Molecular Mechanisms of GSTP1 Reactivation by Green Tea Polyphenols

描述（申请人提供）：异常DNA甲基化和染色质重塑是常见的表观遗传变化，在基因沉默中起关键作用；与前列腺癌的发生和发展有关。其中一种改变是通过5'-调节区CpG岛上脱氧胞苷残基的甲基化，沉默谷胱甘肽s -转移酶-pi （GSTP1）的表达，GSTP1是一种重要的致癌物质防御酶。GSTP1功能的丧失发生在绝大多数（约70%）前列腺肿瘤中，无论其分级或分期如何，这似乎是人类前列腺肿瘤发生的特征。不太可能突变的基因；表观遗传上沉默的基因是完整的，并且是能够“唤醒”这些休眠基因的药物的有吸引力的目标。然而，这些基因的再激活可以通过DNA甲基化和/或组蛋白去乙酰化酶抑制剂来完成；由于严重的副作用和毒性，这些抑制剂的临床应用受到限制。近年来，绿茶及其主要多酚成分表没食子儿茶素-3-没食子酸酯（EGCG）作为一种有前景的前列腺癌化学预防剂受到了广泛关注。这一观点得到了最近的病例对照研究的支持。中华医学杂志（英文版）；临床试验（Cancer Res. 66:1234- 40,2006）和我们的观察表明，在模拟人类疾病的转基因小鼠前列腺腺癌（TRAMP）模型中，口服绿茶多酚（GTP）可显著抑制前列腺癌的发生（Proc. Nail）。学会科学。美国98:10350-5,2001)。在细胞培养系统和有限动物模型中进行的大量实验室研究进一步表明，GTP对多种类型的致癌物具有保护作用，并诱导II期酶活性，从而增强解毒过程。最近，GTP被证明可以抑制DNA甲基转移酶的活性（Cancer Res. 63:7563- 70,2003）。重要的是，如果饮用绿茶可以恢复或补偿前列腺中GSTP1活性的丧失，并可以减缓前列腺癌的发生过程，这可能与预防和/或治疗前列腺癌有关。在我们提出的研究中，我们进一步了解GTP和EGCG如何介导GSTP1基因的再激活，GSTP1基因在前列腺癌发生过程中沉默。通过一系列探索，我们将具体研究这些分子机制是否通过与以下相关的表观遗传途径介导：1)异常甲基化GSTP1基因的去甲基化和激活，2)转录抑制因子结合和表达的调节：甲基CpG结合域（MBD）蛋白，DNA甲基转移酶（dnmt）和组蛋白去乙酰化酶（HDAC）甲基化DNA iii)组蛋白乙酰转移酶（HAT）活性和表达的改变，iv)组蛋白修饰，v)整体低甲基化模式。这些研究将在人前列腺癌LNCaP和MDA PCa 2b细胞中进行，这些细胞具有高甲基化的GSTP1 CpG岛等位基因，缺乏GSTP1 mRNA和蛋白的表达。这将在胸腺裸鼠原位植入前列腺癌细胞中得到进一步验证。该建议的完成将确立绿茶多酚在调节表观遗传事件中的重要作用，并将为开展基于预防的临床试验提供依据。随着“原理证明”，更多与结构相关的无毒化合物可以在未来开发用于预防人类前列腺癌。

DESCRIPTION (provided by applicant): Aberrant DNA methylation and chromatin remodeling are common epigenetic changes that play critical role in gene silencing; implicated in the development and progression of prostate cancer. One such alteration is silencing of the expression of glutathione S-transferase-pi (GSTP1), a critical enzyme of carcinogen defense, through methylation of deoxycytidine residue in CpG islands in the 5'-regulating region. Loss of GSTP1 function occurs in vast majority (>70%) of prostate tumors regardless of grade or stage and appears to be characteristic of human prostate tumorigenesis. Unlikely mutated genes; epigenetically silenced genes are intact and are attractive targets for agents that could 'wake up' these dormant genes. Reactivation of such genes could be accomplished by DNA methylation and/or histone deacetylase inhibitors however; clinical utility of these inhibitors has been limited due to severe side- effects and toxicity. In recent years, green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG) has received much attention as a promising chemopreventive agent for prostate cancer. This notion is supported by recent case-control study (Int. J. Cancer 108:130-5, 2004); clinical trial (Cancer Res. 66:1234-40, 2006) and our observation demonstrating that oral consumption of green tea polyphenols (GTP) at human achievable dose results in significant inhibition of prostate carcinogenesis in transgenic adenocarcinoma of the mouse prostate (TRAMP) model that mimics human disease (Proc. Nail. Acad. Sci. USA 98:10350-5, 2001). Extensive laboratory studies in cell culture systems and in limited animal models have further demonstrated that GTP afford protective effects from diverse types of carcinogens and induce phase II enzyme activity that could lead to enhanced detoxification process. More recently GTP has been shown to inhibit the activity of DNA methyltransferase (Cancer Res. 63:7563-70, 2003). Importantly, if drinking green tea can restore or compensate the loss of GSTP1 activity in the prostate and can slow down the process of prostate carcinogenesis that may have relevance for prevention and/or treatment of this disease. In the proposed studies we further our understanding of how GTP and EGCG mediate reactivation of GSTP1 gene, silenced during prostate carcinogenesis. Through a series of exploration, we will specifically investigate whether these molecular mechanism(s) are mediated through epigenetic pathways related to i) demethylation and activation of aberrantly methylated GSTP1 gene, ii) modulation in binding and expression of transcriptional repressers: methyl CpG binding domain (MBD) proteins, DNA methyltransferases (DNMTs), and histone deacetylases (HDAC) to methylated DNA iii) alteration in activity and expression of histone acetyltransferases (HAT), iv) histone modification, and v) global hypomethylation patterns. These studies will be conducted in human prostate carcinoma LNCaP and MDA PCa 2b cells, which possess hypermethylated GSTP1 CpG island alleles, devoid of GSTP1 mRNA and protein expression. This will be further validated in athymic nude mouse orthotopically implanted with prostate carcinoma cells. Completion of this proposal will establish an important role of green tea polyphenols in modulating epigenetic events and will provide a rationale to conduct prevention-based clinical trial. With the 'proof of principle' more structurally-related non- toxic compounds could be developed in future for prevention of prostate cancer in humans.