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Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells
Life Sciences 92 (2013) 352–358
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Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells Wei-Zhang Wang a, b, c,⁎, 1, Li Li d, 1, Man-Yu Liu a, Xiao-Bao Jin a, Jian-Wen Mao a, Qiao-Hong Pu a, Min-Jie Meng b, Xiao-Guang Chen c, Jia-Yong Zhu a a
Guangdong Province Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, PR China Guangzhou Higher Education Mega Center Health Industrial Science and Technology Park Investment Management Co. Ltd., Guangzhou, PR China School of Pubic Heath and Tropical Medicine, Southern Medical University, Guangzhou, PR China d Department of Hematology, Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA, Guangzhou, PR China b c
a r t i c l e
i n f o
Article history: Received 14 October 2012 Accepted 3 January 2013 Keywords: Curcumin Apoptosis FasL p38 Huh7 cells
a b s t r a c t Aim: The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. Main methods: Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. Key findings: Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. Significance: Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells. © 2013 Elsevier Inc. All rights reserved.
Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the third leading cause of death from cancer globally, with 600,000 deaths per year (Villanueva et al., 2010). As HCC is highly resistant to standard chemotherapy, surgical resection or liver transplantation is still the mainstay of treatment for HCC and provides the consistent long-term survival. However, about 80% of HCC patients present with advanced disease that are not amenable to surgical resection or transplantation, and thus have a poor prognosis (Thomas and Abbruzzese, 2005). Therefore, it remains a challenge to identify safe and effective treatment options for advanced HCC. Curcumin (diferuloylmethane) is a naturally occurring yellow pigment isolated from turmeric (Curcuma longa) and commonly used
⁎ Corresponding author at: Guangdong Province Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Wai Huan Dong Road 280, Guangzhou Higher Education Mega Center, Guangzhou 510006, PR China. Fax: + 86 20 39352617. E-mail addresses: [email protected], [email protected] (W.-Z. Wang). 1 These authors contribute equally to this work. 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.01.013
as flavoring food additive for centuries exhibits anti-inflammatory, antioxidant and anticancer properties (Sharma et al., 2005). The anticancer potential of curcumin stems from its ability to induce apoptosis in a wide variety of cancer cell lines in vitro (Karunagaran et al., 2005), including HCC cell lines (Jiang et al., 1996). Furthermore, curcumin also has the ability to inhibit HCC cell invasion (Lin et al., 1998) and angiogenesis (Yoysungnoen et al., 2005). In vivo, curcumin has been shown to effectively inhibit DEN-induced hepatocarcinogenesis in the mouse model (Chuang et al., 2000). Most importantly, curcumin is safe for humans as evidenced by the phase I clinical trial showing that curcumin is not toxic to humans when taken orally for 3 months at up to 8000 mg/day (Cheng et al., 2001). These data suggest that curcumin has a great potential in HCC prevention and therapy. We have shown previously that curcumin induces apoptosis in hepatocellular carcinoma Huh7 cells (Wang et al., 2008), but the underlying mechanism of curcumin-induced apoptosis remains largely unknown. In the present study, we found that curcumin-induced apoptosis was associated with up-regulation of FasL in a p38dependent manner. Furthermore, by using neutralizing antibody against FasL, we showed that the increase in FasL expression was crucial for the curcumin-induced apoptosis.
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Materials and methods Cell culture Human hepatocellular carcinoma cell line Huh7 (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences) was grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C with 5% CO2 in a fully humidified atmosphere. Antibodies and reagents Sources of antibodies are as follows: the anti-Bcl-2 monoclonal antibody, anti-Bcl-xl monoclonal antibody, anti-Bax monoclonal antibody, anti-Bak monoclonal antibody, anti-Mcl-1 monoclonal antibody, neutralizing anti-human FasL (NOK-2) antibody and isotype-matched control antibody (BD Pharmingen International, San Diego, CA); rabbit polyclonal or monoclonal antibodies against Bid, p38, phospho-p38 (Thr180/Tyr182), JNK, phospho-JNK (Thr183/Tyr185), ERK, phospho-ERK (Thr202/Tyr204), c-Jun, phospho-c-Jun (Ser 73), c-Fos, ATF-2, phospho-ATF-2 (Thr 71), cleaved caspase-3, DR5, TNFR and cleaved PARP (Cell Signaling Technology, Beverly, MA); mouse monoclonal antibodies against phospho-c-Fos (Ser374), β-actin, DR4, Fas and FasL (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-mouse or anti-rabbit IgG horseradish peroxidaseconjugated antibody (DAKO, Carpinteria, CA). The following pharmacologic agents were purchased from the respective vendors: Curcumin (Sigma-Aldrich, Inc., St. Louis, MO); Trizol (Invitrogen, Carlsbad, CA); MMLV reverse transcriptase and p38 inhibitor SB203580 (Promega, Madison, WI); Z-DEVD-FMK (Merck-Calbiochem, Germany); and ECL reagents (Pierce, Rockford, IL). Fluorescence-activated cell sorting (FACS) analysis for apoptosis At the end of indicated treatments, both floating and adherent cells were collected and analyzed. Briefly, the cells were washed twice with
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1 × PBS and collected by trypsinization. After centrifugation at 400 ×g for 5 min at room temperature, the cells were double stained with Annexin V-FITC and propidium iodide (PI) as recommended by the manufacturer (MBL, Woburn, MA). The cell apoptosis distributions were determined on a FACSCalibur flow cytometer (BD, San Jose, CA) and analyzed by CellQuest Pro software program. At least 10,000 cells were measured for each sample. RNA analysis and reverse transcription-polymerase chain reaction (RT-PCR) RNA was extracted from cell pellets using Trizol and quantitated by ultraviolet spectrophometry. Total RNA (2 μg) was reverse transcribed using MMLV reverse transcriptase for RT-PCR using specific primers for Fas and FasL. The primer sequences used for PCR amplification were: (sense) 5′-ATA AGC CCT GTC CTC CAG GT-3′ and (anti-sense) 5′-TGG AAG AAA AAT GGG CTT TG-3′ for Fas; (sense) 5′-GGC CTG TGT CTC CTT GTG AT-3′ and (anti-sense) 5′-TGC CAG CTC CTT CTG TAG GT-3′ for FasL; (sense) 5′-CTC TGC TCC TCC TGT TCG AC-3′ and (anti-sense) 5′-ACG ACC AAA TCC GTT GAC TC-3′ for glyceraldehydes-3-phosphate dehydrogenase (GAPDH). PCR conditions were as follows: an initial denaturation step at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. After a final extension at 72 °C for 7 min, PCR products were then separated on 2% agarose gels and bands were visualized with ethidium bromide (EB). Western blot analysis For protein analysis, both floating and adherent cells after the indicated treatments were collected and lysed with 1 × SDS sample buffer [62.5 mM Tris–HCl (pH 6.8 at 25 °C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue, 2.5 μM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin]. The extracted proteins were loaded onto 8% to 15% SDS-polyacrylamide gels, electrophoretically transferred to nitrocellulose membranes, and then detected by specific antibodies
Fig. 1. Effect of curcumin upon the expression of Bcl-2 family, TNF receptor family and the respective ligands. (A) and (B) Huh7 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were then prepared and analyzed for protein levels of Bcl-2 family (Bcl-2, Bcl-xL, Mcl-1, Bax, Bak and Bid), TNF receptor family (Fas, TNFR, DR4 and DR5) and the respective ligands (FasL and TRAIL) by Western blot using β-actin as loading control. Top panels: Western blot analysis. Bottom panel: quantification of Fas/FasL protein expression from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001, compared with no curcumin treatment). (C) Curcumin induces Fas and FasL mRNA expression. Huh7 cells were treated with 50 μM curcumin for the indicated times, and the total RNA was extracted and examined for expression of Fas and FasL by RT-PCR. GAPDH was used as an internal control to show equal RNA loading.
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Fig. 2. Fas/FasL interaction is required for curcumin-induced apoptosis. Huh7 cells were incubated with or without curcumin for 1 h, followed by treatment with either a neutralizing anti-FasL antibody NOK-2 (25 or 50 μg/ml) or an isotype-matched control IgG (25 or 50 μg/ml) for another 24 h, and then apoptosis was evaluated by FACS. Top panels: representative data showing apoptosis. Bottom panel: statistical analysis of apoptosis (***P b 0.001; n.s., not significant). All data are expressed as the mean ± S.D. of at least three separate experiments. Ctrl, control; Cur, curcumin; Ctrl Ab, control antibody.
and commercial ECL kit. All Western blot results were repeated 3 times, and representative results were selected for inclusion. The quantification of protein was performed by densitometry
analysis using ImageJ software (NIH). Fold changes in protein expression were determined on the basis of the β-actin loading control.
Fig. 3. Role of caspase-3 in curcumin-induced apoptosis. (A) Curcumin induces caspase-3 activation and PARP cleavage. Huh7 cells were treated with 50 μM curcumin for 24 and 48 h, and whole cell extracts were then prepared and analyzed by Western blot for the cleaved caspase-3 and PARP protein levels using β-actin as loading control. Left-hand panels: Western blot analysis. Right-hand panel: quantification of cleaved caspase-3/PARP protein expression from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001, compared with no curcumin treatment). (B) Z-DEVD-FMK inhibits curcumin-induced apoptosis. Huh7 cells were incubated with Z-DEVD-FMK or vehicle for 1 h, followed by treatment with or without curcumin (50 μM) for another 24 h, and then apoptosis was evaluated by FACS (**P b 0.01, compared with curcumin alone treatment). All data are expressed as the mean ± S.D. of at least three separate experiments. Ctrl, control; Cur, curcumin.
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Fig. 4. Effect of NOK-2 on curcumin-induced caspase-3 activation. Huh7 cells were incubated with or without 50 μM curcumin for 1 h, followed by treatment with either NOK-2 (25 or 50 μg/ml) or control IgG (25 or 50 μg/ml) for another 24 h and 48 h, and whole cell extracts were then prepared and analyzed by Western blot for the cleaved caspase-3 protein level using β-actin as loading control. Top panels: Western blot analysis. Bottom panel: quantification of cleaved caspase-3 protein expression from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001; n.s., not significant). Cur, curcumin; Ctrl Ab, control antibody.
Statistical analysis Values represent the mean ± S.D. from at least three independent experiments. Differences between the experimental groups were analyzed using two-sided Student's t-test. A value of P b 0.05 was considered statistically significant. Results The expression of Fas and FasL is significantly up-regulated upon curcumin treatment It has been well demonstrated that apoptosis is mainly mediated through two pathways: the intrinsic (mitochondrial) pathway that is regulated by Bcl-2 family members (Brunelle and Letai, 2009) and extrinsic (death receptor) pathway that is regulated by cell surface receptors (Xu and Shi, 2007). To explore the potential mechanism underlying curcumin-induced apoptosis, Huh7 cells were treated with curcumin (50 μM) for different times (2, 4, 8, 12 and 24 h) and then examined for the expression of Bcl-2 family proteins, death receptors and their respective ligands by Western blot. As shown in Fig. 1A, we found that little effect of curcumin treatment on the expression of Bcl-2, Bcl-xl, Mcl-1, Bax, Bak and Bid proteins at all time points was examined. In contrast, it significantly increased the expression of Fas and FasL at both protein (Fig. 1B) and mRNA levels (Fig. 1C) as early as 2 h, which further sustained over the following 24 h period. The expression of DR4, DR5, TNFR and TRAIL remained unchanged
and the expression of TNF was detected at none of the time points examined after curcumin treatment (Fig. 1B and data not shown). Fas/FasL interaction is required for curcumin-induced apoptosis As activation of Fas/FasL pathway plays a critical role in a variety of drug-induced apoptotic cell death (Friesen et al., 1996; Fulda et al., 2000; Kasibhatla et al., 1998), we next determined whether blocking Fas/FasL interaction influenced curcumin-induced apoptosis in Huh7 cells by treating the cells with NOK-2, an antibody against human FasL, which recognizes and neutralizes both membrane-bound and soluble forms of FasL, thereby preventing their interaction with Fas. Our results showed that curcumin treatment (50 μM, 24 h) induced a remarkable apoptotic cell death of Huh7 cells, neutralization of FasL by NOK-2 protected the cells from curcumin-induced apoptosis in a dose-dependent manner, whereas no significant effect was found with an isotype-matched IgG control (Fig. 2). This data suggests that curcumin-induced apoptosis is highly correlated with Fas/FasL pathway in Huh7 cells. Curcumin-induced apoptosis is dependent upon caspase-3 activity Binding of FasL to Fas triggers a caspase cascade and eventually results in the activation of the critical executioner caspase, caspase-3 and consequent apoptosis (Nagata, 1997). Therefore, we analyzed the protein level of activated caspase-3 by Western blot in Huh7 cells upon curcumin treatment. We observed that curcumin treatment led
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Fig. 5. p38 is required for curcumin-induced FasL up-regulation and apoptosis. Huh7 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were then prepared and analyzed by Western blot for p38, phospho-p38, JNK, phospho-JNK, ERK and phospho-ERK protein levels (A) and ATF-2, phospho-ATF-2, c-jun, phospho-c-jun, c-fos and phospho-c-fos protein levels (B) using β-actin as loading control. Left-hand panels: Western blot analysis. Right-hand panels: quantification of p-p38/p38, p-JNK/JNK, p-ERK/ERK, p-ATF-2/ATF-2, p-c-jun/c-jun and p-c-fos/c-fos from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001 and n.s., compared with no curcumin treatment). (C) Huh7 cells were pretreated with 10 μM p38 inhibitor SB203580 for 2 h, followed by incubation with 50 μM curcumin for another 24 h in the presence of SB203580, and then subjected to detection of p38, phospho-p38, ATF-2, phospho-ATF-2 and FasL expression by Western blot using β-actin as loading control. Left-hand panels: Western blot analysis. Right-hand panels: quantification of p38, phospho-p38, ATF-2, phospho-ATF-2 and FasL from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001 and n.s., compared with no curcumin treatment). (D) Huh7 cells were treated as described in (C) and then subjected to analysis of apoptosis by FACS (**P b 0.01, compared with curcumin alone treatment). All data are expressed as the mean ± S.D. of at least three separate experiments. Cur, curcumin; SB, SB203580; Ctrl, control; n.s., not significant.
to a higher level of active caspase-3 and cleavage of its major substrate PARP in a time-dependent manner (Fig. 3A), suggesting that caspase-3 is activated in response to curcumin in Huh7 cells. To further evaluate the role of caspase-3 in curcumin-induced apoptosis, we pretreated Huh7 cells with Z-DEVD-FMK, a specific caspase-3 inhibitor, prior to and throughout treatment of curcumin and found that Z-DEVD-FMK almost completely inhibited curcumin-induced apoptosis (Fig. 3B), indicating that curcumin-induced apoptosis is dependent upon caspase-3 activity. Moreover, blocking the interaction of FasL and Fas by NOK-2 notably impaired the activation of caspase-3 in a dose- and time-dependent manner (Fig. 4). These results suggest that Fas/FasL signaling-mediated activation of caspase-3 is responsible for curcumin-induced apoptosis.
p38 is required for curcumin-induced FasL up-regulation and subsequent apoptosis To further investigate signaling pathway of curcumin-induced apoptosis in Huh7 cells, we assessed the effect of curcumin on mitogenactivating protein kinases (including p38, JNK and ERK), as they play a critical role in the apoptosis-related signaling pathway. As shown in Fig. 5A, exposure to curcumin resulted in a time-dependent increase in phospho-p38 level, however, under the same condition, we did not detect any significant difference in the phosphorylation of JNK and ERK after curcumin treatment (Fig. 5A). Furthermore, curcumin treatment also led to a time-dependent increase in phosphorylation of ATF-2, a target
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Fig. 6. Effect of NOK-2 on curcumin-induced p38 activation. Huh7 cells were treated as described in Fig. 2, and whole cell extracts were then prepared and analyzed by Western blot for p38 and phospho-p38 protein levels using β-actin as loading control. Top panels: Western blot analysis. Bottom panel: quantification of p-p38/p38 from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001, compared with no curcumin treatment). Cur, curcumin; Ctrl Ab, control antibody.
of p38 signaling pathway, but had no significant effect on c-Jun and c-Fos phosphorylation. These data suggest that p38 but not JNK and ERK is activated in response to curcumin (Fig. 5B). To validate the role of p38, SB203580, a pharmacologic inhibitor of p38 was used to examine the effect of p38 inhibition on curcumin-induced FasL expression and apoptosis. We found that, in contrary to the increased p38 and ATF-2 phosphorylation and FasL expression upon curcumin treatment, pretreatment of the Huh7 cells with SB203580 for 2 h significantly inhibited both FasL protein induction and p38 and ATF-2 phosphorylation (Fig. 5C). Moreover, curcumin-induced apoptosis was dramatically comprised by SB203580 treatment (Fig. 5D). These results indicate that p38 is required for the curcumin-induced FasL expression and apoptosis in Huh7 cells. As binding of FasL to Fas has been shown to activate p38 and its activation is required for apoptosis of CD8+ T cells (Farley et al., 2006), we further determined whether disruption of Fas/FasL interaction influenced p38 activation in Huh7 cells. Our data showed that curcumin could still activate p38 in the presence of NOK-2 (Fig. 6), indicating that blockage of Fas/FasL interaction has no effect on p38 activation and that p38 activation in response to curcumin is an upstream event of Fas/FasL upregulation and interaction in our study model. Discussion HCC has characteristics of rapid growth, early vascular invasion and highly resistant to standard chemotherapy. It has been well established that most cytotoxic agents primarily act by inducing apoptotic cell death in cancer cells. Thus, evasion of apoptosis is believed to be a main contributor to HCC resistance, and targeting apoptosis
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via direct or indirect manipulations of the pro-apoptotic machinery offers a novel strategy for HCC treatment (Kountouras et al., 2003). In the present study, we found that the activation of p38 pathway is highly correlated with curcumin-induced apoptosis. The strong sustained activation of p38 pathway seemed to be a required priming step for curcumin-induced apoptosis; this activation of p38 was correlated with up-regulation of the FasL and accompanied by the induction of apoptosis. Reduction of curcumin-induced apoptosis by the use of either a specific inhibitor of p38 or a neutralizing anti-FasL antibody further underlines the critical role of p38-dependent FasL expression signaling in the induction of apoptosis by curcumin. There are many evidences that curcumin-induced apoptosis mainly involves the mitochondria-mediated pathway in various cancer cells of different tissues of origin (Karunagaran et al., 2005). For example, curcumin induces apoptosis through mitochondrial pathway involving caspase-8, Bid cleavage, cytochrome c release, and caspase-3 activation in human promyelocytic leukemia cell line HL-60 (Anto et al., 2002). However, curcumin also has been found to induce activation of a death receptor pathway in human melanoma cell lines, wherein curcumin induces Fas receptor aggregation in a FasL-independent manner and inhibition of Fas aggregation by low-temperature prevented curcumin-induced cell death (Bush et al., 2001). In the current study, we observed that curcumin increased Fas and FasL expression (Fig. 1B and C) and induced caspase-3 activation and PARP cleavage (Fig. 3A), whereas disruption of the interaction between Fas and FasL suppressed curcumin-induced caspase-3 activation (Fig. 4) and apoptosis (Fig. 2). Taken together, these results suggest that curcumin could also activate death receptor-mediated pathway to induce apoptosis in certain specific type of cancer cell. Despite a large number of in vitro studies on the function of MAPKs, the role of MAPKs in cell death induced by curcumin remains unclear. It has been shown that curcumin induced-apoptosis in human lung adenocarcinoma A549 cells was accompanied by sustained phosphorylation and activation of JNK, p38 and ERK. However, pretreatment with MAPK inhibitors had no effect upon curcumin-induced apoptosis, suggesting that activation of these MAPKs is not associated with curcumin-induced apoptosis in lung cancer (Chen et al., 2010). In human colon cancer HCT116 cells, curcumin treatment also resulted in apoptosis and sustained phosphorylation and activation of JNK and p38. Interestingly, only the JNK-specific inhibitor SP600125, but not p38-specific inhibitor SB203580, could alleviate curcumininduced apoptosis (Collett and Campbell, 2004). Most recently, curcumin has been shown to induce rapid activation of ERK1/2 and JNK as well as apoptosis in human rhabdomyosarcoma Rh30 cells, inhibition of JNK (with SP600125) or ERK1/2 (with U0126) partially prevented curcumin-induced cell death in the cells (Han et al., 2012). In the current study, we found that only p38 was activated by curcumin in human HCC Huh7 cells (Fig. 5A–C) and inhibition of p38 by SB203580 prevented curcumin-induced apoptosis (Fig. 5D). We propose that these discrepancies might be attributed to different functions of MAPKs in response to curcumin. On the one hand, the ERK signaling pathway can be activated by a variety of extracellular stimuli and regulates cell proliferation and cell differentiation (Cagnol and Chambard, 2010). On the other hand, JNK and p38 signaling pathways are responsive to stress stimuli and are involved in cell differentiation and apoptosis (Wagner and Nebreda, 2009). Therefore, the role of JNK or p38 or ERK in curcumin-induced apoptosis may be cell line specific. Our study reveals that induction of FasL is mediated by the activation of p38 in Huh7 cells, as curcumin activates p38 (Fig. 5A–C) and inhibition of p38 abolishes the effect of curcumin on the induction of FasL (Fig. 5C) and apoptosis (Fig. 5D). Similarly, p38 activation is also required for the FasL induction induced by anisomycin in T cells and 293 T cells, suggesting an important role of p38 in FasL regulation (Hsu et al., 1999). Unlike curcumin, the expression of FasL induced by other anti-oxidant agent, e.g. resveratrol, requires JNK rather than p38 (Su et al., 2005). However, both JNK and p38 are necessary for FasL
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induction by cisplatin in ovarian carcinoma cells (Mansouri et al., 2003). These differences might be due to the different transcription factors activated by JNK and p38, inducing expression of FasL. Indeed, some transcription factors which are downstream targets of JNK and/or p38, bind to the FasL promoter and up-regulate its expression in response to extracellular and stress stimuli (Kavurma and Khachigian, 2003). Conclusion We have demonstrated that pro-apoptotic effect of curcumin is mediated by p38 activation, which induces FasL production causing acceleration of apoptosis in Huh7 cells. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments The study is supported by Grants from National Natural Science Foundation of China (81100369), China Postdoctoral Science Foundation (2012M521583), Medical Scientific Research Foundation of Guangdong Province (A2009318), Administration of Traditional Chinese Medicine of Guangdong Province (2009415), and Scientific Research Foundation of Guangdong Pharmaceutical University (2009JCX01). References Anto RJ, Mukhopadhyay A, Denning K, Aggarwal BB. Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 2002;23(1):143–50. Brunelle JK, Letai A. Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 2009;122(Pt 4):437–41. Bush JA, Cheung Jr KJ, Li G. Curcumin induces apoptosis in human melanoma cells through a Fas receptor/caspase-8 pathway independent of p53. Exp Cell Res 2001;271(2):305–14. Cagnol S, Chambard JC. ERK and cell death: mechanisms of ERK-induced cell death— apoptosis, autophagy and senescence. FEBS J 2010;277(1):2-21. Chen Q, Wang Y, Xu K, Lu G, Ying Z, Wu L, et al. Curcumin induces apoptosis in human lung adenocarcinoma A549 cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Oncol Rep 2010;23(2):397–403. Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, et al. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res 2001;21(4B):2895–900. Chuang SE, Kuo ML, Hsu CH, Chen CR, Lin JK, Lai GM, et al. Curcumin-containing diet inhibits diethylnitrosamine-induced murine hepatocarcinogenesis. Carcinogenesis 2000;21(2):331–5.
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Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells Wei-Zhang Wang a, b, c,⁎, 1, Li Li d, 1, Man-Yu Liu a, Xiao-Bao Jin a, Jian-Wen Mao a, Qiao-Hong Pu a, Min-Jie Meng b, Xiao-Guang Chen c, Jia-Yong Zhu a a
Guangdong Province Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, PR China Guangzhou Higher Education Mega Center Health Industrial Science and Technology Park Investment Management Co. Ltd., Guangzhou, PR China School of Pubic Heath and Tropical Medicine, Southern Medical University, Guangzhou, PR China d Department of Hematology, Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA, Guangzhou, PR China b c
a r t i c l e
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Article history: Received 14 October 2012 Accepted 3 January 2013 Keywords: Curcumin Apoptosis FasL p38 Huh7 cells
a b s t r a c t Aim: The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. Main methods: Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. Key findings: Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. Significance: Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells. © 2013 Elsevier Inc. All rights reserved.
Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the third leading cause of death from cancer globally, with 600,000 deaths per year (Villanueva et al., 2010). As HCC is highly resistant to standard chemotherapy, surgical resection or liver transplantation is still the mainstay of treatment for HCC and provides the consistent long-term survival. However, about 80% of HCC patients present with advanced disease that are not amenable to surgical resection or transplantation, and thus have a poor prognosis (Thomas and Abbruzzese, 2005). Therefore, it remains a challenge to identify safe and effective treatment options for advanced HCC. Curcumin (diferuloylmethane) is a naturally occurring yellow pigment isolated from turmeric (Curcuma longa) and commonly used
⁎ Corresponding author at: Guangdong Province Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Wai Huan Dong Road 280, Guangzhou Higher Education Mega Center, Guangzhou 510006, PR China. Fax: + 86 20 39352617. E-mail addresses: [email protected], [email protected] (W.-Z. Wang). 1 These authors contribute equally to this work. 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.01.013
as flavoring food additive for centuries exhibits anti-inflammatory, antioxidant and anticancer properties (Sharma et al., 2005). The anticancer potential of curcumin stems from its ability to induce apoptosis in a wide variety of cancer cell lines in vitro (Karunagaran et al., 2005), including HCC cell lines (Jiang et al., 1996). Furthermore, curcumin also has the ability to inhibit HCC cell invasion (Lin et al., 1998) and angiogenesis (Yoysungnoen et al., 2005). In vivo, curcumin has been shown to effectively inhibit DEN-induced hepatocarcinogenesis in the mouse model (Chuang et al., 2000). Most importantly, curcumin is safe for humans as evidenced by the phase I clinical trial showing that curcumin is not toxic to humans when taken orally for 3 months at up to 8000 mg/day (Cheng et al., 2001). These data suggest that curcumin has a great potential in HCC prevention and therapy. We have shown previously that curcumin induces apoptosis in hepatocellular carcinoma Huh7 cells (Wang et al., 2008), but the underlying mechanism of curcumin-induced apoptosis remains largely unknown. In the present study, we found that curcumin-induced apoptosis was associated with up-regulation of FasL in a p38dependent manner. Furthermore, by using neutralizing antibody against FasL, we showed that the increase in FasL expression was crucial for the curcumin-induced apoptosis.
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Materials and methods Cell culture Human hepatocellular carcinoma cell line Huh7 (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences) was grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C with 5% CO2 in a fully humidified atmosphere. Antibodies and reagents Sources of antibodies are as follows: the anti-Bcl-2 monoclonal antibody, anti-Bcl-xl monoclonal antibody, anti-Bax monoclonal antibody, anti-Bak monoclonal antibody, anti-Mcl-1 monoclonal antibody, neutralizing anti-human FasL (NOK-2) antibody and isotype-matched control antibody (BD Pharmingen International, San Diego, CA); rabbit polyclonal or monoclonal antibodies against Bid, p38, phospho-p38 (Thr180/Tyr182), JNK, phospho-JNK (Thr183/Tyr185), ERK, phospho-ERK (Thr202/Tyr204), c-Jun, phospho-c-Jun (Ser 73), c-Fos, ATF-2, phospho-ATF-2 (Thr 71), cleaved caspase-3, DR5, TNFR and cleaved PARP (Cell Signaling Technology, Beverly, MA); mouse monoclonal antibodies against phospho-c-Fos (Ser374), β-actin, DR4, Fas and FasL (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-mouse or anti-rabbit IgG horseradish peroxidaseconjugated antibody (DAKO, Carpinteria, CA). The following pharmacologic agents were purchased from the respective vendors: Curcumin (Sigma-Aldrich, Inc., St. Louis, MO); Trizol (Invitrogen, Carlsbad, CA); MMLV reverse transcriptase and p38 inhibitor SB203580 (Promega, Madison, WI); Z-DEVD-FMK (Merck-Calbiochem, Germany); and ECL reagents (Pierce, Rockford, IL). Fluorescence-activated cell sorting (FACS) analysis for apoptosis At the end of indicated treatments, both floating and adherent cells were collected and analyzed. Briefly, the cells were washed twice with
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1 × PBS and collected by trypsinization. After centrifugation at 400 ×g for 5 min at room temperature, the cells were double stained with Annexin V-FITC and propidium iodide (PI) as recommended by the manufacturer (MBL, Woburn, MA). The cell apoptosis distributions were determined on a FACSCalibur flow cytometer (BD, San Jose, CA) and analyzed by CellQuest Pro software program. At least 10,000 cells were measured for each sample. RNA analysis and reverse transcription-polymerase chain reaction (RT-PCR) RNA was extracted from cell pellets using Trizol and quantitated by ultraviolet spectrophometry. Total RNA (2 μg) was reverse transcribed using MMLV reverse transcriptase for RT-PCR using specific primers for Fas and FasL. The primer sequences used for PCR amplification were: (sense) 5′-ATA AGC CCT GTC CTC CAG GT-3′ and (anti-sense) 5′-TGG AAG AAA AAT GGG CTT TG-3′ for Fas; (sense) 5′-GGC CTG TGT CTC CTT GTG AT-3′ and (anti-sense) 5′-TGC CAG CTC CTT CTG TAG GT-3′ for FasL; (sense) 5′-CTC TGC TCC TCC TGT TCG AC-3′ and (anti-sense) 5′-ACG ACC AAA TCC GTT GAC TC-3′ for glyceraldehydes-3-phosphate dehydrogenase (GAPDH). PCR conditions were as follows: an initial denaturation step at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. After a final extension at 72 °C for 7 min, PCR products were then separated on 2% agarose gels and bands were visualized with ethidium bromide (EB). Western blot analysis For protein analysis, both floating and adherent cells after the indicated treatments were collected and lysed with 1 × SDS sample buffer [62.5 mM Tris–HCl (pH 6.8 at 25 °C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue, 2.5 μM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin]. The extracted proteins were loaded onto 8% to 15% SDS-polyacrylamide gels, electrophoretically transferred to nitrocellulose membranes, and then detected by specific antibodies
Fig. 1. Effect of curcumin upon the expression of Bcl-2 family, TNF receptor family and the respective ligands. (A) and (B) Huh7 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were then prepared and analyzed for protein levels of Bcl-2 family (Bcl-2, Bcl-xL, Mcl-1, Bax, Bak and Bid), TNF receptor family (Fas, TNFR, DR4 and DR5) and the respective ligands (FasL and TRAIL) by Western blot using β-actin as loading control. Top panels: Western blot analysis. Bottom panel: quantification of Fas/FasL protein expression from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001, compared with no curcumin treatment). (C) Curcumin induces Fas and FasL mRNA expression. Huh7 cells were treated with 50 μM curcumin for the indicated times, and the total RNA was extracted and examined for expression of Fas and FasL by RT-PCR. GAPDH was used as an internal control to show equal RNA loading.
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Fig. 2. Fas/FasL interaction is required for curcumin-induced apoptosis. Huh7 cells were incubated with or without curcumin for 1 h, followed by treatment with either a neutralizing anti-FasL antibody NOK-2 (25 or 50 μg/ml) or an isotype-matched control IgG (25 or 50 μg/ml) for another 24 h, and then apoptosis was evaluated by FACS. Top panels: representative data showing apoptosis. Bottom panel: statistical analysis of apoptosis (***P b 0.001; n.s., not significant). All data are expressed as the mean ± S.D. of at least three separate experiments. Ctrl, control; Cur, curcumin; Ctrl Ab, control antibody.
and commercial ECL kit. All Western blot results were repeated 3 times, and representative results were selected for inclusion. The quantification of protein was performed by densitometry
analysis using ImageJ software (NIH). Fold changes in protein expression were determined on the basis of the β-actin loading control.
Fig. 3. Role of caspase-3 in curcumin-induced apoptosis. (A) Curcumin induces caspase-3 activation and PARP cleavage. Huh7 cells were treated with 50 μM curcumin for 24 and 48 h, and whole cell extracts were then prepared and analyzed by Western blot for the cleaved caspase-3 and PARP protein levels using β-actin as loading control. Left-hand panels: Western blot analysis. Right-hand panel: quantification of cleaved caspase-3/PARP protein expression from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001, compared with no curcumin treatment). (B) Z-DEVD-FMK inhibits curcumin-induced apoptosis. Huh7 cells were incubated with Z-DEVD-FMK or vehicle for 1 h, followed by treatment with or without curcumin (50 μM) for another 24 h, and then apoptosis was evaluated by FACS (**P b 0.01, compared with curcumin alone treatment). All data are expressed as the mean ± S.D. of at least three separate experiments. Ctrl, control; Cur, curcumin.
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Fig. 4. Effect of NOK-2 on curcumin-induced caspase-3 activation. Huh7 cells were incubated with or without 50 μM curcumin for 1 h, followed by treatment with either NOK-2 (25 or 50 μg/ml) or control IgG (25 or 50 μg/ml) for another 24 h and 48 h, and whole cell extracts were then prepared and analyzed by Western blot for the cleaved caspase-3 protein level using β-actin as loading control. Top panels: Western blot analysis. Bottom panel: quantification of cleaved caspase-3 protein expression from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001; n.s., not significant). Cur, curcumin; Ctrl Ab, control antibody.
Statistical analysis Values represent the mean ± S.D. from at least three independent experiments. Differences between the experimental groups were analyzed using two-sided Student's t-test. A value of P b 0.05 was considered statistically significant. Results The expression of Fas and FasL is significantly up-regulated upon curcumin treatment It has been well demonstrated that apoptosis is mainly mediated through two pathways: the intrinsic (mitochondrial) pathway that is regulated by Bcl-2 family members (Brunelle and Letai, 2009) and extrinsic (death receptor) pathway that is regulated by cell surface receptors (Xu and Shi, 2007). To explore the potential mechanism underlying curcumin-induced apoptosis, Huh7 cells were treated with curcumin (50 μM) for different times (2, 4, 8, 12 and 24 h) and then examined for the expression of Bcl-2 family proteins, death receptors and their respective ligands by Western blot. As shown in Fig. 1A, we found that little effect of curcumin treatment on the expression of Bcl-2, Bcl-xl, Mcl-1, Bax, Bak and Bid proteins at all time points was examined. In contrast, it significantly increased the expression of Fas and FasL at both protein (Fig. 1B) and mRNA levels (Fig. 1C) as early as 2 h, which further sustained over the following 24 h period. The expression of DR4, DR5, TNFR and TRAIL remained unchanged
and the expression of TNF was detected at none of the time points examined after curcumin treatment (Fig. 1B and data not shown). Fas/FasL interaction is required for curcumin-induced apoptosis As activation of Fas/FasL pathway plays a critical role in a variety of drug-induced apoptotic cell death (Friesen et al., 1996; Fulda et al., 2000; Kasibhatla et al., 1998), we next determined whether blocking Fas/FasL interaction influenced curcumin-induced apoptosis in Huh7 cells by treating the cells with NOK-2, an antibody against human FasL, which recognizes and neutralizes both membrane-bound and soluble forms of FasL, thereby preventing their interaction with Fas. Our results showed that curcumin treatment (50 μM, 24 h) induced a remarkable apoptotic cell death of Huh7 cells, neutralization of FasL by NOK-2 protected the cells from curcumin-induced apoptosis in a dose-dependent manner, whereas no significant effect was found with an isotype-matched IgG control (Fig. 2). This data suggests that curcumin-induced apoptosis is highly correlated with Fas/FasL pathway in Huh7 cells. Curcumin-induced apoptosis is dependent upon caspase-3 activity Binding of FasL to Fas triggers a caspase cascade and eventually results in the activation of the critical executioner caspase, caspase-3 and consequent apoptosis (Nagata, 1997). Therefore, we analyzed the protein level of activated caspase-3 by Western blot in Huh7 cells upon curcumin treatment. We observed that curcumin treatment led
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Fig. 5. p38 is required for curcumin-induced FasL up-regulation and apoptosis. Huh7 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were then prepared and analyzed by Western blot for p38, phospho-p38, JNK, phospho-JNK, ERK and phospho-ERK protein levels (A) and ATF-2, phospho-ATF-2, c-jun, phospho-c-jun, c-fos and phospho-c-fos protein levels (B) using β-actin as loading control. Left-hand panels: Western blot analysis. Right-hand panels: quantification of p-p38/p38, p-JNK/JNK, p-ERK/ERK, p-ATF-2/ATF-2, p-c-jun/c-jun and p-c-fos/c-fos from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001 and n.s., compared with no curcumin treatment). (C) Huh7 cells were pretreated with 10 μM p38 inhibitor SB203580 for 2 h, followed by incubation with 50 μM curcumin for another 24 h in the presence of SB203580, and then subjected to detection of p38, phospho-p38, ATF-2, phospho-ATF-2 and FasL expression by Western blot using β-actin as loading control. Left-hand panels: Western blot analysis. Right-hand panels: quantification of p38, phospho-p38, ATF-2, phospho-ATF-2 and FasL from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001 and n.s., compared with no curcumin treatment). (D) Huh7 cells were treated as described in (C) and then subjected to analysis of apoptosis by FACS (**P b 0.01, compared with curcumin alone treatment). All data are expressed as the mean ± S.D. of at least three separate experiments. Cur, curcumin; SB, SB203580; Ctrl, control; n.s., not significant.
to a higher level of active caspase-3 and cleavage of its major substrate PARP in a time-dependent manner (Fig. 3A), suggesting that caspase-3 is activated in response to curcumin in Huh7 cells. To further evaluate the role of caspase-3 in curcumin-induced apoptosis, we pretreated Huh7 cells with Z-DEVD-FMK, a specific caspase-3 inhibitor, prior to and throughout treatment of curcumin and found that Z-DEVD-FMK almost completely inhibited curcumin-induced apoptosis (Fig. 3B), indicating that curcumin-induced apoptosis is dependent upon caspase-3 activity. Moreover, blocking the interaction of FasL and Fas by NOK-2 notably impaired the activation of caspase-3 in a dose- and time-dependent manner (Fig. 4). These results suggest that Fas/FasL signaling-mediated activation of caspase-3 is responsible for curcumin-induced apoptosis.
p38 is required for curcumin-induced FasL up-regulation and subsequent apoptosis To further investigate signaling pathway of curcumin-induced apoptosis in Huh7 cells, we assessed the effect of curcumin on mitogenactivating protein kinases (including p38, JNK and ERK), as they play a critical role in the apoptosis-related signaling pathway. As shown in Fig. 5A, exposure to curcumin resulted in a time-dependent increase in phospho-p38 level, however, under the same condition, we did not detect any significant difference in the phosphorylation of JNK and ERK after curcumin treatment (Fig. 5A). Furthermore, curcumin treatment also led to a time-dependent increase in phosphorylation of ATF-2, a target
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Fig. 6. Effect of NOK-2 on curcumin-induced p38 activation. Huh7 cells were treated as described in Fig. 2, and whole cell extracts were then prepared and analyzed by Western blot for p38 and phospho-p38 protein levels using β-actin as loading control. Top panels: Western blot analysis. Bottom panel: quantification of p-p38/p38 from Western blot analysis. Three independent experimental results were analyzed by densitometry (***P b 0.001, compared with no curcumin treatment). Cur, curcumin; Ctrl Ab, control antibody.
of p38 signaling pathway, but had no significant effect on c-Jun and c-Fos phosphorylation. These data suggest that p38 but not JNK and ERK is activated in response to curcumin (Fig. 5B). To validate the role of p38, SB203580, a pharmacologic inhibitor of p38 was used to examine the effect of p38 inhibition on curcumin-induced FasL expression and apoptosis. We found that, in contrary to the increased p38 and ATF-2 phosphorylation and FasL expression upon curcumin treatment, pretreatment of the Huh7 cells with SB203580 for 2 h significantly inhibited both FasL protein induction and p38 and ATF-2 phosphorylation (Fig. 5C). Moreover, curcumin-induced apoptosis was dramatically comprised by SB203580 treatment (Fig. 5D). These results indicate that p38 is required for the curcumin-induced FasL expression and apoptosis in Huh7 cells. As binding of FasL to Fas has been shown to activate p38 and its activation is required for apoptosis of CD8+ T cells (Farley et al., 2006), we further determined whether disruption of Fas/FasL interaction influenced p38 activation in Huh7 cells. Our data showed that curcumin could still activate p38 in the presence of NOK-2 (Fig. 6), indicating that blockage of Fas/FasL interaction has no effect on p38 activation and that p38 activation in response to curcumin is an upstream event of Fas/FasL upregulation and interaction in our study model. Discussion HCC has characteristics of rapid growth, early vascular invasion and highly resistant to standard chemotherapy. It has been well established that most cytotoxic agents primarily act by inducing apoptotic cell death in cancer cells. Thus, evasion of apoptosis is believed to be a main contributor to HCC resistance, and targeting apoptosis
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via direct or indirect manipulations of the pro-apoptotic machinery offers a novel strategy for HCC treatment (Kountouras et al., 2003). In the present study, we found that the activation of p38 pathway is highly correlated with curcumin-induced apoptosis. The strong sustained activation of p38 pathway seemed to be a required priming step for curcumin-induced apoptosis; this activation of p38 was correlated with up-regulation of the FasL and accompanied by the induction of apoptosis. Reduction of curcumin-induced apoptosis by the use of either a specific inhibitor of p38 or a neutralizing anti-FasL antibody further underlines the critical role of p38-dependent FasL expression signaling in the induction of apoptosis by curcumin. There are many evidences that curcumin-induced apoptosis mainly involves the mitochondria-mediated pathway in various cancer cells of different tissues of origin (Karunagaran et al., 2005). For example, curcumin induces apoptosis through mitochondrial pathway involving caspase-8, Bid cleavage, cytochrome c release, and caspase-3 activation in human promyelocytic leukemia cell line HL-60 (Anto et al., 2002). However, curcumin also has been found to induce activation of a death receptor pathway in human melanoma cell lines, wherein curcumin induces Fas receptor aggregation in a FasL-independent manner and inhibition of Fas aggregation by low-temperature prevented curcumin-induced cell death (Bush et al., 2001). In the current study, we observed that curcumin increased Fas and FasL expression (Fig. 1B and C) and induced caspase-3 activation and PARP cleavage (Fig. 3A), whereas disruption of the interaction between Fas and FasL suppressed curcumin-induced caspase-3 activation (Fig. 4) and apoptosis (Fig. 2). Taken together, these results suggest that curcumin could also activate death receptor-mediated pathway to induce apoptosis in certain specific type of cancer cell. Despite a large number of in vitro studies on the function of MAPKs, the role of MAPKs in cell death induced by curcumin remains unclear. It has been shown that curcumin induced-apoptosis in human lung adenocarcinoma A549 cells was accompanied by sustained phosphorylation and activation of JNK, p38 and ERK. However, pretreatment with MAPK inhibitors had no effect upon curcumin-induced apoptosis, suggesting that activation of these MAPKs is not associated with curcumin-induced apoptosis in lung cancer (Chen et al., 2010). In human colon cancer HCT116 cells, curcumin treatment also resulted in apoptosis and sustained phosphorylation and activation of JNK and p38. Interestingly, only the JNK-specific inhibitor SP600125, but not p38-specific inhibitor SB203580, could alleviate curcumininduced apoptosis (Collett and Campbell, 2004). Most recently, curcumin has been shown to induce rapid activation of ERK1/2 and JNK as well as apoptosis in human rhabdomyosarcoma Rh30 cells, inhibition of JNK (with SP600125) or ERK1/2 (with U0126) partially prevented curcumin-induced cell death in the cells (Han et al., 2012). In the current study, we found that only p38 was activated by curcumin in human HCC Huh7 cells (Fig. 5A–C) and inhibition of p38 by SB203580 prevented curcumin-induced apoptosis (Fig. 5D). We propose that these discrepancies might be attributed to different functions of MAPKs in response to curcumin. On the one hand, the ERK signaling pathway can be activated by a variety of extracellular stimuli and regulates cell proliferation and cell differentiation (Cagnol and Chambard, 2010). On the other hand, JNK and p38 signaling pathways are responsive to stress stimuli and are involved in cell differentiation and apoptosis (Wagner and Nebreda, 2009). Therefore, the role of JNK or p38 or ERK in curcumin-induced apoptosis may be cell line specific. Our study reveals that induction of FasL is mediated by the activation of p38 in Huh7 cells, as curcumin activates p38 (Fig. 5A–C) and inhibition of p38 abolishes the effect of curcumin on the induction of FasL (Fig. 5C) and apoptosis (Fig. 5D). Similarly, p38 activation is also required for the FasL induction induced by anisomycin in T cells and 293 T cells, suggesting an important role of p38 in FasL regulation (Hsu et al., 1999). Unlike curcumin, the expression of FasL induced by other anti-oxidant agent, e.g. resveratrol, requires JNK rather than p38 (Su et al., 2005). However, both JNK and p38 are necessary for FasL
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induction by cisplatin in ovarian carcinoma cells (Mansouri et al., 2003). These differences might be due to the different transcription factors activated by JNK and p38, inducing expression of FasL. Indeed, some transcription factors which are downstream targets of JNK and/or p38, bind to the FasL promoter and up-regulate its expression in response to extracellular and stress stimuli (Kavurma and Khachigian, 2003). Conclusion We have demonstrated that pro-apoptotic effect of curcumin is mediated by p38 activation, which induces FasL production causing acceleration of apoptosis in Huh7 cells. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments The study is supported by Grants from National Natural Science Foundation of China (81100369), China Postdoctoral Science Foundation (2012M521583), Medical Scientific Research Foundation of Guangdong Province (A2009318), Administration of Traditional Chinese Medicine of Guangdong Province (2009415), and Scientific Research Foundation of Guangdong Pharmaceutical University (2009JCX01). References Anto RJ, Mukhopadhyay A, Denning K, Aggarwal BB. Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 2002;23(1):143–50. Brunelle JK, Letai A. Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 2009;122(Pt 4):437–41. Bush JA, Cheung Jr KJ, Li G. Curcumin induces apoptosis in human melanoma cells through a Fas receptor/caspase-8 pathway independent of p53. Exp Cell Res 2001;271(2):305–14. Cagnol S, Chambard JC. ERK and cell death: mechanisms of ERK-induced cell death— apoptosis, autophagy and senescence. FEBS J 2010;277(1):2-21. Chen Q, Wang Y, Xu K, Lu G, Ying Z, Wu L, et al. Curcumin induces apoptosis in human lung adenocarcinoma A549 cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Oncol Rep 2010;23(2):397–403. Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, et al. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res 2001;21(4B):2895–900. Chuang SE, Kuo ML, Hsu CH, Chen CR, Lin JK, Lai GM, et al. Curcumin-containing diet inhibits diethylnitrosamine-induced murine hepatocarcinogenesis. Carcinogenesis 2000;21(2):331–5.
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