The Methylated-(3”)-epigallocatechin gallate: MethylEGCG inhibits cell growth both in vitro and in vivo in Huh7 hepatoma cell line

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It is generally agreed that many of the chemoprevention effects of green tea catechin ((-)-epigallocatechin gallate; EGCG) are mediated by polyphenols which have an anti-oxidization effect. We herein report a new finding, namely, the anti-proliferate potential of MethylEGCG and attempt to clarify its mechanism of action against a Huh7 hepatoma cell line. We observed the cell proliferation after treatment at various concentrations by means of BrdU uptake ELISA. Next, the cell cycle distribution was investigated after treatment at the lowest concentration to demonstrated cell growth inhibition by flow cytometry. In addition, we determined the cell numbers using a cell counter. MethylEGCG was found to induce cell proliferation at a very low concentration (5μM). G1 cell arrest occurred after treatment at this concentration. The number of cells also decreased in comparison to the control. Next, we attempted to investigate the anti-oxidation effect of MethylEGCG. We processed cells in hydrogen peroxide and measured the increase in oxidation stress using DHA-DF, which is a marker of oxidation after treatment with saline, 5μM EGCG, or MethylEGCG. The intensity of DHA-DF by flow cytometry reflects the strength of oxidation stress. MethylEGCG suppressed the increase in oxidation stress at this concentration compared with the control. Oxidation stress has been reported to activate the PI3K-Akt cell growth signal. We observed the expression of phosphorylated Akt (pAkt) which shows that PI3K-Akt signal is activated and whole Akt by Western blotting after treatment with MethylEGCG at various concentrations. The expression of pAkt was down-regulated after treatment at concentrations under 5μM. We next observed the time dependent expression of pAkt and Cyclin D at this concentration. Cyclin D is down-stream of PI3K-Akt signal. And the decrease of the expression suppresses the progress of the cell cycle. The expressions of pAkt and Cyclin D also decreased after treatment which is more than of 3 hours. We observed the in vivo growth of a tumor graft model (5×10⁶ Huh7 cells grafted in the skin of nude mice). We injected a serum control, 1mg/1kg EGCG, 0.1mg/kg, and 1mg/1kg MethylEGCG in the peritoneum every day. The tumor growth of the 1mg/1kg MetylEGCG group showed a significant difference (after 2 weeks, vs. control; p<0.05 and after 3 weeks, vs control; p<0.01(n=12)). In summary, these findings suggest that MethylEGCG may be an important chemoprevention agent for the treatment of hepatoma.