Effects Of Stability Of Epigallocatechin-3-gallate On The Proliferation Inhibition Of Cancer Cells

Tea is one of the most popular beverages worldwide. Among the biological activities of tea, the cancer-preventive activity has attracted the greatest attention. The active constituents and molecular mechanisms of the cancer-preventive activity, however, are not well understood. Among the green tea constituents, (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and most active constituent in inhibiting experimental carcinogenesis and related reactions.Much of the mechanistic information on the possible biological activities of tea was derived from studies in cell lines with EGCG. In many of the experiments in vitro, measurements were usually made a few hours or even days after the addition of EGCG to the culture media, and it was difficult to identify the primary targets of action of the compound and to elucidate the mechanisms of its actions. Stadies showed that EGCG was auto-oxidized under cell culture conditions, and the half-life of EGCG is rather short. The reaction produces superoxide radicals, which can be converted by SOD.In present study, effects of EGCG on the growth of human colonic cancer cells HT-29, human breast cancer MCF-7 and human cervical cancer Hela were investigated by MTT assay; Cytotoxicity of EGCG on these cells was detected by determining the activity of LDH in culture medium; Induction of apoptosis in HT-29 cell after the EGCG treatment was analyzed by flow cytometry. All studies above were compared in the presence or absence of SOD. Stability of EGCG in culture medium was determined by HPLC and LC/MS.MTT results showed that EGCG were screened for growth inhibition of HT-29, MCF-7 and Hela cells in dose-dependent manner. Adding SOD to the cell culture medium enhenced the effect of EGCG against the three cell lines. The activity of LDH in the culture medium increased after adding SOD to the culture medium, and it suggest that the cytotoxicity of EGCG was enhanced in the presence of SOD. SOD also increased HT-29 apoptotic rate which is low and induced by EGCG. The half-life of EGCG in culture medium in the presence of these three cancer cells is about 1 h or 45 min. After added SOD in the medium, the half-life of EGCG inceased to 8 h. The result proposed that the stability of EGCG under culture conditions was increased by SOD. Results of LC/MS showed that the product of EGCG auto-oxidation include P2 (Mt 884) and dehydrodimers (Mt 912).So the stabilization of EGCG by SOD in the culture medium potentiated the proliferation inhibition of cancer cells. In furore studies of EGCG in cell culture, instability and auto-oxidation of EGCG can not be ignored and SOD may be added to stabilize EGCG.