Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3 '-digallate

Our previous study showed that tea polyphenols inhibited MAP kinase and AP- 1 activities in mouse epidermal JB6 cells and the corresponding H-ras-trans formed cell line 30.7b Ras 12. The present study investigated the mechanism s of this inhibition. The cells were incubated with (-)-epigallocatechin-3- gallate (EGCG) or theaflavin-3,3'-digallate (TFdiG) (20 muM) for different times, and the cell lysate was analyzed by immunoblotting. EGCG treatment d ecreased the levels of phospho-Erk1/2 and -MEK1/2 time-dependently (by 60% at 60 min). TFdiG lowered their levels by 38%-50% at 15 min. TFdiG effectiv ely decreased total Raf-1 protein levels, most likely through lysosomal deg radation. EGCG did not affect protein levels or the activity of Raf-1 signi ficantly but decreased its association with MEK1 as determined by co-immuno precipitation. In addition, EGCG and TFdiG (10 muM) inhibited the phosphory lation of Elk-1 by isolated phospho-Erk1/2 in vitro. This inhibition of Erk 1/2 activity is Elk-1 concentration-dependent and ATP concentration-indepen dent, which suggests that EGCG and TFdiG interfere with the binding of the protein substrate to the kinase. The presently demonstrated specific mechan isms of inhibition of MAP kinases by EGCG and TFdiG may help us to understa nd the effects of tea consumption on cancer, inflammatory diseases, and car diovascular diseases.