autophagy, which appears to represent a major form of cell death in glioblastoma. The association of such combined chemotherapies with radiotherapy could be useful for the management of these hard-to-treat malignancies. PMID:23645729
In Salvia miltiorrhiza, phenolic acids possess protective properties against amyloid ?-induced cytotoxicity, and tanshinones act as acetylcholinesterase inhibitors.
Zhou, Yongqiang; Li, Weize; Xu, Lei; Chen, Lvyi
Radix Salvia miltiorrhiza (RSM), a traditional Chinese medicinal herb, has been alleged to possess therapeutic effects against senile dementia, also known as Alzheimer's disease (AD). However, the effects of the major components in RSM on cytotoxicity induced by amyloid-? peptide (A?) and on acetylcholinesterase activity have not been studied in depth to date. In this report, the effects of RSM aqueous/ethanol extracts, total polyphenols, total tanshinones and 3 phenolic compounds against toxicity mediated by A?(25-35) were tested with PC-12 cells. The results showed that A?(25-35)-induced cytotoxicity was revised by RSM aqueous/ethanol extracts and total polyphenols and that danshensu and salvianolic acid B could protect PC-12 cells by blocking A?(25-35)-induced Ca(2+)-intake, lactate dehydrogenase release, cell viability decrease and apoptosis. In addition, the activities of RSM extracts and relevant constituents in their inhibition of acetylcholinesterase were investigated using rat brain homogenates as an enzyme resource. Galanthamine hydrobromide, an accepted acetylcholinesterase inhibitor, was employed as a positive control agent. Our preliminary studies demonstrated that RSM ethanol extract, total tanshinones, tanshinone I and dihydrotanshinone I had remarkable inhibition effects on acetylcholinesterase in vitro. These findings suggest that both tanshinones and polyphenols in RSM are the active constituents responsible for the beneficial effects of this herb in AD treatment. PMID:21787715
Duodenal ulcers induced by diethyldithiocarbamate, a superoxide dismutase inhibitor, in the rat: role of antioxidative system in the pathogenesis.
Takeuchi, K; Nishiwaki, H; Niida, H; Ueshima, K; Okabe, S
Pathogenesis of duodenal ulcers induced by diethyldithiocarbamate (DDC), a superoxide dismutase (SOD) inhibitor, was investigated in the rat. Repeated s.c. administration of DDC (750 mg/kg) every 12 hr induced duodenal ulcers in the fed rats, and the severity of the ulcers reached the maximum after three injections. DDC not only reduced basal acid output but also impaired duodenal alkaline secretion. These ulcers were significantly prevented by antioxidative agents such as SOD (50000 units/kg, s.c.), allopurinol (50 mg/kg, s.c.) or glutathione (200 mg/kg, s.c.) as well as the antisecretory agent cimetidine (100 mg/kg, s.c.). The impaired HCO3- response caused by DDC was partially but significantly reversed by either SOD (15000 units/kg/hr, i.v.), allopurinol or glutathione; and SOD by itself significantly elevated the rate of basal alkaline secretion. 16,16-Dimethyl prostaglandin E2 (10 micrograms/kg, s.c.) increased duodenal HCO3- output in the presence of DDC and significantly prevented the development of duodenal ulcers in response to DDC. These results suggest that the mucosal antioxidative system including SOD may play a role in the regulatory process of alkaline secretion and contribute to the mucosal defensive ability in the duodenum. The insufficiency of this system may be involved in the pathogenesis of DDC-induced duodenal ulcers. PMID:1667532
Effect of iNOS inhibitor S-methylisothiourea in monosodium iodoacetate-induced osteoathritic pain: implication for osteoarthritis therapy.
More, Amar S; Kumari, Rashmi R; Gupta, Gaurav; Lingaraju, Madhu C; Balaganur, Venkanna; Pathak, Nitya N; Kumar, Dhirendra; Kumar, Dinesh; Sharma, Anil K; Tandan, Surendra K
Much information is available on the role of nitric oxide (NO) in osteoarthritis (OA). However, its role has not been studied in the monosodium iodoacetate (MIA)-induced model of osteoarthritic pain. The present study was undertaken in rats to investigate the effect of iNOS inhibitor S-methylisothiourea (SMT) in MIA-induced osteoathritic pain and disease progression in rats. Osteoarthritis was produced by single intra-articular injection of the MIA in the right knee joint on day 0. Treatment groups were orally gavazed with different doses of SMT (10, 30 and 100mg/kg) and etoricoxib (10mg/kg) daily for 21 days. On days 0, 3, 7, 14 and 21, pain was measured and histopathology of right knee joint was done on day 21. SMT produced analgesia in a dose-dependent manner as shown by mechanical, heat hyperalgesia, knee vocalization, knee squeeze test, and spontaneous motor activity test. SMT reduced NO production in synovial fluid. Histopathological findings indicated that SMT reduced disease progression as evident from complete cartilage formation in rats treated with SMT at 30 mg/kg. In conclusion, the results indicate that SMT attenuates the MIA-induced pain and histopathological changes in the knee joint. The antinociceptive and antiarthritic effects of SMT were mediated by inhibiting cartilage damage and suppression of NO in synovial fluid. It is suggested that SMT has potential as a therapeutic modality in the treatment of osteoarthritis. PMID:23287799
Matrine induces the apoptosis of lung cancer cells through downregulation of inhibitor of apoptosis proteins and the Akt signaling pathway.
Niu, Huiyan; Zhang, Yifei; Wu, Baogang; Zhang, Yi; Jiang, Hongfang; He, Ping
Lung cancer is the leading cause of cancer?related mortality in humans. The prognosis for advanced lung cancer patients is extremely poor. Current standard care is rather ineffective for prolonging patient life while preserving satisfactory quality of life due to adverse side-effects. Matrine extracted from the traditional Chinese herbal plant Sophora flavescens was shown to induce cancer cell death inĀvitro. The aim of this study was to investigate the effect of matrine on the proliferation and apoptosis of lung cancer cells and the molecular basis of matrine-induced apoptosis. The results showed that matrine inhibited cell proliferation and induced apoptosis in lung cancer A549 and 95D cells in a dose- and time-dependent manner. The apoptotic effects of matrine on lung cancer cells appeared to act via the phosphatidylinositolĀ3-kinase/Akt/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathway and downregulation of the expression of the inhibitor of apoptosis protein (IAP) family proteins. Matrine exerts its cancer-killing effect via promoting apoptosis in lung cancer cells and may be a useful adjuvant therapeutic scheme for treating advanced lung cancer patients. PMID:24969052
Zinc-deficient culture medium and protein kinase C inhibitors impair phytohemagglutinin-induced proliferation of murine splenocytes
Schroeder, J.J.; Cousins, R.J. (Univ. of Florida, Gainesville (United States))
Zinc deficiency inhibits mitogen-induced proliferation of T-lymphocytes. The role of protein kinase C (PKC) in this process is being evaluated by culturing splenocytes from C57Bl/6 mice in medium containing 5% Chelex-treated fetal bovine serum and the T-cell mitogen, phytohemagglutinin (PHA). PHA induces proliferation measured by ({sup 3}H)thymidine incorporation in a concentration-dependent manner with a maximal induction at 2.5 {mu}g/ml. The PKC inhibitors staurosporine and H-7 inhibit PHA-stimulated proliferation in concentration-dependent manners with IC{sub 50} values of 2.6 nM and 15 {mu}M, respectively. PHA has little or not effect on proliferation of cells cultured in medium containing 0.8 {mu}M zinc. However, increasing the medium zinc concentration to 16 {mu}M dramatically increases PHA-stimulated proliferation over control cultures. The results suggest that PHA-induced proliferation of murine T-cells is mediated by PKC. It is hypothesized that zinc deficiency inhibits mitogen-stimulated proliferation by preventing PKC coupling to plasma membranes. The results of these studies may provide a mechanism to explain impaired immunocompetence and other clinical problems associated with zinc deficiency.
The JNK inhibitor, SP600125, potentiates the glial response and cell death induced by methamphetamine in the mouse striatum.
Urrutia, Andres; Granado, Noelia; Gutierrez-Lopez, Maria Dolores; Moratalla, Rosario; O'Shea, Esther; Colado, Maria Isabel
This study investigates the effect of the selective Jun NH2-terminal kinase 1/2 (JNK1/2) inhibitor, (SP600125) on the striatal dopamine nerve terminal loss and on the increased interleukin-15 (IL-15) expression and glial response induced by methamphetamine (METH). Mice were given repeated low doses of METH (4Āmg/kg, i.p., three times separated by 3Āh) and killed 24Āh or 7 d after the last dose. SP600125 (30Āmg/kg, i.p) was administered 30Āmin before the last METH injection. Results indicate that METH produced dopaminergic axonal neurotoxicity reflected as a marked decrease in the striatal density of tyrosine hydroxylase-immunoreactive (TH-ir) fibres and dopamine transporter-immunoreactivity (DAT-ir) 24Āh after dosing. These effects were not modified by SP600125. This compound also failed to prevent the long-term loss of dopamine levels and DAT observed 7 d following METH injection. Nevertheless, SP600125 potentiated METH-induced striatal cell loss reflected by an increase in Fluoro-Jade immunostaining, cleaved capase-3 immunoreactivity and the number of terminal deoxyncleotidyl transferase-mediated dUTP nick end labelling (TUNEL) positive cells. In line with a deleterious effect of JNK1/2 inhibition, SP600125 increased the astroglial and microglial response induced by METH and interfered with drug-induced IL-15 expression. Together these data indicate that, not only does SP600125 fail to protect against the dopaminergic damage induced by METH but also, in fact, it potentiates the glial response and the non-dopaminergic striatal cell loss caused by the drug. PMID:24103647
Effects of peroxisome proliferator-activated receptor alpha activation on pathways contributing to cholesterol homeostasis in rat hepatocytes.
Le Jossic-Corcos, Catherine; Duclos, Sandrine; Ramirez, Leyla C; Zaghini, Isabelle; Chevillard, Grķgory; Martin, Pascal; Pineau, Thierry; Bournot, Paulette
Peroxisome proliferator-activated receptor alpha (PPARalpha) activation by fibrates controls expression of several genes involved in hepatic cholesterol metabolism. Other genes could be indirectly controlled in response to changes in cellular cholesterol availability. To further understand how fibrates may affect cholesterol synthesis, we investigated in parallel the changes in the metabolic pathways contributing to cholesterol homeostasis in liver. Ciprofibrate increased HMG-CoA reductase and FPP synthase mRNA levels in rat hepatocytes, together with cholesterogenesis from [(14)C] acetate and [(3)H] mevalonate. The up-regulation observed in fenofibrate- and WY-14,643-treated mice was abolished in PPARalpha-null mice, showing an essential role of PPARalpha. Among the three sterol regulatory element-binding protein (SREBP) mRNA species, only SREBP-1c level was significantly increased. In ciprofibrate-treated hepatocytes, cholesterol efflux was decreased, in parallel with cholesteryl ester storage and bile acids synthesis. As expected, AOX expression was strongly induced, supporting evidence of the peroxisome proliferation. Taken together, these results show that fibrates can cause cholesterol depletion in hepatocytes, possibly in part as a consequence of an important requirement of cholesterol for peroxisome proliferation, and increase cholesterogenesis by a compensatory phenomenon afterwards. Such cholesterogenesis regulation could occur in vivo, in species responsive to the peroxisome proliferative effect of PPARalpha ligands. PMID:15238219
The histone deacetylase inhibitor sodium butyrate modulates acquisition and extinction of cocaine-induced conditioned place preference
Raybuck, Jonathan D.; McCleery, Ellen J.; Cunningham, Christopher L.; Wood, Marcelo A.; Lattal, K. Matthew
Despite decades of research on treatments for cocaine dependence, relapse rates following many behavioral and drug-based therapies remain high. This may be in part because cocaine-associated cues and contexts can invoke powerful drug cravings years after quitting. Recent studies suggest that drugs that promote cognitive function can enhance the formation of memories involving cocaine and other substances. One target of these drugs is facilitating histone acetylation to promote learning by increasing gene transcription that supports memory formation. Here, we investigate the effects of the histone deacetylase (HDAC) inhibitor sodium butyrate (NaBut) on cocaine-induced conditioned place preference (CPP) in C57BL/6 mice. After establishing a graded dose-response curve (2, 5, & 20 mg/kg) for cocaine-induced CPP, we examined the effects of different doses of NaBut (0, 0.3, 0.6, & 1.2 g/kg) on conditioning, extinction, and post-extinction reconditioning of CPP. A high dose of NaBut (1.2 g/kg) enhanced initial acquisition of cocaine CPP, but there were no effects of NaBut on reconditioning of extinguished CPP. Effects of NaBut on extinction were more complex, with a low-dose (0.3 g/kg) facilitating extinction and a high dose (1.2 g/kg) weakening extinction evident by preference at a retention test. These findings suggest that HDAC inhibition may have dose dependent effects on different components of cocaine CPP, with implications for (1) involvement of histone acetylation in context-drug learning, (2) interpretation of acute and chronic drug effects, and (3) the targeting of different types of learning in therapeutic application of HDAC inhibitors. PMID:23454534
p62/sequestosome 1 as a regulator of proteasome inhibitor-induced autophagy in human retinal pigment epithelial cells
Viiri, Johanna; Hyttinen, Juha M. T.; Ryhõnen, Tuomas; Rilla, Kirsi; Paimela, Tuomas; Kuusisto, Erkki; Siitonen, Ari; Urtti, Arto; Salminen, Antero
Purpose The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress-induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the functional roles of p62 and HSP70 were evaluated in conjunction with proteasome inhibitor¢induced autophagy in human RPE cells (ARPE-19). Methods The p62, HSP70, and ubiquitin protein levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results Proteasome inhibition evoked the accumulation of perinuclear aggregates that strongly colocalized with p62 and HSP70. The p62 perinuclear accumulation was time- and concentration-dependent after MG-132 proteasome inhibitor loading. The silencing of p62, rather than Hsp70, evoked suppression of autophagy, when related to decreased LC3-II levels after bafilomycin treatment. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently, we showed that hsp70 mRNA depletion increased cell death in ARPE-19 cells. Here, we demonstrate that p62 mRNA silencing has similar effects on cellular viability. Conclusions Our findings open new avenues for understanding the mechanisms of proteolytic processes in retinal cells, and could be useful in the development of novel therapies targeting p62 and HSP70. PMID:20680098
CXC Receptor 1 and 2 and Neutrophil Elastase Inhibitors Alter Radiation-induced Lung Disease in the Mouse
Fox, Jessica [Department of Medicine and the Meakins-Christie Laboratories, McGill University, Montreal, Quebec (Canada)] [Department of Medicine and the Meakins-Christie Laboratories, McGill University, Montreal, Quebec (Canada); Haston, Christina K., E-mail: firstname.lastname@example.com [Department of Medicine and the Meakins-Christie Laboratories, McGill University, Montreal, Quebec (Canada)
Purpose: We previously reported increased numbers of neutrophils to be associated with the development of the radiation-induced lung responses of alveolitis (pneumonitis) and fibrosis in mice. In the present study we investigated whether CXC receptor 1 and 2 antagonism with DF2156A, a small molecule inhibitor of neutrophil chemotaxis, or the neutrophil elastase inhibitor sivelestat decreases the lung response to irradiation. Methods and Materials: KK/HIJ mice received 14 Gy whole-thorax irradiation, and a subset of them received drug treatment 3 times per week from the day of irradiation until they were killed because of respiratory distress symptoms. Results: Irradiated mice receiving sivelestat survived 18% longer than did mice receiving radiation alone (73 vs 60 days for female mice, 91 vs 79 days for male mice), whereas postirradiation survival times did not differ between the group of mice receiving DF2156A and the radiation-only group. The numbers of neutrophils in lung tissue and in bronchoalveolar lavage fluid did not differ among groups of irradiated mice, but they significantly exceeded the levels in unirradiated control mice. The extent of alveolitis, assessed histologically, did not differ between irradiated mice treated with either drug and those receiving radiation alone, when assessed at the end of the experiment, but it was significantly reduced, as were the neutrophil measures, in sivelestat-treated mice at the common kill time of 60 days after irradiation. Mice treated with radiation and DF2156A developed significantly less fibrosis than did mice receiving radiation alone, and this difference was associated with decreased expression of interleukin-13 in lung tissue. Conclusions: We conclude that neutrophil elastase inhibition affects alveolitis and prolongs survival, whereas CXCR1/2 antagonism reduces radiation-induced fibrotic lung disease in mice without affecting the onset of distress.
Peroxisomal changes during hiberation of jerboa (Jaculus orientalis)
M. Kabine; M Cherkaoui-Malki; C.-C. Clķmencet; M. S. El Kebbaj; N. Latruffe
As a member of the order of Rodentia, jerboa (Jaculus orientalis) is a natural deep hibernator and lives in subdesert highland\\u000a in many parts of the world, including Morocco. Its small size (adult body weight ?100 g), availability in the wild, tolerance\\u000a to laboratory conditions, and some unique peroxisomal properties make it a suitable research subject for exploring peroxisome\\u000a biogenesis
Peroxisomes in zebrafish: distribution pattern and knockdown studies
Olga Krysko; Mieke Stevens; Tobias Langenberg; Marc Fransen; Marc Espeel; Myriam Baes
Peroxisomes are organelles that are essential for normal development in men and mice. In order to explore whether zebrafish\\u000a could be used as a model system to study the role of peroxisomes, we examined their distribution pattern in developing and\\u000a adult zebrafish and we tested different approaches to eliminate them during the first days after fertilization. In 4-day-old\\u000a embryos, catalase-containing
Peroxisomal and Mitochondrial Status of Two Murine Oligodendrocytic Cell Lines (158N, 158JP): Potential Models for the Study of Peroxisomal Disorders Associated with
Paris-Sud XI, Universitķ de
1 Peroxisomal and Mitochondrial Status of Two Murine Oligodendrocytic Cell Lines (158N, 158JP title: Peroxisomal and Mitochondrial Status of Two Differentiated Murine Oligodendrocytic Cell Lines, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood
Bindarit, an Inhibitor of Monocyte Chemotactic Protein Synthesis, Protects against Bone Loss Induced by Chikungunya Virus Infection
Chen, Weiqiang; Foo, Suan-Sin; Taylor, Adam; Lulla, Aleksei; Merits, Andres; Hueston, Linda; Forwood, Mark R.; Walsh, Nicole C.; Sims, Natalie A.; Herrero, Lara J.
ABSTRACT The recent global resurgence of arthritogenic alphaviruses, in particular chikungunya virus (CHIKV), highlights an urgent need for the development of therapeutic intervention strategies. While there has been significant progress in defining the pathophysiology of alphaviral disease, relatively little is known about the mechanisms involved in CHIKV-induced arthritis or potential therapeutic options to treat the severe arthritic symptoms associated with infection. Here, we used microcomputed tomographic (?CT) and histomorphometric analyses to provide previously undescribed evidence of reduced bone volume in the proximal tibial epiphysis of CHIKV-infected mice compared to the results for mock controls. This was associated with a significant increase in the receptor activator of nuclear factor-?B ligand/osteoprotegerin (RANKL/OPG) ratio in infected murine joints and in the serum of CHIKV patients. The expression levels of the monocyte chemoattractant proteins (MCPs), including MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, were also highly elevated in joints of CHIKV-infected mice, accompanied by increased cellularity within the bone marrow in tibial epiphysis and ankle joints. Both this effect and CHIKV-induced bone loss were significantly reduced by treatment with the MCP inhibitor bindarit. Collectively, these findings demonstrate a unique role for MCPs in promoting CHIKV-induced osteoclastogenesis and bone loss during disease and suggest that inhibition of MCPs with bindarit may be an effective therapy for patients affected with alphavirus-induced bone loss. IMPORTANCE Arthritogenic alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), cause worldwide outbreaks of polyarthritis, which can persist in patients for months following infection. Previous studies have shown that host proinflammatory soluble factors are associated with CHIKV disease severity. Furthermore, it is established that chemokine (C-C motif) ligand 2 (CCL2/MCP-1) is important in cellular recruitment and inducing bone-resorbing osteoclast (OC) formation. Here, we show that CHIKV replicates in bone and triggers bone loss by increasing the RANKL/OPG ratio. CHIKV infection results in MCP-induced cellular infiltration in the inflamed joints, and bone loss can be ameliorated by treatment with an MCP-inhibiting drug, bindarit. Taken together, our data reveal a previously undescribed role for MCPs in CHIKV-induced bone loss: one of recruiting monocytes/OC precursors to joint sites and thereby favoring a pro-osteoclastic microenvironment. This suggests that bindarit may be an effective treatment for alphavirus-induced bone loss and arthritis in humans. PMID:25339772
PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Influence of Aminoguanidine, an Inhibitor of Inducible Nitric Oxide Synthase, on the Pulmonary Hypertensive Response to Microparticle Injections in Broilers1
R. F. Wideman; O. T. Bowen; G. F. Erf; M. E. Chapman
The pulmonary hypertensive response to pulmonary vascular obstruction caused by intravenously injected microparticles is amplified by pretreatment with N?nitro-L-arginine methyl ester (L-NAME). The L-NAME prevents the synthesis of the potent vasodilator nitric oxide (NO) by inhibiting both the constitutive (endothe- lial NO synthase (eNOS or NOS-3)) and inducible (induc- ibleNOsynthase(iNOSorNOS-2))formsofNOsynthase. In the present study we used the selective iNOS inhibitor
Lovastatin-induced cardiac toxicity involves both oncotic and apoptotic cell death with the apoptotic component blunted by both caspase-2 and caspase-3 inhibitors
Simon W Rabkin; Jennifer Y Kong
The objective of this study was to evaluate the cardiac toxicity of the HMG¢CoA reductase inhibitors by testing the hypothesis that lovastatin induces apoptotic and\\/or oncotic cell death in the myocyte element of the heart and further that cell death is mediated through interruption of the mevalonate pathway and that apoptosis is induced through activation of caspase-2 and caspase-3. Cardiomyocytes
Dimethylfumarate is an Inhibitor of Cytokine-Induced Nuclear Translocation of NF-?B1, But Not RelA in Normal Human Dermal Fibroblast Cells
Marc Vandermeeren; Sophie Janssens; Hilde Wouters; Inge Borghmans; Marcel Borgers; Rudi Beyaert; Johan Geysen; Vandermeeren Marc
We previously demonstrated that the oral antipsoriatic dimethylfumarate is an inhibitor of cytokine-induced adhesion molecule expression in endothelial HUVEC cells. We now report the inhibitory effect of dimethylfumarate on tumor-necrosis-factor-?- or interleukin-1?-induced intercellular adhesion molecule 1 expression in normal human dermal fibroblasts. Western blots of normal human dermal fibroblast cytoplasmic extracts showed that dimethylfumarate has minor effects on the I?B?,
Novel diacylglycerol kinase inhibitor selectively suppressed an U46619-induced enhancement of mouse portal vein contraction under high glucose conditions
Nobe, Koji; Miyatake, Mari; Nobe, Hiromi; Sakai, Yasushi; Takashima, Junko; Momose, Kazutaka
Diacylglycerol kinase (DG kinase) is a key enzyme in vascular contraction; however, alterations of the regulatory mechanisms in vascular dysfunction are poorly understood. In this study, the effect of a novel DG kinase inhibitor, stemphone, on vascular contraction was investigated. The conventional DG kinase inhibitor, 6-[2-(4-[(4-fluorophenyl)phenyl-methylene]-1-piperidinyl)ethyl]-7-methyl-5H-thiazolo [3,2-?] pyrimidine-5-one (R59022) (0.1¢30 ?M), inhibited thromboxane A2 analogue 9,11-dideoxy-11?,9?-epoxymethanoprostaglandin F2? (U46619)-induced sustained contractions in mouse aorta and porcine coronary artery in a dose-dependent manner. Treatment with stemphone did not affect contractions in these tissues. However, stemphone significantly inhibited (>0.3 ?M) U46619-induced spontaneous phasic contraction in mouse portal vein. This inhibitory effect was not detected following R59022 treatment in portal vein. Therefore, stemphone demonstrated selectivity in terms of portal vein contraction. Under high glucose (22.2 mM) conditions, U46619-induced contraction was enhanced in these three types of vascular tissue. Inhibitory effects of R59022 were attenuated under these conditions; however, effects of stemphone were observed. These results indicated that stemphone could inhibit portal vein contraction under high glucose conditions, for example, diabetes. These data suggested the possibility that DG kinase may be a target of hyperportal pressure. Total mass of DG was enhanced under high glucose conditions. DG was derived from incorporated glucose via de novo synthesis in the absence of phospholipase C pathway mediation. This enhanced DG under high glucose conditions activated a calcium-independent protein kinase C (PKC). This PKC was associated with calcium-independent DG kinase activation. Treatment with stemphone also inhibited calcium-independent DG kinase. These signal transduction pathways were distinguishable from a DG¢PKC pathway under normal glucose conditions. The present investigation suggested that stemphone selectively inhibited overcontraction of portal vein induced by high glucose levels. This phenomenon was attributable to inhibition of calcium-independent DG kinase activation that occurred under high glucose conditions mediated by both DG synthesized from glucose and calcium-independent PKC activation. PMID:15289283
Synthesis and identification of chiral aminomethylpiperidine carboxamides as inhibitor of collagen induced platelet activation.
Anil Kumar, K S; Misra, Ankita; Siddiqi, Tanveer Irshad; Srivastava, Stuti; Jain, Manish; Bhatta, Rabi Sankar; Barthwal, Manoj; Dikshit, Madhu; Dikshit, Dinesh K
A series of chiral lactam carboxamides of aminomethylpiperidine were synthesized and investigated for the collagen induced inĀvitro anti-platelet efficacy and collagen plus epinephrine induced inĀvivo pulmonary thromboembolism. The compound 31a (30Ā?M/kg) displayed a remarkable antithrombotic efficacy (60% protection) which was sustained for more than 24Āh and points to its excellent bioavailability. The compounds 31a (IC50Ā=Ā6.6Ā?M) and 32a (IC50Ā=Ā37Ā?M), as well as their racemic mixture 28i (IC50Ā=Ā16Ā?M) significantly inhibited collagen-induced human platelet aggregation inĀvitro. Compound 34c displayed dual mechanism of action against both collagen (IC50Ā=Ā3.3Ā?M) and U46619 (IC50Ā=Ā2.7Ā?M) induced platelet aggregation. The pharmacokinetic study of 31a indicated very faster absorption, prolonged and constant systemic exposure and thereby exhibiting better therapeutic response. PMID:24859764
Xanthine Oxidase Inhibitor, Allopurinol, Prevented Oxidative Stress, Fibrosis, and Myocardial Damage in Isoproterenol Induced Aged Rats
Sagor, Md. Abu Taher; Tabassum, Nabila; Potol, Md. Abdullah; Alam, Md. Ashraful
We evaluated the preventive effect of allopurinol on isoproterenol (ISO) induced myocardial infarction in aged rats. Twelve- to fourteen-month-old male Long Evans rats were divided into three groups: control, ISO, and ISO + allopurinol. At the end of the study, all rats were sacrificed for blood and organ sample collection to evaluate biochemical parameters and oxidative stress markers analyses. Histopathological examinations were also conducted to assess inflammatory cell infiltration and fibrosis in heart and kidneys. Our investigation revealed that the levels of oxidative stress markers were significantly increased while the level of cellular antioxidants, catalase activity, and glutathione concentration in ISO induced rats decreased. Treatment with allopurinol to ISO induced rats prevented the elevated activities of AST, ALT, and ALP enzymes, and the levels of lipid peroxidation products and increased reduced glutathione concentration. ISO induced rats also showed massive inflammatory cells infiltration and fibrosis in heart and kidneys. Furthermore, allopurinol treatment prevented the inflammatory cells infiltration and fibrosis in ISO induced rats. In conclusion, the results of our study suggest that allopurinol treatment is capable of protecting heart of ISO induced myocardial infarction in rats probably by preventing oxidative stress, inflammation, and fibrosis. PMID:26137187
Thymic alterations induced by Plasmodium berghei: expression of matrix metalloproteinases and their tissue inhibitors.
Lima, Alliny Carolina Dionete; Francelin, Carolina; Ferrucci, Danilo Lopes; Stach-Machado, Dagmar Ruth; Verinaud, Liana
The thymus plays a crucial role in the generation of T-cells, and so our laboratory has been interested in the study of the intrathymic events that occur during infection diseases and may cause disruption in its functions. Previously, we showed that thymus from experimentally Plasmodium berghei-infected mice present histological alterations with high levels of apoptosis, changes in cell migration-related molecules, and premature egress of immature thymocytes to periphery. In addition, parasites were found inside the thymus. In this work we investigated alterations in the expression pattern and activity of matrix metalloproteinases MMP-2 and -9, and their tissue inhibitors, TIMP-1 and TIMP-2. Our results show enhanced expression and widespread distribution of these molecules in thymus from infected animals. Also, the presence of active MMP-2 was detected. These data are suggestive of MMPs and TIMPs importance in the earlier observed changes in the extracellular matrix during thymic alterations after plasmodium infection. PMID:23089194
The peroxisome proliferator-activated receptor-? (PPAR-?) agonist, AVE8134, attenuates the progression of heart failure and increases survival in rats
Wolfgang Linz; Paulus Wohlfart; Manuel Baader; Kristin Breitschopf; Eugen Falk; Hans-Ludwig Schõfer; Martin Gerl; Werner Kramer; Hartmut R³tten
Aim:To investigate the efficacy of the peroxisome proliferator-activated receptor-? (PPAR?) agonist, AVE8134, in cellular and experimental models of cardiac dysfunction and heart failure.Methods:In Sprague Dawley rats with permanent ligation of the left coronary artery (post-MI), AVE8134 was compared to the PPAR? agonist rosiglitazone and in a second study to the ACE inhibitor ramipril. In DOCA-salt sensitive rats, efficacy of AVE8134
Largazole, a class I histone deacetylase inhibitor, enhances TNF-?-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts
Ahmed, Salahuddin, E-mail: firstname.lastname@example.com [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V. [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Bhansali, Pravin; Tillekeratne, L.M. Viranga [Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States)
In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-? (TNF-?)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1¢5 ?M) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5¢5 ?M) inhibited the constitutive expression of HDAC1 (0¢30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ? 220% with a concomitant decrease in HDAC5 [30¢58%] expression in RA synovial fibroblasts. SAHA (5 ?M), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-?-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-?-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-?-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-? + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-?-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-?Bp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-? in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: Ģ Largazole enhances TNF-?-induced ICAM-1 and VCAM-1. Ģ Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. Ģ Largazole also induces the expression of phospho-p38 and phospho-Akt. Ģ A selective HDAC isoform inhibitor may be more effective than a class inhibitor. Ģ Further studies are required to understand the role of class II HDACs in RA.
Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8
Ogura, Hirotsugu; Tsukumo, Yoshinori [Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Sugimoto, Hikaru [Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Igarashi, Masayuki [Bioactive Molecular Research Group, Microbial Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan); Nagai, Kazuo [Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Department of Biological Chemistry, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501 (Japan); Kataoka, Takao [Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan)], E-mail: email@example.com
The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in a dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-C