Investigation of pro-apoptoti?c and anti-apoptotic proteins in the apoptotic mechanism triggered by egcg in HCT-116 colon cancer cells

EGCG, a catechin derivative polyphenol, exist in green tea, is easy-to-handle and non-toxic. Its potential chemoteraputic usage is studied recently because it is ability to trigger programmed-cell death. However, its effect may change according to cell type and apoptotic mechanism has not been explained yet. Especially, the sudies on colon carcinoma are limited and any study about the effect of EGCG on pro-and apoptotic gene expression profile does not exist yet. According to this information, in this study, it is aimed to reveal the effects of apoptosis-triggering dose of EGCG on pro-and apoptotic gene expression and to investigate the importance of overexpressed apoptotic proteins by gene-silencing. Beside of this, oxidative influence of EGCG, produced in a cell by its prooxidant property, was intended for measurement with rective oxygen species.On this aim, HCT- 116Bax+/+ ve HCT116Bax-/- human colorectal tumor cell lines were used in the study. The effect of EGCG on cell viability is determined with MTT method, cell death type is examined by flow cytometric analyses. The changes in protein expression levels of caspases is determined by western-blotting method. The effect on pro-and apoptotic gene expression is analysed by Real-Time PCR method. The initiator caspases of intrinsic and extrinsic apoptosis pathways, caspase-8 and caspase-9 is selected for gene-silencing by siRNA. Reactive oxygen species produced by oxidative effects of EGCG was determined by flow cytometry.According to obtained gene and protein expression data, it is claimed that the apoptotic effect of EGCG occurs via caspase 8 enzyme-mediated extrinsic pathway, but caspase 9 enzyme-mediated intrinsic pathway enhances this effect indirectly and intracellular ROS production by EGCG has also roles in the mechanism.Key Words: EGCG, Colon cancer, Intrinsic apoptosis, Extrinsic apoptosis