Chemopreventive mechanisms of green tea catechins and black tea theaflavins: Modulation of the Ras-activated signal transduction pathway

The popular beverage, green tea and black tea, has been extensively studied over the past two decades for their cancer preventive properties in many animal carcinogenesis models. The results from these studies showed strong inhibition of tumor formation and growth. To investigate the anti-carcinogenic effects of green and black tea, the potential active components, the catechins and theaflavins were used for the present study.; Green tea catechins, (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin, (−)-epicatechin-3-gallate, (−)epicatechin, and their epimers along with black tea theaflavins, theaflavin, theaflavin-3-gallate, theaflavin-3 ′-gallate, and theaflavin-3,3′-digallate (TFdiG) were examined for their ability to inhibit the growth of H- ras-transformed cells. This cell line was derived from mouse epidermal JB6 cells by a stable transfection of a mutant H-ras gene to mimic carcinogenesis in vitro. These transformed cells exhibit anchorage independent growth and high endogenous activator protein 1 (AP-1) activity, which can be measured by a stable AP-1 luciferase reporter. The AP-1 inhibitory effects by the individual tea polyphenols were also examined.; All of the tea polyphenols with the exception of (−)-epicatechin showed strong inhibition of growth and AP-1 activity. Structure activity analysis among the green tea catechins showed the importance of both the galloyl structure on the B ring and the gallate moiety. The structures of black tea theaflavins with the gallate moiety showed strong growth inhibitory activity, whereas all theaflavins were strong inhibitors of AP-1 activity. Mechanistic studies showed the inhibition of AP-1 activity and growth may be due to the decreased levels of phospho-c-jun and -ERK1/2 protein levels by EGCG and TFdiG, which led to the decrease of c-jun and fra-1 protein levels by EGCG and TFdiG, respectively. Further investigation into the mechanisms by which EGCG and TFdiG inhibited the activity of these signaling proteins showed that EGCG and TFdiG strongly and time-dependently decreased both phospho-ERK1/2 and -MEK1/2 protein levels. TFdiG was more potent in the inhibition of phospho-ERK1/2 and MEK1/2 levels than EGCG. TFdiG effectively decreased Raf-1 protein levels through the lysosomal degradation pathway. EGCG was unable to promote the degradation of Raf-1 nor affect its activity, but the mechanism of inhibition of phospho-MEK1/2 by EGCG was through the inhibition of Raf-1 and MEK1 association via the proline rich region on MEK1, as determined by immunoprecipitation with antibody against MEK1. These results suggest that tea polyphenols inhibited AP-1 activity and cell proliferation by rapidly inhibiting the Ras-activated signal transduction pathway, however, the mechanisms for green tea catechins and black tea theaflavins may be different.