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3-Bromopyruvate sensitizes human breast cancer cells to TRAIL-induced apoptosis via the phosphorylated AMPK-mediated upregulation of DR5

Previous studies have indicated that the sensitivity of breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is associated with the expression of death receptors on the cell membrane. However, drug resistance limits the use of TRAIL in cancer therapy. Numerous studies have indicated that death receptors, which induce apoptosis, are upregulated by the endoplasmic reticulum (ER) stress response. 3-Bromopyruvate (3-BP), an anticancer agent, inhibits cell growth and induces apoptosis through interfering with glycolysis. In the present study, it was demonstrated that 3-BP synergistically sensitized breast cancer cells to TRAIL-induced apoptosis via the upregulation of death receptor 5 (DR5). Furthermore, we found that the protein levels of glucose-related protein 78 (GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP) increased following treatment with 3-BP. The expression of Bax (in MCF-7 cells) and caspase-3 (in MDA-MB-231 cells) increased following co-treatment with 3-BP and TRAIL, whereas the expression of the anti-apoptotic protein Bcl-2 decreased. In order to investigate the molecular mechanism regulating this effect, the expression of adenosine monophosphate-activated protein kinase (AMPK), activated by 3-BP, was determined. It was demonstrated that phosphorylated-AMPK was upregulated following treatment with 3-BP. Notably, Compound C, an AMPK inhibitor, reversed the effects of 3-BP. Finally, a synergistic antitumor effect of 3-BP and TRAIL was observed in MCF-7 cell xenografts in nude mice. In conclusion, these results indicated that 3-BP sensitized breast cancer cells to TRAIL via the AMPK-mediated upregulation of DR5.


Introduction
Breast cancer is the leading cause of cancer-related deaths among females and is the most frequently diagnosed type of cancer in women. The incidence of breast cancer in China is increasing by 2.2% per year (1). The currently available treatments for patients with breast cancer includes chemotherapy, surgical resection and radiotherapy. Considering that drug resistance is often associated with targeted therapies, there is a requirement for novel therapeutic strategies for the treatment of cancer (2).
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has a crucial role in cancer therapy; by binding to death receptor (DR)4 and DR5, it selectively induces apoptosis in cancer cells. TRAIL has reduced toxicicity compared with other members of the tumor necrosis factor (TNF) protein family, such as TNFα and Fas ligand (CD95L) (3). Pre-clinical trials have determined that TRAIL effectively inhibits tumor growth without inducing toxicity in both mice and non-human primates (4,5). However, cancer cells may aquire resistence to TRAIL-induced apoptosis via mutation or deficiency of DR4 or DR5. A number of studies have investigated whether the use of recombinant TRAIL may reverse this effect, by enabling the interaction of TRAIL with mutated receptors, or whether the combination of treatment with TRAIL and

3-Bromopyruvate sensitizes human breast cancer cells to TRAIL-induced apoptosis via the phosphorylated AMPK-mediated upregulation of DR5
Chinese herbology (6) may increase the therapeutic efficacy and decrease the toxicity of TRAIL (7). The expression of TRAIL receptor 2 (TRAIL-R2 or DR5) is crucial in TRAIL-induced apoptosis in cancer cells (8), and is upregulated following the inhibition of ATP synthesis in cancer cells (9). Cancer cells and healthy cells may be characterized according to their different energy metabolisms (10). In 1956, Warburg demonstrated that cancer cells produce energy predominantly via fermentation rather than through oxidative respiration as exhibited by healthy cells, even when sufficient oxygen is present (11). However, fermentation is associated with the higher consumption of glucose relative to oxidative respiration, as well as the greater production of lactate.
3-Bromopyruvate (3-BP), a hexokinase II inhibitor, can induce apoptosis in hepatocellular carcinoma cells by inducing endoplasmic reticulum (ER) stress (12). ER stress occurs upon accumulation of misfolded proteins in the ER, and results in the activation of the ER stress chaperone protein glucose-related protein 78 (GRP78) and the pro-apoptotic transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP). Previous studies have indicated that DR5 is upregulated during the ER stress response (13)(14)(15). Adenosine monophosphate-activated protein kinase (AMPK) is a nutrient and energy marker in the cells, and can also induce apoptosis (16)(17)(18). Notably, numerous studies have suggested that c-Jun N-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is potentially a regulator of AMPK-induced apoptosis. JNK regulates apoptosis via the phosphorylation of apoptosis related-proteins, such as B-cell f lymphoma 2 (Bcl-2), or via the activation of activator protein-1 (AP-1) (19). In addition, 3-BP suppresses the levels of cellular adenosine tri-phosphate (ATP) and activates AMPK, as well as the AMPK-mediated upregulation of Bcl-2-associated X protein (Bax), in which subsequently induces cell death via the mitochondrial pathway (20).
Our previous study indicated that the hexokinase inhibitor 3-BP induced cell death in colon cancer cells via a number of different mechanisms (21). Therefore, we aimed to investigate whether 3-BP upregulated DR5, thus enhancing the anticancer effect of TRAIL. The results of the present study demonstrated that AMPK was induced by 3-BP to act as a regulator of apoptosis. Thus, we hypothesized that AMPK played a role in the ER stress response, and subsequent upregulation of CHOP and DR5. Furthermore, the results of the present study revealed that co-treatment with 3-BP and TRAIL induced apoptosis in breast cancer cells in a Bax-and caspase-dependent manner.
Cell culture. The human breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (100 U/ml). Cells were maintained at 37˚C in a humidified atmosphere containing 5% CO 2 .
PI staining. Cells were seeded in 12-well plates (1.5x10 5 cells/well) and incubated for 24 h, until the cells reached exponential phase. Subsequently, cells were treated for 24 h with various concentrations of 3-BP, TRAIL or both. Cells were subsequently stained using propidium iodide (PI; 600 µl/well) for 2 h and then analyzed using flow cytometry (Accuri™ C6 system; BD Biosciences, Franklin Lakes, NJ, USA).
ATP quantification. CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) was used to investigate ATP levels, according to the manufacturer's instructions. Cells (1.5x10 5 /well) were seeded in 12-well plates for 24 h and then treated with different concentrations of 3-BP for 4 h at 37˚C. Following this, cells were collected and then lysed using radioimmunoprecipitation assay (RIPA) lysis buffer for 10 min on ice. Cell lysates were subsequently centrifuged at 13,225 x g for 5 min at 4˚C. A nucleotide-releasing buffer (100 µl/well), ATP-monitoring enzyme (10 µl/well), and cell lysate (30 µl/well) were added to 96-well plates. Developed signals were then detected using a Luminoskan luminometer and a Varioskan™ Flash spectral scanning multimode reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Cell surface staining. Cells (2.5x10 5 /ml) were seeded in 6-well plates, treated with PBS or 3-BP (80 µmol/l), and then incubated for 24 h. Subsequently, non-specific antibody binding sites were blocked using PBS containing 10% FBS for 20 min. Cells were then washed with PBS, re-suspended in 200 µl PBS, aliquoted into two tubes and incubated with 20 µl of an antibody-containing solution (rabbit anti-DR5 in PBS with 1% FBS; dilution 1:1,000) for 30 min on ice. Subsequently, the cells were washed twice with PBS, pelleted and incubated with 100 µl FITC-conjugated goat anti-rabbit IgG (1:100 dilution; cat. no. BL033A; Biosharp) for 30 min on ice. Cell surface staining was investigated via flow cytometry using the Accuri™ C6 flow cytometer (BD Biosciences).
Western blot analysis. Harvested cells were washed with PBS and then lysed using RIPA buffer for 30 min on ice. Subsequently, cell lysates were centrifuged at 13,225 x g for 30 min at 4˚C. The proteins were determined by BCA assay. Subsequently, proteins (50 µg) were loaded per lane, and then separated on a 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were then blocked using 5% skimmed milk in PBS for 4 h at 20-25˚C and incubated overnight at 4˚C with the primary antibodies. After washing with Tris-buffered saline containing 1% Tween ® -20 (Beyotime Institute of Biotechnology, Haimen, China), membranes were incubated with the corresponding secondary antibodies. β-actin was used as the loading control.
In vivo experiments. In order to investigate the antitumor effect of 3-BP and TRAIL, female nude mice (BALB/c; 4-5-weeks old and 18-20 g) were used. Mice were purchased from the Animal Experimental Center of Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained under specific pathogen-free conditions (26-28˚C, air pressure difference was 10-20 kPa, 10-h light/14-h dark cycle, food and water were taken ad libitum). Mice were injected subcutaneously with MCF-7 cells (4x10 6 cells/mouse) to induce tumor formation. A total of 20 mice with tumors of >100 mm 3 were randomly divided into four groups (five per group), and injected intraperitoneally with either PBS (0.2 ml), 3-BP (8 mg/kg), TRAIL (0.1 mg/kg) or both 3-BP (8 mg/kg) and TRAIL (8 mg/kg) every 4 days. Body weight was monitored prior to each injection. Tumor volume was calculated using the following formula: Length x width 2 /2. Mice were sacrificed by cervical dislocation after 28 days of treatment. Following treatment for 28 days, the solid tumors were resected from mice, stored in 4% formalin solution, cut into sections and subsequently subjected to with hematoxylin and eosin (H&E) or TUNEL staining. All procedures performed in this study involving animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Bengbu Medical College.
Statistical analysis. Data are expressed as the mean ± the standard error of the mean (SEM). Statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The difference among groups was calculated by one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test. P<0.05 was considered to indicate a statistically significant difference.

3-BP enhances TRAIL-induced apoptosis in breast cancer
cells. MCF-7 and MDA-MB-231 cells, were selected as they are estrogen/progesterone receptor positive and negative, respectively, which frequently exhibit different drug sensitivities. To investigate the effects of 3-BP and TRAIL on cell viability, MCF-7 and MDA-MB-231 cells were treated with various concentrations of 3-BP and TRAIL for 24 h. The results of the MTT assay and PI staining analyses demonstrated that 3-BP (80 and 160 µmol/l) and TRAIL (400 ng/ml) significantly inhibited cell viability (Fig. 1A). Notably, concomitant treatment with 3-BP (80 µmol/l) and TRAIL (200 ng/ml) inhibited cell viability to a greater extent compared with treatment with 3-BP or TRAIL alone (P<0.05, Fig. 1B and C). Therefore, the results indicated that co-treatment with 3-BP and TRAIL synergistically induced apoptosis in breast cancer cells.

3-BP inhibits ATP generation and upregulates the expression of DR5.
Taking the above-mentioned results into consideration, we investigated whether 3-BP affected the activity and/or expression of one or more mediators in the enhancement of the anticancer activity of TRAIL in breast cancer cells. To investigate this, we determined whether 3-BP affected ATP levels and/or the expression of DR5. It was demonstrated that treatment with 3-BP induced ATP depletion in breast cancer cells ( Fig. 2A). Similarly, DR5 staining revealed that the expression of DR5 was enhanced in breast cancer cells treated with 3-BP compared with control cells (Fig. 2B).

3-BP upregulates CHOP, GRP78 and the phosphorylation of AMPK and augments TRAIL-induced Bax and caspase-3 levels.
Previous studies have demonstrated that 3-BP induced apoptosis in hepatocellular carcinoma cells via ER stress (12). Therefore, the present study aimed to investigate whether 3-BP induced ER stress in breast cancer cells by determining the expression of ER stress-associated proteins in cells treated with 3-BP. The results of western blot analysis revealed that CHOP and GRP78 levels were enhanced in breast cancer cells following treatment with 3-BP (Fig. 3A). In addition, previous studies have demonstrated that 3-BP induced apoptosis via the disruption of energy metabolism (20). Therefore, the present study aimed to investigate the level of p-AMPK in cells treated with 3-BP. The results of western blot analysis revealed that treatment with 3-BP increased levels of p-AMPK in a dose-dependent manner, as well as the AMPK-α levels overall (Fig. 3A). Furthermore, the expression levels of Bax in MCF-7 cells and caspase-3 in MDA-MB-231 cells were increased in breast cancer cells following co-treatment with 3-BP and TRAIL. However, co-treatment with 3-BP and TRAIL decreased the expression of Bcl-2 in breast cancer cells (Fig. 3B).
T he A M PK inhibitor Compou n d C reduces the TRAIL-synergizing effect of 3-BP on cell growth inhibition and apoptosis. Subsequently, we investigated whether 3-BP sensitized breast cancer cells to growth inhibition and apoptosis by TRAIL via the AMPK activation. MCF-7 and MDA-MB-231 cells were treated with Compound C (an AMPK inhibitor), and 3-BP, TRAIL or both 3-BP and TRAIL. The results of the MTT assays demonstrated that the viability of cells treated with either 3-BP, TRAIL or 3-BP and TRAIL and Compound C did not exhibit a significant difference compared with the control group (Fig. 4A). Similar results were obtained using PI staining, which indicated the number of apoptotic cells upon each treatment (Fig. 4B). Therefore, the results suggested that Compound C attenuated the TRAIL-synergistic effect of 3-BP.

Compound C inhibits 3-BP-induced upregulation of DR5, GRP78, CHOP and p-AMPK apoptosis-associated proteins.
To investigate the regulatory role of AMPK in mediating the effects of 3-BP associated with the upregulation of ER stressand apoptosis-associated proteins, MCF-7 and MDA-MB-231 cells were treated with Compound C. Membrane receptor   staining revealed that the fluorescence intensity of DR5 did not increase in breast cancer cells following treatment with Compound C (Fig. 5A). In addition, the results demonstrated that DR5, CHOP, GRP78 and p-AMPK levels did not significantly increase in cells treated with various concentrations of 3-BP and Compound C compared with the control cells (Fig. 5B). Similarly, the expression of Bax and Bcl-2 in MCF-7 cells and caspase-3 levels in MDA-MB-231 cells treated with Compound C, 3-BP, TRAIL, or 3-BP and TRAIL did not demonstrate a significant increase compared to with the control cells (Fig. 5C). These results suggested that AMPK activation may be involved in the ER stress response induced by 3-BP in breast cancer cells.

Antitumor efficacy of 3-BP and TRAIL in tumor xenografts.
MCF-7 cells were inoculated hypodermically into the forelimb of nude mice to induce the formation of xenograft tumors. Once the tumors reached a mean volume of ~100 mm 3 , mice matched for tumor volumes were divided into 4 groups and treated with either PBS, 3-BP, TRAIL or 3-BP and TRAIL.
Following 28 days of treatment, the tumor volumes of mice treated with either PBS, 3-BP, TRAIL, or 3-BP and TRAIL were ~1,200±100, ~850±71, ~700±77 and ~232±40 mm 3 , respectively (Fig. 6A). To investigate the hepatotoxicity and nephrotoxicity of the aforementioned treatments, the levels of serological markers including aspartate aminotransferase (AST), alanine-aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cr) were determined. The results revealed that mice did not exhibit marked levels of toxicity following treatment with either PBS, 3-BP, TRAIL or 3-BP and TRAIL, as the expression of these markers was unchanged (Fig. 6B). The results of H&E staining demonstrated that necrosis occurred in the tumors of groups treated with 3-BP, TRAIL or both 3-BP and TRAIL, and the necrotic area in the group treated with both 3-BP and TRAIL was larger compared with the other treatment groups (Fig. 6C). H&E staining of the liver and kidney revealed that no evident damage was induced following the treatments (Fig. 6C). Finally, TUNEL staining demonstrated that the number of apoptotic cells in the group treated with 3-BP and TRAIL was increased compared with the other groups (Fig. 6D). Therefore, the results suggested that the antitumorigenic effect of 3-BP and TRAIL was associated with low hepatotoxicity and nephrotoxicity in vivo.

Discussion
Breast cancer is an aggressive malignancy. It is the most frequently diagnosed cancer and its incidence has been increasing in both economically developed and developing countries (22). Numerous therapeutic strategies are available for the treatment of patients with breast cancer, including chemotherapy, surgery and radiotherapy (23). However, the mortality rate for patients with breast cancer remains high. Therefore, novel and effective therapies for the treatment of patients with breast cancer are required. It has been demonstrated that TRAIL is an effective anticancer agent that inhibits the proliferation of cancer cells and tumor growth in xenograft models (24)(25)(26). However, loss or mutation of the death receptors targeted by TRAIL may lead to drug resistance (27). Therefore, it is important to determine the molecular mechanisms underlying TRAIL-induced apoptosis in order to develop novel therapeutic agents that circumvent resistance (28,29).
The ER stress response pathway regulates cancer cell fate, and may induce autophagy, senescence, or apoptosis (30). Protein sensors such as PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activator transcription factor 6 (ATF6) initiate the ER stress response and regulate the unfolded protein response (UPR) under both pathological and physiological conditions (31). Specifically, the PERK/eukaryotic initiation factor 2 (eIF2α)/activator transcription factor 4 (ATF4) axis upregulates CHOP, which subsequently induces apoptosis (32,33). The expression of DR5 is regulated by CHOP, the predominant regulator of the ER stress response. Several previous studies point in this direction: Lu et al revealed that ER stressors regulated the transcription of DR5 via the UPR mediator, CHOP (15). In addition, Guo et al demonstrated that tunicamycin sensitized human colon cancer cells to TRAIL-induced apoptosis via the JNK-CHOP-mediated upregulation of DR5 expression (13). Chen et al determined that the expression of DR5 in human esophageal cancer cells is regulated by the ATF4-CHOP-DR5 axis (14). In addition, treatment with caffeic acid phenethyl ester upregulated the protein level of DR5 and promoted apoptosis in hepatocarcinoma Hep3B cells through CHOP (34). While ER stress sensors and mediators have been revealed to be involved in the regulation of death receptors, the molecular mechanisms underlying 3-BP and TRAIL-induced apoptosis in human breast cancer cells have not yet been clarified. In the present study, it was demonstrated that treatment with 3-BP and TRAIL in MCF-7 and MDA-MB-231 breast cancer cells is associated with the suppression of cell viability in a dose-dependent manner. Furthermore, the results revealed that co-treatment with 3-BP and TRAIL significantly increased apoptosis compared with 3-BP or TRAIL alone, thus suggesting that 3-BP and TRAIL exhibit a synergistic anticancer effect both in vitro and in a tumor xenograft model. Furthermore, 3-BP was demonstrated to induce the ER stress response and subsequently to upregulate GRP78 and CHOP levels in MCF-7 and MDA-MB-231 cells. Simultaneously, 3-BP was revealed to increase the protein expression level of DR5, which subsequently enhanced the sensitivity of cells to TRAIL. These data suggested that the anticancer efficacy of 3-BP in human breast cancer cells is regulated via the activa- tion of ER stress and the upregulation of DR5, which, in turn, sensitizes cells to TRAIL treatment.
There are three types of cell death, namely apoptosis, autophagic cell death and necrosis, largely defined by the morphology of the dying cells (35). The death receptor and mitochondrial pathways play major roles in apoptotic cell death despite the existence of additional regulatory pathways associated with apoptosis. DR5 is upregulated in ER stress-induced apoptosis and is an important factor in this mechanism (35). DR5 binds to its ligands and then induces apoptosis via recruitment of the caspase-activation platform. UPR sensitizes cancer cells to TRAIL-induced apoptosis by enhancing the enzymatic activity of caspase-8 and caspase-3/7. Conversely, mitochondrial outer membrane permeabilization (MMOP) has an important role in the mitochondrial pathway of apoptosis (36). MMOP is regulated by members of the Bcl-2 family. Bcl-2 proteins are classified into three groups: The pro-apoptotic effector proteins (e.g., Bax and Bak), anti-apoptotic Bcl-2 proteins (e.g., Bcl-2, Bcl-xL and Mcl-1), and BH3-only proteins (e.g., Bad, Bim, Bid and Noxa) (37). The results of the present study suggested that co-treatment with 3-BP and TRAIL decreased the expression of Bcl-2 in MCF-7 cells and upregulated the expression of Bax in MCF-7 cells and caspase-3 in MDA-MB-231 cells. Bax expression was investigated in MCF-7 cells, as these cells do not express caspase-3 (38). The results of the present study indicated that the co-treatment with 3-BP and TRAIL may be involved in caspase-3-and Bcl-2 family-mediated apoptosis in breast cancer cells.
AMPK is a sensor of cellular energy status that regulates metabolism in cellular processes. AMPK is activated by changes in the adenosine mono-phosphate (AMP)/ATP or adenosine di-phosphate (ADP)/ATP ratio (16,17). Numerous studies indicated that activated AMPK induced apoptosis in pancreatic cancer cells, renal carcinoma Caki cells, neuroblastoma and colorectal cancer cells (39)(40)(41)(42). Furthermore, previous studies demonstrated that the inhibition of glycolysis, or caloric restriction may promote AMPK-induced apoptosis in cancer cells (43,44), which indicated that the activation and phosphorylation of AMPK could be used as a therapeutic strategy for the treatment of cancer. We hypothesized that 3-BP, an inhibitor of glycolysis, activated AMPK through an effect on cellular energy status. Western blot assays demonstrated that levels of p-AMPK were increased following treatment with 3-BP in a concentration-dependent manner. However, the expression of AMPK-α did not show a significant difference post-treatment. To investigate the association between p-AMPK and ER stress response, MCF-7 and MDA-MB-231 cells were treated with Compound C, an inhibitor of AMPK. Western blot analysis revealed that the increased expression levels of ER stress response associated proteins following treatment with 3-BP and TRAIL was attenuated following treatment with Compound C. In addition, the viability of cells treated with Compound C and 3-BP and/or TRAIL significantly decreased, while the expression of Bcl-2 family proteins (Bcl-2 and Bax) and caspase-3 did not significantly differ compared with the control group. These results indicated that the 3-BP induced ER stress response may be associated with the activation and phosphorylation of AMPK.
In conclusion, the results of the present study demonstrated that 3-BP, a hexokinase II inhibitor that interferes with glycolysis, upregulated GRP78, CHOP and DR5 expression levels and enhanced TRAIL-induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. These results revealed that the ER stress response had a crucial role in sensitizing human breast cancer cells to TRAIL-induced apoptosis, potentially via the activation and phosphorylation of AMPK. Further studies are required to determine whether the simultaneous treatment with 3-BP and TRAIL may represent a novel therapeutic strategy for patients with cancer.