Introduction
Hepatocellular carcinoma (HCC) is a malignant tumor with a high prevalence in Asia and Africa (1). HCC is the third most common cause of cancer-related mortalities (500,000 mortalities per year) and the fifth most common cancer worldwide (2). Treatments for HCC include percutaneous ethanol injection therapy, transcatheter arterial chemoembolization, liver transplantation and surgical intervention. Of these treatments, surgical intervention is the most effective for improving the 5-year survival rate of patients (3). One major obstacle for the optimum treatment of HCC is the high expression of anti-apoptotic genes, which contributes to an extremely poor prognosis (4,5). Therefore, novel or additional therapeutic strategies for HCC are required to circumvent this challenge.
Catechins constitute ~40% of the dry weight of green tea, and epigallocatechin-3-gallate (EGCG) is the most common catechin (6–8). EGCG has great potential as an inexpensive, bioavailable chemotherapeutic agent for cancer prevention and complementary treatment (9). The anticancer role of EGCG has been studied epidemiologically in in vitro and in vivo systems, and in clinical trials (10–12). In vitro studies have demonstrated that EGCG inhibits cancer by inducing apoptosis (11–13). Several studies have also reported its role in cancer prevention via the induction of apoptosis, alteration of protein expression and inhibition of intracellular communication pathways (14–17). Apoptosis is driven by two major pathways: The cell death receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway. However, the precise mechanism by which EGCG exerts its anticancer effects remains unknown.
There is a lack of studies describing the effect of EGCG on HCCs, compared to other cancers (18–20). To the best of our knowledge, no comparative studies of the effects of EGCG on HCCLM6 (a human HCC metastatic cell line) and HL-7702 (a non-cancerous, normal human liver cell line) cells have been reported. The present study investigated the potential effect of EGCG on these cell lines and the molecular mechanism of EGCG induced apoptosis of HCCLM6 cells.
Materials and methods
Antibodies and reagents
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). EGCG (≥98% purity), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4′,6-diamidino-2-phenylindole (DAPI) were provided by Sigma (St. Louis, MO, USA). The fluorescein isothiocyanate (FITC)-conjugated anti-Annexin V and propidium iodide (PI) Dye Apoptosis Detection kit, the JC-1 Mitochondrial Membrane Potential Detection kit, the Cell Cycle Detection kit and the BCA Protein Assay kit were purchased from Nanjing KeyGen Biotech., Co., Ltd. (Nanjing, China). The SuperSignal® West Pico Trial kit was purchased from Thermo Scientific (Rockford, IL, USA). Mouse anti-human β-actin (sc-1496), mouse anti-human caspase-3 (sc-65497), mouse anti-human Bax (sc-7480), rabbit anti-human B-cell lymphoma 2 (Bcl-2) (sc-783) and horseradish peroxidase (HrP)-conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-human p53 (AP062), rabbit anti-human nuclear factor-κB (NF-κB) (AN365), mouse anti-human caspase-9 (AC062), mouse anti-human cytochrome c (AC909), rabbit anti-human cyclooxygenase (COX) IV (AC610) were purchased from Beyotime Institute of Biotechnology (Beijing, China).
Cells and cell culture
HCCLM6 cells (ATCC, Rockefeller, MD, USA) and HL-7702 liver cells (Shenke Biological Technology, Co., Ltd., Shanghai, China) were grown in DMEM supplemented with 10% FBS, 0.1% benzyl penicillin and streptomycin. The cells were maintained at 37°C in a humidified incubator containing 5% CO2. The cells were treated with varying concentrations of EGCG dissolved in serum-free medium.
Cell viability assay
HCCLM6 and HL-7702 cells (6×103 cells/well) were incubated in 96-well plates for 24 h. This was followed by 24 h incubation of HCCLM6 cells with 0, 5, 10, 20, 30, 40, 60, 80, and 100 μg/ml EGCG and incubation of HL-7702 cells with 0, 5, 10, 20, 40, 60, 80, 100, 140, 180, 220, and 260 μg/ml EGCG. At the end of the treatment, the culture supernatant was replaced with fresh complete medium containing 0.5 mg/ml MTT, and the cells were incubated at 37ºC for 4 h. The medium was discarded and replaced by dimethyl sulfoxide, and the cells were subjected to 10 min of gentle agitation. Absorbance was subsequently measured at 490 nm. The assay was repeated three times. The growth inhibition rates of the cells were calculated according to the formula: Inhibition rate (%) = (1 - mean absorbance of treated group/mean absorbance of untreated group) × 100%.
Characterization of nuclear morphology
A nuclear DAPI dihydrochloride-staining assay was conducted to detect apoptosis. HCCLM6 and HL-7702 cells (1×105 cells/ml) were seeded onto 1-cm plates and incubated at 37°C overnight. The cells were treated with 0, 10 and 30 μg/ml EGCG and incubated for an additional 24 h. The cells were washed once with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, washed with PBS and air-dried. The cells were stained with DAPI at room temperature for 20 min, washed with PBS and covered with Anti-Fade Fluoromount-G (Beyotime, Shanghai, China). Stained cells were observed and images captured at magnification, ×400, with a fluorescent microscope (Leica, ELS, Wetzlar, Germany) equipped with a Nikon camera (Nikon, Tokyo, Japan).
In vitro apoptosis assay
HCCLM6 and HL-7702 cells (3×105 cells/well) were seeded into 6-well plates. At 80% confluency, the cells were treated with 0, 10, and 30 μg/ml EGCG and incubated for 24 h. The cells were harvested, washed with PBS and stained with the FITC-conjugated anti-Annexin V and PI Dye Apoptosis Detection kit according to the manufacturer’s protocol. The apoptotic stage of the cells was analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Cell cycle analysis
HCCLM6 and HL-7702 cells were seeded into 6-well plates (6×105 cells/well). At 80% confluency, the cells were treated with EGCG (0, 10 and 30 μg/ml) and incubated for 24 h. The cells were harvested, washed with PBS and fixed in a 70% methanol solution at 4°C for 24 h. Following fixation, the cells were washed with PBS and stained using the Cell Cycle Detection kit according to the manufacturer’s instructions. The cell cycle distribution was analyzed using a flow cytometer (BD Biosciences).
Mitochondrial membrane potential (MMP) determination
HCCLM6 and HL-7702 cells were seeded into 6-well plates (6×105 cells/well). At 80% confluency, the cells were treated with EGCG (0, 10 and 30 μg/ml) and incubated for 24 h. Cells were harvested, washed with PBS and stained with the JC-1 Mitochondrial Membrane Potential Detection kit according to the manufacturer’s instructions. The MMP was analyzed using a flow cytometer (BD Biosciences).
Western blot analysis
HCCLM6 cells (1×107) were seeded into a T-25 culture flask and incubated until 80% confluency was achieved. The cells were treated with 0, 10 and 30 μg/ml EGCG and incubated for 24 h. Total cell protein was extracted from the treated cells and quantified with the BCA Protein Assay kit. Bovine serum albumin was used as the standard. Protein (80 μg) was separated on a 12% SDS-PAGE gel and transferred to nitrocellulose membranes with a semi-dry transfer unit (Jim-X Biotechnology, Co., Ltd., Dalian, China). The membranes were treated with a 5% skimmed dry milk-Tris-buffered saline with Tween 20 (TBST) solution at 37°C for 2 h. Antibodies against p53, NF-κB, caspase-3, caspase-9, Bcl-2, Bax, cytochrome c, COX IV and β-actin in 5% skimmed dry milk-TBST solution were employed to probe their respective proteins. Incubation was performed at 4°C overnight. Membranes were washed with TBST (3×10 min washes) and incubated with HRP-conjugated secondary antibodies (1:1,000) of the appropriate species (mouse or rabbit) at room temperature for 2 h. Following thorough washing of membranes, immunoreactive bands were visualized using the SuperSignal® West Pico Trial kit and detected with the ECL Gel Documentation and Analysis System (Bio-Rad, Hercules, CA, USA).
Statistical analysis
The data from all the experiments were expressed as the mean ± standard deviation. Statistical analyses were performed with SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Effects of EGCG on cell viability
To study the growth inhibitory effect of EGCG, HCCLM6 cells and HL-7702 liver cells were treated with 5–100 μg/ml and 5–260 μg/ml EGCG, respectively (Fig. 1). EGCG significantly inhibited the growth of HCCLM6 cells in a dose-dependent manner at concentrations between 10 and 100 μg/ml (P<0.01; Fig. 1A). The IC50 for HCCLM6 cells was 62 μg/ml. By contrast, a much higher minimum dose of EGCG (80 μg/ml) was required to significantly inhibit the growth of HL-7702 liver cells (P<0.01; Fig. 1B), and the IC50 was 227 μg/ml.
Cell death assessment
To assess the type of cell death induced by EGCG, the nuclei of EGCG-treated (0, 10 or 30 μg/ml) and untreated cells were stained with DAPI. The nuclei of EGCG-treated non-viable HCCLM6 cells were predominantly condensed with nuclear morphological changes and DNA fragmentation (Fig. 2A-b and c) compared to untreated cells (Fig. 2A-a). This indicates that these cells underwent apoptotic-like cell death. Notably, HL-7702 cells that received a similar treatment were round in shape and had stable nucleus morphology with intact DNA integrity (Fig. 2B-a to c). This indicates that the HL-7702 cells did not undergo apoptotic-like cell death.
Effect of EGCG on cell apoptotic rate
To confirm the type of cell death induced by EGCG in HCCLM6 cells and to further evaluate the effects of EGCG on HL-7702 cells, a double-staining assay was conducted with Annexin V-FITC and PI. Annexin V-positive and PI-negative (Fig. 3, lower right quadrant) cell staining indicated early apoptosis. The cells that were strongly positive for Annexin V and PI (upper right quadrant) underwent late apoptosis/necrosis (23). EGCG treatment significantly increased the percentage of apoptotic HCCLM6 cells in a dose-dependent manner compared with untreated cells (P<0.05; Fig. 3A and B). HCCLM6 cells exposed to 10 μg/ml EGCG exhibited significant early apoptosis (P<0.01; Fig. 3B). Significant late apoptosis (P<0.05; Fig. 3B) occurred only at 30 μg/ml EGCG. By contrast, neither 10 nor 30 μg/ml EGCG caused significant early or late apoptosis in HL-7702 cells compared to untreated cells (Fig. 3C and D).
Effect of EGCG on the cell cycle
HCCLM6 cells treated with 30 μg/ml EGCG exhibited significant G0/G1 phase arrest compared to untreated cells (Fig. 4 and Table I). HL-7702 cells, however, did not exhibit phase arrest upon EGCG treatment (Fig. 4B and Table I). This indicates that EGCG is able to selectively induce cell cycle phase arrest in cancerous HCCLM6 cells but spares non-cancerous cells under similar treatment conditions.
Measurement of ΔΨm in HCCLM6 and HL-7702 cells
MMP was measured using the mitochondria-specific lipophilic cationic fluorescent dye, JC-1. As a monomer, JC-1 is capable of selectively entering mitochondria. Under normal conditions, JC-1 aggregates within mitochondria and emits red fluorescence. However, when the MMP collapses during apoptosis, JC-1 emits green fluorescence. The dissipation of ΔΨm was measured as an increase in green fluorescence.
Fig. 5A and C show the analysis of MMP in HCCLM6 and HL-7702 cells that were incubated with or without EGCG. Following 10 and 30 μg/ml EGCG treatment, HCCLM6 cells showed a significantly lower ratio of JC-1 aggregation in mitochondria compared to untreated cells (Fig. 5B). However, there was no significant change in the ΔΨm of HL-7702 cells, as indicated by the fairly constant JC-1 aggregation in the mitochondria of EGCG-treated and untreated cells (Fig. 5D).
Effect of EGCG on apoptotic proteins
Western blot analysis showed significantly lower expression of Bcl-2 and NF-κB (Fig. 6A; P<0.01) in EGCG-treated HCCLM6 cells. Conversely, the expressions of Bax, p53, caspase-3 and caspase-9 were significantly increased (P<0.05) in a dose-dependent manner (Fig. 6A). EGCG treatment significantly reduced the expression of cytochrome c in mitochondria but increased its expression in the cytosol of HCCLM6 cells (Fig. 6A). As shown in Fig. 6B, EGCG treatment did not significantly affect the expression of any of these proteins in HL-7702 cells, even at high EGCG concentrations.
Discussion
Apoptosis, a physiological death mechanism that preserves homeostasis, is the most common form of eukaryotic cell death (21). There are two major apoptotic pathways: The receptor-mediated (extrinsic) pathway and the mitochondrial-mediated (intrinsic) pathway (22). In the present study, the anticancer activity of EGCG was investigated. EGCG-induced cell death was observed to be apoptotic-like and characterized by cellular shrinkage, DNA fragmentation and the reduction of MMP (Figs. 1, 2 and 5). EGCG induced apoptosis of malignant HCCLM6 cells without inducing a similar effect on non-cancerous HL-7702 cells that received similar treatment (Figs. 3 and 4).
Apoptotic stimuli resulted in the loss of MMP and the release of cytochrome c from the mitochondrial inner membrane space into the cytosol (23–26). The released cytochrome c, a potent caspase-activating protein, initiated the caspase-dependent apoptotic cascade. EGCG treatment caused a significant increase in the cytochrome c levels in the cytosol and a concomitant reduction in the levels in the mitochondria of the HCCLM6 cells (Fig. 6A). The significantly increased expression of caspase-3 and -9 (Fig. 6) may be the direct result of the increased cytosolic cytochrome c levels induced by EGCG in HCCLM6 cells but not in HL-7702 cells (Fig. 6A and B). Thus, apoptosis is increased in HCCLM6 cells.
Bax is present in its inactive form in the cytosol or loosely attached to intracellular membranes as a monomer. In apoptotic cells, activated Bax is translocated and integrated into mitochondrial membranes (27,28). This event causes membrane permeabilization and release of cytochrome c from the mitochondria. Cytochrome c is subsequently complexed with apoptotic protease activating factor-1 and procaspase-9 to form the apoptosome. Cytochrome c recruits procaspase-3, which is cleaved and activated by caspase-9 to induce apoptosis. The ratio of Bax/Bcl-2 is critical for cell survival such that an increase in Bax levels could shift the ratio in favor of apoptosis. EGCG induced an increase in Bax expression but a reduction in Bcl-2 (a major anti-apoptotic protein) expression in HCCLM6 cells. Thus, a higher Bax/Bcl-2 ratio favored the apoptosis of these cells compared to non-cancerous HL-7702 cells (Fig. 6A and B).
EGCG was found to effectively decrease NF-κB translational activity in human HCCLM6 cells (Fig. 6A). The NF-κB protein stimulates cell survival and promotes cell proliferation, and increased NF-κB activity is associated with numerous cancers, including HCC. A previous study also indicates that NF-κB activates the expression of the anti-apoptotic protein Bcl-2 (29).
The increase in p53 expression in EGCG-treated HCCLM6 cells (Fig. 6A) was associated with increased cytosolic cytochrome c and Bax expression. The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair and programmed cell death. This gene stimulates the release of cytochrome c by transcriptionally increasing cytosolic Bax levels in certain human cell lines (29,30). Ahmed et al (31) reported in 2000 that EGCG induces G0/G1 phase cell cycle arrest, which is an irreversible process that ultimately leads to apoptotic death of human carcinoma cells. Cell cycle perturbations are an important component of the cellular response to DNA damage (32,33). In addition, p53 plays an important role in controlling the G1/S checkpoint. Some cells undergo cell cycle arrest in the G1 phase in response to DNA damage (34). The present study results indicate that EGCG-induced G0/G1 cell cycle phase arrest of HCCLM6 cells occurs via increased expression of p53, which eventually results in apoptotic cell death.
The data indicated that EGCG treatment results in upregulation of Bax via p53 and downregulation of Bcl-2 via NF-κB. The overexpression of Bax and low expression of Bcl-2 eventually led to mitochondrial membrane disintegration and the release of cytochrome c. During apoptosis, cytochrome c release caused the activation of caspase-9 through the formation of the multimeric apoptosome complex. Caspase-9 activated caspase-3, which resulted in apoptosis (Fig. 7). Additionally, EGCG caused G0/G1 phase arrest of the HCCLM6 cell cycle via increased expression of p53.
In conclusion, EGCG affects HCCLM6 cells by inducing apoptosis, predominantly through the mitochondria-mediated apoptosis pathway. Although the concentrations of EGCG used in the present study are higher than those achievable by drinking tea or through EGCG oral administration (35,36), higher plasma and/or organ-specific EGCG bioavailability is achievable through intravenous administration (37). The present study indicates that EGCG is an anticancer agent in the treatment of HCC. EGCG selectively induced apoptosis in cancerous cells with a minimal effect on non-cancerous cells. However, the present study only approximately assessed the role of mitochondrial dysfunction in EGCG-induced apoptosis. Therefore, further studies are required to investigate other pathways that may induce apoptosis in EGCG-treated cells, the effect of EGCG on diseased and healthy tissues in vivo and the relevant mechanisms for maintaining cell viability of non-tumor cells.