Induction Of Apoptosis By Epigallocatechin-3-gallate Via Activating Caspase-dependent Mitochondrial Signaling In Human Gastric Cancer Cells

OBJECTIVE To elucidate the mechanism of apoptosis induced by Epigallocatechin-3-gallate (EGCG) on human gastric cancer BGC823 cells via caspase-dependent mitochondrial pathway.MEHTODS The survival rate of BGC823 cells was detected by MTT assay. Cell mitochondrial membrane potential (?ψm) and apoptosis rate was measured by flow cytometry (FCM) Rh123/PI staining. The activity of caspase-9 was detected by caspase-9 Colorimetric Assay Kit after treatment with the EGCG.Immunocytochemical method and Western blot was adopted to analyze the expression of proteins related to apoptosis mitochondrial signal transduction pathway, such as Bax, Bcl-2, caspase-9, cytochrome c and caspase-3, in BGC823 cells after EGCG treatment.RESULTS EGCG inhibited proliferation of BGC823 cells in a time- and dose- dependent manner. Rh123/PI staining FCM detection showed that, treated by EGCG with 20μg/ml, 40μg/ml and 80μg/ml for 24 h, ?ψm decreased and the apoptosis rate increased in BGC823, Ac-LEHD-CHO, the specificity blocking agent of caspase-9, with EGCG treatment group, ?ψm and apoptosis rate had no notable variability compared with the control, ?ψm was restrained and the apoptosis rate increased after treatment of EGCG with 40μg/ml for 12 h, 24 h, 48 h. The results above indicated that EGCG induced BGC823 cell apoptosis in a dose- and time- dependent manner, Ac-LEHD-CHO could block EGCG-mediated apoptosis. EGCG fostered the activity of caspase-9 in a dose- and time- dependent manner, tested by caspase-9 Colorimetric Assay Kit, Ac-LEHD-CHO could suppress the activity of caspase-9 encouraged by EGCG. EGCG could promote the releasing of cytochrome c(Cyt c), raise the expression of Bax, caspase-9, caspase-3 protein, and depress the expression of Bcl-2 protein in a dose-dependent manner, while Ac-LEHD-CHO could interrupt these function induced by EGCG; treated with EGCG 40μg/ml for 12h, 24h and 48h in BGC823 cells, the releasing of Cyt c was enhanced, the expression of Bax, caspase-9, caspase-3 protein was upregulated, and expression of Bcl-2 protein was reduced in a time-dependent manner, by immunocytochemical method and Western blot.CONCLUSION EGCG can inhibit proliferation and induces apoptosis via activating caspase-dependent mitochondrial signal transduction pathway in BGC823 cells.