Abstract
Capsaicin, a pungent ingredient of red pepper, has been reported to possess antitumor activities. In this study, the effects of capsaicin on human HepG2 cells were investigated. Capsaicin reduced viability by PI incorporation in HepG2 cells in a dose and time dependent manner. Capsaicin promoted intracellular Ca2+ production and reactive oxygen species (ROS). The ΔΨm significantly decreased after capsaicin treatment for 24 h. Co-treatment of HepG2 cells with capsaicin and BAPTA (an intracellular Ca2+ chelator) significantly reduced intracellular Ca2+ levels, prevented ΔΨm disruption and inhibited apoptosis induction. The protein levels of Bcl-2 decreased and Bax increased in the mitochondrial fraction while the Bax protein decreased, and p53 and cytochrome c protein levels increased in the cytosolic fraction in HepG2 cells after capsaicin treatment for 24 h by Western blot. Immunostaining and confocal microscopic analysis also showed that capsaicin promoted cytoplasmic GADD153 expression and GRP78 nuclear translocation. The caspase-3 activity significantly increased after capsaicin treatment for 24 h. Our results indicated that the capsaicin-induced apoptosis in HepG2 cells may result from the elevation of intracellular Ca2+ production, ROS, disruption of ΔΨm, regulation of Bcl-2 family protein expression and caspase-3 activity.
Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is a pungent ingredient (0.1-1%) in varieties of red pepper of the genus Capsicum (1). Previous studies have shown the effect of capsaicin on genotoxicity and mutagenicity (2). Recently, many studies have focused on its chemopreventive effects, including regulation of cell proliferation and apoptosis in cancer cells (3). The expression of interleukin 1-beta converting enzyme family, Bcl-2 family and PI-3K/Akt signal transduction pathways are well known to induce mechanisms of apoptosis (4). Capsaicin suppressed the expression of inhibitor of caspase activated DNase to induce human melanoma cells apoptosis (3), and reduced the intracellular ratio of antiapoptotic Bcl-2 to pro-apoptotic Bax, and increased caspase-3 activity in hepatocarcinoma cells (5). In addition, capsaicin induced apoptosis through increased intracellular reactive oxygen species (ROS) and calcium levels in a carcinoma cell model (6).
In the induced pathway of apoptosis, mitochondria play a very important role, through the opening of their megachannel pores and the release of apoptogenic factors such as cytochrome c and apoptotic-inducing factor (7). Cytochrome c is an important regulating factor in the generation of mitochondrial membrane potential (ΔΨm). Outer membrane permeability resulting in cytochrome c release (8) is regulated by the Bcl-2 family proteins (9) which cause swelling of the mitochondrial matrix leading to the opening of the pores or channels in the outer mitochondrial membrane (10).
In this study, the involvement of intracellular Ca2+, ROS, ΔΨm, Bcl-2 family, cytochrome c protein expression, and caspase-3 activity were investigated in hepatoma cells undergoing capsaicin-induced apoptosis.
Materials and Methods
Chemicals and reagents. Capsaicin, propidium iodide (PI), Triton® X-100, Tris-HCl, trypan blue and ribonuclease-A were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Potassium phosphates, dimethyl sulfoxide (DMSO) and TE buffer were purchased from Merck Co. (Darmstadt, Germany). Ham's Dulbecco's modified Eagle's medium (HDMEM), L-glutamine, fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were obtained from Gibco BRL (Grand Island, NY, USA).
Cell culture. HepG2 cell line (human hepatoma cells) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC). The HepG2 cells were placed into 75 cm3 tissue culture flasks and grown at 37°C under a humidified 5% CO2 atmosphere in 90% HDMEM with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate and supplemented with 10% fetal bovine serum, and 2% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin) (Gibco BRL).
Cell viability determinations. HepG2 cells were plated in 12-well plates at a density of 2×105 cells/well and grown for 24 h. Different concentrations of capsaicin were then added to cells at a final concentration of 10, 50, 100, or 200 μM, while only adding DMSO (as solvent) for the control regimen and grown at 37°C, 5% CO2 and 95% air for different periods of time. Cells were harvested by centrifugation. To determine cell viability, the flow cytometric assay was used as described previously (11).
Apoptosis analysis. Apoptosis of HepG2 cells treated with different concentrations of capsaicin were examined by using flow cytometry analysis. Approximately 5×105 cells/well of HepG2 cells in 12-well plates with concentrations of 10, 50, 100, or 200 μM of capsaicin were incubated for different time periods. The zero concentration was defined as control. Cells were harvested by centrifugation and were fixed gently by addition of 70% ethanol at 4°C overnight and then re-suspended in phosphate-buffered saline (PBS) containing 40 μg/ml PI and 0.1 mg/ml RNase and 0.1% Triton® X-100 in a dark room. After 30 minutes at 37°C, the cells were analyzed with a flow cytometer (Becton-Dickinson, San Jose, CA, USA) equipped with an argon ion laser at 488 nm wavelength. Apoptosis was determined and analyzed (11). Annexin V-FITC and PI double staining kit from PharMingen (San Diego, CA, USA) were used for apoptotic cell quantification (11, 12).
Reactive oxygen species (ROS) and intracellular Ca2+ assays. The level of ROS in HepG2 cells was examined by flow cytometry (Becton Dickinson FACS Calibur), using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma). Approximately 5×105 cells/well of HepG2 cells in 12-well plates with 0, 50, 100, 150 or 200 μM capsaicin were incubated for 1 h to detect ROS changes. The cells were harvested and washed twice, re-suspended in 500 μl of DCFH-DA (10 μM) incubated at 37°C for 30 min and analyzed by flow cytometry (11, 13-14).
The level of Ca2+ in HepG2 cells was determined by flow cytometry (Becton Dickinson FACS Calibur) using Indo 1/AM (Calbiochem, La Jolla, CA, USA). Approximately 2×105 cells/well of HepG2 cells in 12-well plates with different concentrations (0, 50, 100, 150 and 200 μM) of capsaicin with or without 5 μM 1,2-bis (O-amino phenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were incubated for 24 h to detect the changes of Ca2+ concentration (9, 15).
Mitochondrial membrane potential (ΔΨm) measurement. The level of ΔΨm in HepG2 cells was determined by flow cytometry (Becton Dickinson FACS Calibur) using DiOC6 (4 μmol/l). Approximately 5×105 cells/well in 12-well plates with different concentrations (0, 50, 100, 150 or 200 μM) of capsaicin with or without 5 μM BAPTA were incubated for 24 h to detect the changes of ΔΨm. The cells were harvested and washed twice, re-suspended in 500 μl of DiOC6 (4 μmol/l) and incubated at 37°C for 30 min then analyzed by flow cytometry (14).
Caspase-3 activity determination. Approximately 5×105 HepG2 cells/well in 12-well plates with concentrations of 0, 50, 100, 150 or 200 μM of capsaicin were incubated for different time periods. Cells were harvested by centrifugation and the media removed. A volume of 50 μl of 10 μM substrate solution (PhiPhilux is a unique class of substrates for caspase-3) was added to the cell pellet (1×105 cells per sample); the cells were not vortex mixed. Cells were incubated at 37°C for 60 minutes, then washed once by adding 1 ml of ice-cold PBS and were re-suspended in 1 ml fresh PBS. Cells were analyzed with a flow cytometer (Becton-Dickinson) equipped with an argon ion laser at 488 nm wavelength. Caspase-3 activity was determined and analyzed (14).
Western blotting assay of apoptosis-regulating proteins. Approximately 5×105 HepG2 cells/well in 12-well plates with concentrations of 0, 50, 100, 150 and 200 μM of capsaicin were incubated for different time periods. After treatment cells were washed twice in PBS and suspended in homogenizing buffer (containing 20 mM Hepes-KOH at pH 7.5, 10 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulphonyl-fluoride (PMSF), 2 mg/ml aprotinin, 10 mg/ml leupeptin and 5 mg/ml pepstatin) and homogenized by a Dounce homogenizer (Bellco Biotechnology, Vineland, NJ, USA). After centrifugation at 23,100 ×g for 30 min at 4°C, the supernatant was collected as the cytosolic fraction. The pellet containing mitochondria was re-suspended in lysis buffer (containing 150 mM NaCl, 0.5% Triton® X-100, 50 mM Tri-HCl at pH 7.4, 20 mM EGTA, 1 mM DTT, 1 mM sodium orthovanadate) as the mitochondrial fraction for further analysis or was frozen at -70°C until use. Cytosolic p53, GADD153, GRP78, Smac/DIABLO, xIAP, cIAP1, Bax and cytochrome c and mitochondrial Bax and Bcl-2 expressions were determined by SDS-PAGE and immunoblot assay. Equal amounts of cytosol proteins were subjected to 10% polyacrylamide gel electrophoresis. Following electrophoresis, proteins separated on SDS-polyacrylamide gels were transferred to polyvinyldiene difluoride membranes. To block non-specific binding, membranes were incubated at 4°C overnight with 5% skim milk, followed by a 30 min incubation at 37°C with antiserum containing antibodies against p53, GADD153, GRP78, Smac/DIABLO, xIAP, cIAP1, Bax, Bcl-2 and cytochrome c from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The blots were developed using the enhanced chemiluminescence (ECL) kit (Amersham Life Science, USA). The intensity of each band was determined using an Image analyzer (AlphaImagerTM 2200; Alpha Innotech Co., USA) (11, 14).
Immunofluorescence microscopy. HepG2 cells (5×105 cells/well) plated on 4-well chamber slides were treated with or without 100 μM capsaicin for 24 hours then cells were fixed in 4% formaldehyde in PBS for 15 min, permeabilized with 0.3% Triton®-X 100 in PBS for 1 hour with blocking of non-specific binding sites using 2% bovine serum albumin (BSA). Fixed cells were then incubated with antihuman GADD153 and GRP78 antibody (1:100 dilution) overnight and then exposed to the secondary antibody (FITC-conjugated goat anti-mouse IgG at 1:100 dilution) from Santa Cruz Biotechnology followed by mitochondrial staining with MitiTracker. Cells were photographed under a Leica TCS SP2 Confocal Spectral Microscope (16).
Statistical analysis. Statistical analysis was performed using Student's t-test. Tukey's multiple comparison was used to determine significant differences among group means (p<0.05).
Results
Effects of capsaicin on HepG2 cell viability. As shown in Figure 1A, when HepG2 cells were treated with 10, 50, 100 and 200 μM capsaicin for 24 h, the cell viability significantly decreased by 15, 37, 73 and 96%, respectively of that of the control group (p<0.05). After HepG2 cells were treated with 100 μM capsaicin for 12, 18 or 24 h, the cell viability significantly decreased (p<0.05) (Figure 1B). These results demonstrated that capsaicin reduced the percentage of viable cells in a dose- and time-dependent manner.
Effects of capsaicin on apoptosis. Figure 2A shows the induction of apoptosis by different concentrations of capsaicin in HepG2 cells. The cell cycle progress was significantly arrested in the sub-G1 phase (i.e. apoptosis occurred) when HepG2 cells were treated with 100 or 200 μM capsaicin for 24 h as compared with the control group (p<0.05). The percentages of induction of apoptosis were 41.5 and 73.4% of total cells, respectively (Figure 2B).
Effects of capsaicin on intracellular ROS, Ca2+ levels, ΔΨm, and caspase-3 activity. When HepG2 cells were treated with different concentrations of capsaicin, the changes of cells being stained for ROS and Ca2+, ΔΨm and caspase-3 activity were as shown in Table I. As compared with the control group, the proportion of cells stained for ROS significantly increased after HepG2 cells were treated with 10, 50, 100 or 200 μM capsaicin (p<0.05). When HepG2 cells were treated with 200 μM capsaicin, the proportion of cells stained for Ca2+ significantly increased as compared with the control group (p<0.05). The proportion of HepG2 cells stained for ΔΨm however, significantly decreased after 100 or 200 μM capsaicin treatment, as compared with the control group (p<0.05). In addition, the activity of caspase-3 on HepG2 cells significantly increased after 100 and 200 μM capsaicin treatment as compared with the control group (p<0.05).
Effects of capsaicin on the levels of apoptosis-regulating proteins. As shown in Figure 3, the expression of apoptosis-regulation proteins in cytosolic and mitochondrial fractions of HepG2 cells were examined by Western blotting assay. Figure 3A indicated that in the mitochondrial fraction of HepG2 cells treat with 100 or 200 μM capsaicin significantly increased the level of Bax protein, as compared with the control group. The cytosolic Bax protein levels decreased after the cells were treated with 100 or 200 μM capsaicin, respectively, as compared with the control group (Figure 3B). In addition, the p53 levels increased 1.3-, 1.6- and 2.1-fold, respectively, that of the control group in the cytosolic fraction of HepG2 cells after 50, 100 or 200 μM capsaicin treatment for 24 h (data not shown). The levels of cytosolic cytochrome c increased after treatment with different concentrations of capsaicin for 24 h (Figure 3B). The modulation of capsaicin on Bcl-2 family protein and cytochrome c expression occurred in a dose-dependent manner. In addition, as shown in Figure 3C, when cytosolic and mitochondrial fractions were isolated before and after capsaicin treatment, cytochrome c was shown to be released from mitochondria after capsaicin treatment.
Effects on endoplasmic reticulum stress protein in HepG2 cells after exposure to capsaicin. As shown in Figure 4, the levels of GADD153, GRP78 and Smac/DIABLO proteins were increased in HepG2 cells after treatment with capsaicin but the levels of xIAP and cIAP1 proteins decreased. GADD153 and GRP78 are a hallmark of ER stress. xIAP and cIAP1 proteins are inhibitors of the caspase-3 pathway. Therefore, these results also confirmed that capsaicin induced apoptosis via caspase-3 activation.
Effects of capsaicin with or without BAPTA on intracellular Ca2+ levels, MMP, apoptosis and the protein levels of p53 and cytochrome c. To explore whether the Ca2+ was required for induction of apoptosis by capsaicin. HepG2 cells were co-treated with Ca2+ chelator (BAPTA) and capsaicin. Figure 5A shows that when HepG2 cells were co-treated with capsaicin and BAPTA, the intracellular Ca2+ concentration significantly decreased as compared with the capsaicin only group (p<0.05). At the same time, the ΔΨm and apoptosis significantly decreased after HepG2 cells were co-treated with capsaicin and BAPTA as compared with the capsaicin only treated group (p<0.05) (Figures 5B, 5C, respectively). As shown in Figure 5D, when HepG2 cells were treated with capsaicin and BAPTA, the p53 protein levels were lower than that of the capsaicin only group. A similar response was obtained on the cytochrome c protein levels after co-treatment with capsaicin and BAPTA, and the cytochrome c protein level was lower than that of the capsaicin only group.
Effects of capsaicin on GRP78 nuclear translocation and cytoplasmic GADD153 expression in HepG2 cells. As in previous studies (17, 18), HepG2 cells (5×104 cells/well) treated or without 100 μM capsaicin for 24 h were plated on 4-well chamber slides. The results from immunostaining by confocal laser microscopic analysis indicated that the GRP78 protein was translocated into nuclei (Figure 6A) and cytoplasmic GADD153 expression was promoted (Figure 6B) in examined HepG2 cells. When compared to the control groups, both GRP78 and GADD153 were higher in the treated group.
Discussion
The actual signal pathway of apoptosis induced by capsaicin is still controversial. Irreversible damage due to oxidative stress on cellular macromolecules, e.g. protein, lipid, DNA and RNA, is due to multiple mechanisms (19).
Several studies showed that capsaicin induced apoptosis via increased Ca2+ and ROS levels, and reduced ΔΨm in different carcinoma cells models (7, 15, 20). How capsaicin induced endoplasmic reticulum (ER) stress and the associated signal pathway of apoptosis is not clear. When capsaicin was added with BAPTA, an intracellular chelator of Ca2+, the intracellular Ca2+ concentration and apoptosis induction significantly decreased (p<0.05) in this study. This means that the release of intracellular Ca2+ may be an important regulatory factor on early apoptosis induced by capsaicin. Mitochondrial Ca2+ sequestration is a prerequisite for ROS production and mitochondrial dysfunction (22). In this study, a dramatic ROS burst was observed in the early period of capsaicin treatment. It was reported that cancer chemopreventive agents induce apoptosis in part with generation of ROS and the disruption of redox homeostasis (23, 24). High levels of ROS not only induced cell death (25) but also caused DNA damage and genomic instability (26). HepG2 cells were pretreated with NAC then treated with capsaicin which led to reduced ROS production and also decreased the percentage of apoptosis (data not shown). These data suggest that apoptosis induced by capsaicin is dependent on ROS production. Our results also showed that capsaicin reduced ΔΨm in HepG2 cells. Mitochondria were reported to play a key role in the regulation of apoptosis (27). Mitochondrial dysfunctions, including the loss of ΔΨm, permeability transition, and release of cytochrome c from the mitochondrion into the cytosol, are associated with apoptosis (28). Thus, a mitochondrial damage-dependent pathway might be involved in capsaicin-induced apoptosis in HepG2 cells. The results also showed capsaicin promoted caspase-3 activation (Table I).
Cytosolic p53 protein levels significantly decreased and cytochrome c release from mitochondria significantly decreased after HepG2 cells were co-treated with capsaicin and BAPTA in this study. These results may indicate that DNA damage decreased after reduced ROS production under BAPTA chelating intracellular Ca2+. P53 is involved in the activation of apoptosis after DNA damage induced by such agents as cisplatin. P53 could increase the transcription of many genes, such as p21 or members of the Bcl-2 family, and the pattern of transcriptional regulation is critical in determining the cellular response to DNA damage (20, 29). In addition, intracellular Ca2+ levels may also regulate the expression of the Bcl-2 family, a protein family present in the mitochondrial outer membrane, nuclear envelope and endoplasmic reticulum (ER) (30). Our results also showed that capsaicin promoted the levels of cytoplasmic GADD153 and caused GRP78 nuclear translocation, which are indicators of ER stress, followed by Ca2+ released from the ER then finally causing apoptosis. Capsaicin also reduced the levels of xIAP and cIAP1; in the HepG2 cells, both proteins have been shown to inhibit caspase-3 activation. Mitochondria play an important role in the induction of apoptosis via increasing the permeability of the outer mitochondrial membrane and decreasing the ΔΨm (31, 32). There are many apoptosis regulatory factors, including Bcl-2 family members, cytochrome c, apoptosis protease-activating factor-1 (Apaf-1), which exist in mitochondria. Once the apoptosis signal is triggered, mitochondrial permeability transition is regulated and this leads to a decrease of mitochondrial membrane potential (33). In this study, capsaicin significantly affected the expression of Bcl-2 family members present in cytosolic and mitochondrial fractions. The levels of Bax, a pro-apoptotic Bcl-2 family member, increased in mitochondria after capsaicin treatment. In contrast, the levels of Bcl-2, an antiapoptotic Bcl-2 family member, decreased in mitochondria. In addition, cytochrome c release to the cytosol increased after capsaicin treatment. Previous studies have shown that cytochrome c release from mitochondria could be controlled by Bax. The translocation of Bax can alter the outer mitochondrial membrane permeability, leading to cytochrome c release from the mitochondria to the cytosol (31, 34-35). It was reported that insertion of Bax to the outer mitochondrial membrane and formation of Bax oilgomers may provide channels conducting large proteins, allowing transport of cytochrome c (36).
In conclusion, our results, as shown in Figure 7, clearly demonstrated that capsaicin induces apoptosis through elevating the levels of intracellular ROS and Ca2+, promoting the levels of Bax, GADD153 and GRP78, decreasing ΔΨm, Bcl-2, xIAP and cIAP1 and then increasing caspase-3 activation in HepG2 cells. ROS play an important role in the induction of apoptosis in HepG2 cells by capsaicin.
Acknowledgements
This work was supported by grant CMU-95-083 from the China Medical University, Taichung, Taiwan, R.O.C.
Footnotes
- Received April 8, 2008.
- Revision received July 24, 2008.
- Accepted November 3, 2008.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved