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Adenanthin targets peroxiredoxin I/II to kill hepatocellular carcinoma cells.

Adenanthin, a natural diterpenoid isolated from the leaves of Isodon adenanthus, has recently been reported to induce leukemic cell differentiation by targeting peroxiredoxins (Prx) I and II. On the other hand, increasing lines of evidence propose that these Prx proteins would become potential targets to screen drugs for the prevention and treatment of solid tumors. Therefore, it is of significance to explore the potential activities of adenanthin on solid tumor cells. Here, we demonstrate that Prx I protein is essential for the survival of hepatocellular carcinoma (HCC) cells, and adenanthin can kill these malignant liver cells in vitro and xenografts. We also show that the cell death-inducing activity of adenanthin on HCC cells is mediated by the increased reactive oxygen species (ROS) levels. Furthermore, the silencing of Prx I or Prx II significantly enhances the cytotoxic activity of adenanthin on HCC, whereas the ectopic expression of Prx I and Prx II but not their mutants of adenanthin-bound cysteines can rescue adenanthin-induced cytotoxicity in Prxs-silenced HCC cells. Taken together, our results propose that adenanthin targets Prx I/II to kill HCC cells and its therapeutic significance warrants to be further explored in HCC patients.

Peroxiredoxins (Prxs) belong to an important superfamily of small nonseleno peroxidases that scavenge hydrogen peroxide (H 2 O 2 ) and organic hydroperoxide, and are essential for maintaining reactive oxygen species (ROS) homeostasis in the cells. 1 On the basis of the numbers of conserved cysteine (Cys) residues participating in the redox reaction, Prxs are divided into the typical 2-Cys Prxs (including Prx I À IV), atypical 2-Cys Prx (Prx V) and 1-Cys (Prx VI). 2 The typical Prxs form homodimers through an intersubunit disulfide bond, whereas the atypical Prx V forms a monomer with intramolecular disulfide bond during H 2 O 2 catalysis. Besides their cytoprotective function against oxidative stress, Prxs also contribute to the regulation of H 2 O 2 signaling in cellular responses to stimulators such as growth factors and tumor necrosis factors. [2][3][4][5] Emerging evidence has implicated that the aberrant expression of 2-Cys Prx isoform proteins are correlated with several human diseases including cancers, and the elevated Prx expressions are discovered in various cancer tissues, which is possibly due to the adaptive response to oxidative stress facilitating cancer cell growth. 6,7 On the other hand, knockout of Prx I gene causes spontaneous tumor formation in aging mice, proposing that Prx I can also function as tumor suppressor. Further, it has been shown that Prx I interacts with c-Myc and PTEN, thereby inhibiting c-Myc transcriptional activity and protecting PTEN, respectively, from oxidation-induced inactivation. [8][9][10] Moreover, modulated expression of Prxs with small interfering RNA (siRNA) or transfection can potentiate or attenuate, respectively, the sensitivity of radiotherapy and chemotherapy linked with clinical outcomes and overall patient survival. 11 These studies suggest that Prxs would be potential therapeutic targets for cancer prevention and treatment. 12 Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality and the fifth most common cancer worldwide. 13 HCC is often diagnosed at an advanced stage so that patients lose the opportunity of surgical resection. 14,15 There has been an explosion in our understanding of the pathophysiology and molecular alterations occurring in HCC and a series of molecular biomarkers that have potential prognostic significance and may be therapeutic targets have been identified over the past decades. [16][17][18][19] However, sorafenib is regarded as the only effective agent available in clinic for these kinds of patients, but it is not completely satisfactory in HCC patients with extrahepatic spread or macrovascular invasion. 20 Thus, other effective agents need to be further explored.
Previously, our group reported that adenanthin, a diterpenoid isolated from the leaves of Isodon adenanthus, directly targets Prx I and Prx II and inhibits their peroxidase activities, resulting in differentiation induction of acute myeloid leukemia cells. 21,22 In this work, we investigated the effect of adenanthin on HCC cells, and find that the natural agent can kill malignant liver cells in vitro and xenografts through targeting Prx I and Prx II and thus increasing ROS level. These findings validate that Prx I/II are the effective drug targets for killing cancer cells and that therapeutic significance of adenanthin warrants to be further explored in HCC patients.

Results
Adenanthin effectively kills malignant liver cells. We first detected the possible effect of adenanthin on the growth of three human HCC cells (SMMC-7721, Bel-7402 and HepG2) and two human immortal hepatic cell lines (QSG-7701 and HL-7702). For this purpose, these cells were exposed into different concentrations of adenanthin for 24  We then carried out the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to assess the cell death with VP16 treatment as controls. As depicted in Figure 1b, VP16 similarly induced all five cell lines to undergo cell death in a dose-dependent manner. Thus, 50 mM of VP16 was used as a positive control in the following experiments for cell death assay. Under light microscope, less living cells were seen in HepG2 cells under treatment of adenanthin at 3 mM and SMMC-7721 and Bel-7402 cells under adenanthin at 9 mM for 48 h (data not shown). Therefore, we used 3 and 9 mM of adenanthin to, respectively, treat HepG2 and other four cells for 24 and 48 h, followed by TUNEL assay. The results showed that adenanthin for 24 or 48 h induced death of three HCC cell more sensitively than those in two immortal hepatic cell lines (Figure 1c). In line with this notion, adenanthin induced caspase-3 activation with the cleavage of its substrate poly-(adenosine diphosphate ribose) polymerase (PARP) in HCC cells in concentration-and time-dependent manners, which was not found in QSG-7701 and HL-7702 cells (Figures 1d and 1e). Collectively, these results suggest that adenanthin more effectively kills malignant liver cells.
ROS mediates adenanthin-induced cell death of HCC cells. It is believed that adaptation to the enhanced cellular stress (such as oxidative stress) is required for cancer cells to survive, and consequently, cancer cells may become addicted to non-oncogenes that do not ordinarily perform such a vital function in normal cells. 23,24 To address whether the oxidative stress contributes to responses of HCC and human immortal hepatic cells to adenanthin, all five liver cell lines were treated with adenanthin at 3 mM (for HepG2) or 9 mM (for other cells) for 1 h, followed by incubation with fluorophore dichlorodihydrofluorescein diacetate (DCFDA), a dye to monitor intracellular ROS, for 30 min. As consistent with the previous reports, 25 all three HCC cells displayed higher basal ROS levels than QSG-7701 and HL-7702 cells, as assessed by the fluorescent signal intensity on flow cytometry (Figures 2a and b). More intriguingly, adenanthin significantly increased ROS production in all three HCC cells rather than two human immortal hepatic cell lines tested (Figures 2a and b). We also applied DCP-Bio1, a probe to detect cysteine sulfenic acid of oxidative proteins and therefore the oxidative modification of proteins in the cellular context, 26 into cell lysates from HL-7702 and SMMC-7721 with and without adenanthin treatment. Western blot analysis showed that adenanthin-treated SMMC-7721 but not HL-7702 cells contained significantly increased protein oxidization signaling compared with vehicle-treated control cells (Figure 2c), suggesting the increased ROS stress in HCC cells by adenanthin.
To address whether increased ROS production mediates killing of adenanthin to cancer cells, these cells were pretreated by a general ROS scavenger N-acetyl-l-cysteine (NAC). Although NAC treatment did not substantially lower the basal ROS level of HCC cells, as reported, 27 it did effectively inhibit adenanthin-induced ROS accumulation and protein oxidization in all three HCC cells (Figures 2a-c). Accordingly, NAC pretreatment could completely abrogate adenanthin-induced cell death of three HCC cells, as determined by morphologic observations, TUNEL assay and caspase-3 activation/PARP cleavage (Figures 2d-f). Considering the possibility that NAC likely protects cells by a direct chemical reaction with adenanthin in analogy to the proposed mechanism for adenanthin's actions, that is, covalent reaction with cysteine 173 of Prx I, 21 we also pre-treated three HCC cells with two additional thiol antioxidants including glutathione and dithiothrietol and two non-thiol antioxidants, vitamin E analog tiron (sodium 4,5-dihydroxybenzene-1,3disulfonate) 28 and a superoxide dismutase mimetic tempol. 29 The results demonstrated that like NAC, all other four antioxidants could significantly inhibit adenanthin-induced ROS production and cell death ( Figure 2g). These data suggest that increased ROS is critical for adenanthin-induced death of HCC cells.
Prx I is required for HCC cell survival. Previously, we reported that adenanthin targets Prx I and Prx II, and inhibit their peroxidase activities, resulting in ROS accumulation of leukemic cells. 21,22 More recently, Muchowicz et al. 30 also reported that adenanthin can bind with intracellular Prx I and Prx II. On the basis of this notion, we used siRNA specifically against Prx I (designated siPrx I#1) to knock Prx I down in HCC and immortal hepatic cell lines. Our preliminary experiments showed that, on 6-8 days after transfection of siPrx I#1, all HCC cells underwent death. Thus, we further transfected with GFP-tagged siPrx I#1 and a non-specific siRNA as a negative control (NC) into HCC and immortal hepatic cell lines for 48 h. Then, GFP-positive cells were sorted. As depicted in Figure 3a, siPrx I#1 but not NC completely eliminated the endogenous Prx I but not Prx II protein expression in all five cell lines. HCC cells but not immortal hepatic cells underwent cell death on the sixth day after sorting (Figure 3b). These results suggest that Prx I is essential for the survival of HCC cells.
We further transfected another pair of siRNAs specifically against Prx I (designated siPrx I#2) and one pair of siRNAs against Prx II (siPrx II) together with a NC siRNA into two human immortal hepatic cell lines. As depicted in Figure 3c, siPrx I#2 partially silenced Prx I rather than Prx II expression. Vice versa, siPrx II suppressed Prx II but not Prx I expression, proposing the specificity of these siRNAs against Prx I and Prx II. As expected, the silencing of Prx I or Prx II did not accumulate ROS in these cells in the presence or absence of adenanthin ( Figure 3d). Accordingly, they also failed to trigger these cells to undergo death, as evidenced by CCK-8 assay (Figure 3e), TUNEL assay ( Figure 3f) and caspase-3 activation (data not shown). All these data support that Prx I and Prx II are not essential for survival of these immortal hepatic cell lines.
Prx I/II suppression significantly enhances adenanthininduced HCC cell death. On the other hand, siPrx I#2 expression partially knocked down Prx I whereas siPrx II remarkably silenced Prx II expression in HCC cells tested (Figure 4a). These siRNAs' expressions did not significantly affect the growth and viability of these HCC cells. We also tried to establish double silencing of Prx I and Prx II in HCC cells, but we found that double depletion of Prx I and Prx II could trigger cell death so as that a stable Prx I/II doubledeleted cell line could not be established, which was similar to the effect of complete elimination of Prx I. The results support that there is compensation between Prx I and Prx II for survival of HCC cells. Therefore, we only assessed the effects of adenanthin on two HCC cells (SMMC-7721 and Bel-7402) with the stable expression of siPrx I#2 or siPrx II alone. Compared with NC-transfected cells, partial suppression of Prx I increased intracellular ROS accumulation in HCC cells to a degree but with statistical significance and remarkably potentiated adenanthin-induced ROS accumulation in HCC cells (Figure 4b). In line with the notion, partial suppression of Prx I significantly enhanced cytotoxic effects of adenanthin on SMMC-7721 and Bel-7402 cells, as assessed by CCK8 (Figure 4c), caspase-3 activation/PARP cleavage ( Figure 4d) and TUNEL assays (Figure 4e). Likewise, the significant suppression of Prx II by siPrx II also enhanced 9 mM adenanthin-induced ROS accumulation and cell death in HCC cells (Figures 4b-d and f). Totally, the decreased Prx I or Prx II expression increases the sensitivity of HCC cells to adenanthin.
Re-introduction of Prx I/II abrogates the activity of adenanthin on Prxs-silenced HCC cells. To further validate the role of Prx I and Prx II in adenanthin-induced HCC cell death, we infected lentiviruses carrying Flag-tagged and siRNA-resistant wild type (WT) Prx I or Prx II and their mutants with adenanthin-targeted cysteines (C173S-Prx I and C172S-Prx II), 21 together with the empty vector as control, into SMMC-7721 and Bel-7402 cells in the presence of Prx I and Prx II knockdown, respectively, by siPrx I#2 and siPrx II, as shown in Figure 4a. The expression of Flag-tagged WT-Prx I/II and C173S-Prx I or C172S-Prx II were confirmed by western blot (Figures 5a and b). These cells were, respectively, treated with and without adenanthin at indicated concentrations for 24 (5c, e) or 18 (5d, f) h. The results showed that re-expression of WT-Prx I but not C173S-Prx I could abrogate adenanthin-induced cell death in siPrx I#2-expressing SMMC-7721 and Bel-7402 cells. Of note, C173S-Prx I overexpression potentiated adenanthininduced cell death (Figures 5c and d). In addition, reexpression of WT-Prx II but not C172S-Prx II could rescue adenanthin-induced cell death in siPrx II-expressing SMMC-7721 and Bel-7402 cells (Figures 5e and f). Taken together, these results further confirm that Prx I and Prx II directly contribute to adenanthin-induced cytotoxicity of HCC cells.
Adenanthin exhibits significant therapeutic activity in HCC xenografts. Finally, we subcutaneously injected SMMC-7721 cells into BALB/c nude mice. Two weeks later, these mice were intraperitoneally injected with adenanthin (10 or 20 mg/kg) or saline as control by the protocol described in materials and methods. Administration of adenanthin at 10 mg/kg did not present observable toxic effects on mice, but 20 mg/kg adenanthin decreased body weight of mice compared with the control and 10 mg/kg adenanthin-treated mice (Figure 6a). At the end of therapy, autopsies showed that adenanthin-treated mice had much smaller tumor masses (Figure 6b). The tumor volumes and average tumor weights with the treatment of adenanthin of 10 Figure 3 The effects of Prx I or Prx II silencing and/or adenanthin on human hepatic and HCC cells. (a, b) The cells were infected with EGFP-tagged siPrx I#1 for 2 days, followed by sorting for EGFP-positive cells. Prx I and Prx II proteins were examined by western blots with b-tubulin as a loading control (a). TUNEL þ cells % (b) were observed and analyzed when the sorted cells were cultured for 4 (for HepG2) or 6 days (for other cells). The symbol * indicates Po0.01 compared with NC siRNA-expressing cells. (c-f) Two human immortal hepatic cell lines were stably infected with siPrx I#2 or siPrx II, together with NC as controls. Prx I and Prx II proteins were examined by western blots with b-tubulin as a loading control(c). Fluorescent intensities of DCFDA staining were quantified on flow cytometry for the cells treated with and without adenanthin at 9 mM for 1 h. Data represent means with bar as S.D. of triplicate samples in an independent experiment (d). These siRNA-infected cells were treated with and without indicated concentrations (e) or 9 mM adenanthin (f) for 24 h, followed by CCK-8 (e) and TUNEL assays (f). All experiments were repeated three times with the same results Figure 2 The role of ROS in adenanthin-induced cell death of HCC cells. (a, b) After pretreatment with and without 2 mM NAC for 4 h, cells were treated with or without adenanthin at 3 mM (for HepG2) or 9 mM (for the other cells) for 1 h. Then, fluorescent intensity of DCFDA staining was analyzed on flow cytometry (a) and quantified data were shown (b). Data represent means with bar as S.D. of triplicate samples in an independent experiment. The symbols * and # stand for Po0.01 compared, respectively, with the vehicle-and adenanthin-treated cells. (c) Cell lysates of HL-7702 and SMMC-7721 cells with treatment as performed in panel (a) were probed with DCP-Bio1, followed by western blot with streptavidin. Coomassie blue staining was used as protein loading control. Null indicates the cell lysates without probe incubation. (d-f) After pretreatment with and without 2 mM NAC for 4 h, three HCC cells were incubated with and without adenanthin at 3 (for HepG2) or 9 mM (for other two cells) for 48 h, followed by morphological observation (d), TUNEL assay (e) and western blots for the indicated proteins (f). The scale bars in panel (d) are 100 mm. In panel (e), the symbol * presents Po0.01 compared with the vehicle-treated cells. All experiments were repeated three times with the same results. (g) After pretreatment with and without 2 mM NAC, 1 mM dithiothrietol, 5 mM glutathione, 1 mM tiron or 2 mM tempol for 4 h, the cells as indicated were treated with or without adenanthin at 3 (for HepG2) or 9 mM (for SMMC-7721 and Bel-7402) for 1 (upper part) or 48 h (lower part). The fluorescent intensity of DCFDA staining were analyzed and quantified on flow cytometry (upper part) and TUNEL-positive cells % was calculated (lower part). All data represent means with bar as S.D. of triplicate samples in an independent experiment. The symbol * stands for Po0.01 compared with the adenanthin alone-treated cells and 20 mg/kg group were significantly decreased in comparison with control mice, although 10 and 20 mg/kg of adenanthin appeared to present the similar therapeutic activity (Figures 6c and d).

Discussion
Ent-kauranes are the largest and widely distributed group of the known Isodon diterpenoids, and have recently been shown as therapeutic agents against bacterial infection, tumor, inflammation and feeding. 31 Here, we report that a natural diterpenoid designated as adenanthin can induce cell death of HCC cells more effectively than that of two human immortal hepatic cell lines. Accordingly, the agent can also significantly inhibit the growth of xenografts of HCC cells without significant toxic effects. ROS, such as H 2 O 2 , arise during normal aerobic metabolism. They are constantly generated during electron transfer in the respiratory chain, in peroxisomes, and as products of a growing list of NADPH oxidases. 4,32 The increase in ROS generation is one common biochemical change in cancer cells. Conventionally, increased ROS in cancer cells is considered as an adverse factor, because ROS increase appears to play an important role in maintaining cancer phenotype because of their stimulating effects on cancer cell proliferation, genetic instability and senescence evasion. 33,34 However, increasing lines of evidence show that the excessive levels of ROS stress may be beneficial to the clearance of cancer cells, in which cells are likely to be more vulnerable to damage when further ROS insults are induced by exogenous agents. 35,36 Therefore, manipulating ROS levels by redox modulation would become an ideal way to the prevention and treatment of cancers. 37 On the basis of these notions, we show that adenanthin moderately increases ROS accumulation in HCC cell lines, which exhibit a significantly higher basal ROS level than human hepatic cells. On the other hand, all three thiol and non-thiol antioxidants significantly block death-inducing activity of adenanthin. These data support that adenanthin kills HCC cells through the increased ROS, which is reminiscent of piperlongumine and b-phenylethyl isothiocyanate. These two small molecules were reported to selectively kill cancer cells through ROSmediating mechanisms. 35,36 Accumulating evidence showed that Prxs protect cells and tissues from oxidative damage through their peroxidase activities. Prx I and II have also been found to have antiapoptotic functions through direct or indirect interactions with the key cell death regulators, such as apoptosis signal  40 Previously, we reported that adenanthin can target to and inhibit peroxidase activities of Prx I and Prx II. 21,22 Therefore, we asked whether adenanthin increases ROS production in HCC cells through targeting Prx I and Prx II.
Although several lines of evidence demonstrated that the expressions of Prx I and Prx II were higher in HCC tissues compared with those in adjacent non-tumorous liver tissues or pre-HCC tissues, [41][42][43] here we found that all three HCC and two human immortal hepatic cell lines have the similar Prx I or Prx II levels. Even so, HCC cells are more sensitive to adenanthin than human immortal hepatic cells, suggesting that Prx I/II exert a critical function for survival of HCC but not other two immortal hepatic cells. Indeed, we show that Prx I is necessary for the survival of HCC, because the siRNAmediated complete suppression of Prx I expression can directly trigger HCC cells to undergo death. Hence, we used adenanthin to treat HCC and immortal hepatic cells with the partial suppression of Prx I and great inhibition of Prx II, under which all cells can survive. Our results show that the suppression of Prx I or Prx II expression significantly enhances ROS-accumulating and killing effects of adenanthin on HCC cells, which can be abrogated by the ectopic expressions of Prx I and Prx II but not their mutants with adenanthin-bound sites. It should be pointed out that partial decrease of Prx I protein by siPrx I#2 did not elevate ROS levels substantially, whereas adenanthin elevated ROS to two to three folds, suggesting that adenanthin might elevate ROS by mechanisms beyond modulation of Prx I, which remains to be further explored. Actually, a recent report showed that adenanthin can also bind with thioredoxin-1 (Trx-1) and inhibits the activity of Trxthioredoxin reductase system and protein disulfide isomerase in vitro and in cultured cells. 30 Additionally, adenanthin was also reported to inhibit NF-kB signaling in experimental autoimmune encephalomyelitis. 44 Therefore, we also evaluated whether NF-kB signaling is involved in adenanthin-induced HCC cell death. The results showed that the overexpression of p65, one subunit of NF-kB, did not affect adenanthin-induced death of three HCC cells (data not shown).
Collectively, our findings propose that Prx I and II mediate the death-inducing selectivity of adenanthin on HCC, and adenanthin can serve as the natural lead compound for the development of Prx I-and Prx II-targeted selective therapeutic agents for HCC patients. Detection of ROS by flow cytometry. Cells were washed with phosphate-buffered saline and then re-suspended in pre-warmed phosphatebuffered saline (37 1C), followed by incubation with 10 mM 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (C-6827, CM-H2-DCFDA, Invitrogen, Carlsbad, CA, USA) for 30 min at 37 1C. Next, the cells were washed with phosphate-buffered saline twice and scraped into 0.3 ml of ice-cold phosphate-buffered saline. CM-H2-DCFDA fluorescence was determined by measuring 10 000 events per sample following excitation with a 488 nm wavelength laser and reading through a 530/30 filter (FACSCalibur, BD Bioscience, San Jose, CA, USA).
Plasmids and transfection. Human Prx I and Prx II cDNAs were amplified from HEK293T cells by reverse transcription-PCR, and then cloned into pLVX-IRES-ZsGreen vector (PT4064-5, Clontech, Palo Alto, CA, USA) for generating Prx I and Prx II expression plasmids. SiRNA-resistant wide-type and cysteinemutated Prx I/II (C173S and C172S) plasmids were generated by QuickChange Lightning Site-Directed Mutagenesis Kit (210518, Stratagen, La Jolla, CA, USA). The plasmids we made were further confirmed by DNA sequencing (Biosune, Shanghai, China). Transient transfection was performed with Hilymax transfection reagents according to the manufacturer's instructions (H357, Dojindo Molecular Technologies).
Animal experiments. Four-week-old male BALB/c nude mice were obtained from Shanghai laboratory of animal research center, Shanghai, China. SMMC-7721 cells (3 Â 10 6 ) were subcutaneously injected in one side of ventral flanks. When tumor volumes reached to 100 mm 3 , the mice with tumor burden were randomly grouped and injected intraperitoneally with adenanthin (10 mg/kg and 20 mg/kg) or saline (injection daily for 3 days and resting 1 day). The total treatment period is 15 days. Tumor growth was monitored by caliper rule every 3 days. 45 At the end of adenanthin treatment, mice were killed and tumors were picked, photographed, size measured and weighed. All animals were handled in strict accordance to the 'Guide for the Care and Use of Laboratory Animals' and the 'Principles for the Utilization and Care of Vertebrate Animals', and all animal work was approved by the Institutional Animal Care and Use Committee at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine.
Statistical analysis. Student's t-test was used to evaluate differences between two groups. A P-value of less than 0.05 or 0.01 was considered statistically significant.

Conflict of interest
The authors declare no conflict of interest.