Hastak Kedar, Agarwal Mukesh K, Mukhtar Hasan, Agarwal Munna L
Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106, USA.
FASEB J. 2005 May;19(7):789-91. doi: 10.1096/fj.04-2226fje. Epub 2005 Mar 11.
Treatment with epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, results in activation of p53 and induction of apoptosis in prostate cancer LnCaP cells. However, no direct evidence has delineated the role of p53 and p53-dependent pathways in EGCG-mediated apoptosis. To understand the mechanism of negative growth regulation of prostate cancer cells by EGCG we undertook a genetic approach and generated an 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, which exacerbated both G1 arrest and apoptosis. This response was accompanied by an increase in the levels of p21 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. This observation was revalidated in another prostate cancer LNCaP cells harboring wild-type p53. Inactivation of p53 using small interfering RNA (siRNA) rendered these cells resistant to EGCG-mediated apoptosis. Because p53 activation led to increase in p21 and Bax, we investigated whether these two proteins are important in this process. Ablation of p21 protein by 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. In summary, using isogenic cell lines and siRNA, we have clearly demonstrated 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.
表没食子儿茶素-3-没食子酸酯（EGCG）是绿茶中的一种多酚化合物，用其处理可导致前列腺癌LnCaP细胞中的p53激活并诱导细胞凋亡。然而，尚无直接证据阐明p53及p53依赖途径在EGCG介导的细胞凋亡中的作用。为了解EGCG对前列腺癌细胞负性生长调节的机制，我们采用遗传学方法，通过稳定导入编码野生型p53的cDNA，构建了一对同基因的前列腺癌细胞PC3（p53-/-）。用EGCG处理所得细胞PC3-p53后，如先前在LnCaP细胞中报道的那样，p53蛋白增加，这加剧了G1期阻滞和细胞凋亡。这种反应伴随着p21和Bax水平的升高。缺乏p53的细胞继续循环，在用相似浓度的EGCG处理后未发生凋亡，从而确定了EGCG以p53依赖的方式发挥作用。这一观察结果在另一种含有野生型p53的前列腺癌LNCaP细胞中得到再次验证。使用小干扰RNA（siRNA）使p53失活，使这些细胞对EGCG介导的细胞凋亡产生抗性。由于p53激活导致p21和Bax增加，我们研究了这两种蛋白在此过程中是否重要。用siRNA消除p21蛋白可阻止PC3-p53细胞中的G1期阻滞和细胞凋亡。p53依赖的Bax表达增加改变了Bax/Bcl-2比率，并与caspase 9和3的激活以及PARP的裂解平行。用Bax siRNA转染细胞消除了这些效应并抑制了细胞凋亡，但不影响细胞在G1期的积累。总之，使用同基因细胞系和siRNA，我们清楚地证明，EGCG主要通过p53依赖途径激活生长阻滞和细胞凋亡，该途径涉及p21和Bax的功能，因此任一分子的下调都赋予细胞生长优势。
