Epigallocatechin-3-gallate induces apoptosis in human hepatocellular carcinoma cells

Abstract

To investigate the effects of epigallocatechin-3-gallate (EGCG) on human hepatocellular carcinoma (HCC) cells and mechanism thereof. Human HCC cells of the lines HepG2 and SMMC-7721 were cultured and treated with of EGCG of the concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 microg/ml respectively for 24 h and 48 h. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Trypan blue staining was used to count the cells. Flow cytometry was conducted to detect the cell apoptosis. The protein levels of Bcl-2, an anti-apoptosis factor, and cyclooxygenase-2 (COX-2), an up-regulator of Bcl-2. The activities of caspase-9 and caspase-3 hat promote the apoptosis of HCC cells, were measured using colorimetric method. RT-PCR was used to detect the mRNA expression of COX-2 and Bcl-2 family. The viabilities of the HepG2 and SMMC-7721 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 93.8% +/- 2.8%, 62.3% +/- 5.4%, 33.9% +/- 2.5%, and 17.6% +/- 3.2% respectively, all significantly lower than that of the control group [(100.0% +/- 2.8%), all P < 0.05]; and the viabilities of the SMMC-772 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 49.6% +/- 3.5%, 30.3% +/- 3.8%, 17.7% +/- 2.2%, and 13.0% +/- 2.5% respectively, all significantly lower than that of the control group [(100.0% +/- 0.8%), all P < 0.05]. After treatment with 100 microg/ml EGCG for 24 h, 48 h, 72 h, and 96 h, the live HepG2 cell numbers were (8.0 +/- 1.5), (22.0 +/- 3.1), (37.0 +/- 5.4), and (61.0 +/- 8.7) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]; and the live SMMC-7721 cell numbers were (7.0 +/- 2.2), (13.0 +/- 2.5), (20.0 +/- 3.7), and (31.0 +/- 4.0) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]. The apoptotic rates of HepG2 cells treated with EGCG of the concentrations of 50, 100, and 200 microg/ml for 12 h were 8.7% +/- 0.4%, 18.1% +/- 1.1%, and 22.1% +/- 1.8% respectively, all significantly higher than that of the control group (3.3% +/- 0.3%, P < 0.05); and the apoptotic rates of SMMC-7721 cells were 5.9% +/- 0.3%, 7.8% +/- 0.6%, and 12.2% +/- 0.8% respectively, all significantly higher than that of the control group (3.7% +/- 0.4%, P < 0.05). After treatment with EGCG of the concentrations of 100 and 200 microg/ml for 12 h, the caspase-9 activities of the HepG2 cells increased to (1.8 +/- 0.4) and (2.5 +/- 0.4) respectively, both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05); and the caspase-3 activities of the HepG2 cells increased to (2.0 +/- 0.4) and (2.8 +/- 0.5) respectively, both significantly higher than that of the control group (1.0 +/- 0.2, P < 0.05) ; and the caspase-9 activities of the SMMC-7721 cells increased to (1.7 +/- 0.4) and (2.5 +/- 0.4), both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05), and the caspase-3 activities of the SMMC-7721 cells increased to (1.9 +/- 0.4) and (2.6 +/- 0.3) respectively, both significantly higher than that of the control group [ (1.0 +/- 0.2), both P < 0.05]. When the concentration of EGCG was over 200microg/ml, it down-regulated the expression of COX-2 and Bcl-2 in both cell lines, however, EGCG resulted in no significant changes of Bcl-xl, Bax, Bad, and Bid. EGCG induces apoptosis in HCC cells through down-regulation of COX-2 and Bcl-2 and consequently activating caspase-9 and caspase-3.