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Several lines of compelling evidence have established that green tea is an effective chemopreventive agent against many cancer types. The biochemical and pharmacological activities of epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea is primarily responsible for the chemopreventive effects of green tea against tumorigenesis and tumor growth. EGCG exerts antiproliferative effect by inducing G1 arrest and apoptosis, and the manifestations of these effects are more pronounced in cancer cells as compared to normal cells. Treatment with EGCG results into activation of p53 and induction of apoptosis in cells. However, there has been no direct evidence to delineate the role of p53 and p53 dependent pathways in EGCG mediated apoptosis. In order to understand the mechanism of negative growth regulation of prostate cancer cells by EGCG we undertook a genetic approach and generated isogenic pair of prostate carcinoma cells PC3 (p53^-/-) by stably introducing a cDNA encoding wild type p53. Treatment of the resultant cells, PC3-p53, with EGCG led to, as reported earlier in LnCaP cells, an increase in p53 protein, exacerbated both G1 arrest and apoptosis and this response was accompanied by an increase in the levels of p21^WAF1 and Bax. The cells lacking p53 continued to cycle and did not undergo apoptosis upon treatment with similar concentrations of EGCG, thus establishing the action of EGCG in a p53-dependent manner. Since p53 activation leads to increase in p21 and Bax, we investigated if these two proteins are important in this process. Ablation of p21 protein by small interfering RNA (siRNA) prevented G1 arrest and apoptosis in PC3-p53 cells. The p53 dependent increase in Bax expression altered the Bax/Bcl-2 ratio and paralleled the activation of caspase 9 and 3 and cleavage of PARP. Transfection of cells with Bax siRNA abolished these effects and inhibited apoptosis but did not affect the accumulation of the cells in G1. These observations, thus, demonstrate that EGCG activates growth arrest and apoptosis primarily via p53 dependent pathway that involves the function of both p21 and Bax such that down regulation of either molecule confers a growth advantage to the cells.

In order to further dissect the p53 signaling in cells treated with EGCG we investigated the role of protein tyrosine phosphatase, SHP-2. MEFs from SHP-2 knock out mice were much more sensitive to the treatment of EGCG compared to the normal cells. The treatment of SHP-2-null cells with EGCG resulted into increased phosphorylation of p53 at serine 6 and 15 with not much of induction of total p53 protein. Furthermore, expression of two p53 transcriptional target proteins, p21 and mdm2 was much more increased in absence of SHP-2. Thus presence of SHP-2 negatively regulates the activation of p53 and consequently p53-dependent apoptosis. The mechanism of this novel connection between SHP-2 and p53 is being investigated and will be discussed.