Cancer, 2010, 116(1):66–76.

Genistein reverses hypermethylation and induces active histone modifications in tumor suppressor gene B-Cell translocation gene 3 in prostate cancer

Shahana Majid, Rajvir Dahiya, et al

Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, California; et al

BACKGROUND: B-cell translocation gene 3 (BTG3/ANA/APRO4) is a candidate tumor suppressor gene in some malignancies. We report here that B-cell translocation gene 3 (BTG3) is transcriptionally down-regulated in prostate cancer and the mechanism of inactivation is through promoter hypermethylation.

METHODS: Prostate cancer and normal cell lines were treated with different doses of genistein and 5-aza-2'-deoxycytidine (5Aza-C). BTG3 messenger ribonucleic acid (mRNA) expression was determined by quantitative real-time polymerase chain reaction in tissues and cell lines. Bisulfate-modified polymerase chain reaction, cloning and sequencing were used to examine promoter methylation in tumor samples and cell lines. Enzyme activity/inhibition assays were done to check the effect of genistein and 5Aza-C on DNA methyltransferases. ChIP assay was performed to analyze chromatin modifications caused by genistein treatment.

RESULTS: BTG3 mRNA expression was down-regulated in cancer tissues and cells. Genistein and 5Aza-C induced BTG3 mRNA expression in all PC cell lines. Complete methylation of BTG3 promoter in tumor samples and cancer cell lines was observed. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Genistein and 5Aza-C increased levels of acetylated histones 3, 4, histone 3 dimethylated at lysine 4, histone 3 trimethylated at lysine 4, and RNA polymerase II, decreased DNA methyl transferase and methyl-binding domain protein 2 activity, and increased histone acetyl transferase (HAT) activity.

CONCLUSIONS: This is the first report to show that BTG3 is silenced in prostate cancer and can be reactivated by genistein-induced promoter demethylation and active histone modification. Genistein showed similar effects to that of 5Aza-C, which is currently undergoing phase 2 clinical trials as a treatment for prostate cancer. Because genistein is a natural, nontoxic, and dietary isoflavone, these results indicate that genistein is a novel, advantageous therapeutic agent for treating prostate cancer.

美国《癌症》，2010年1月

对于前列腺癌，染料木黄酮在肿瘤抑癌基因 B 细胞易位基因3中逆转高甲基化并且诱导活性组蛋白修饰

沙哈纳  马吉德，罗杰 戴哈亚等

加利福尼亚州退伍军人事务医学中心泌尿外科和加利福尼亚大学等

背景︰ B 细胞易位基因 3 (BTG3/ANA/APRO4) 在某些恶性肿瘤中是一种候选的抑癌基因。这里，我们报道了在前列腺癌中 B 细胞易位基因 3 (BTG3) 转录性下调，并且通过启动子甲基化发生失活机制。

方法︰ 用不同剂量的染料木素和 5-氮杂-2'-脱氧胞苷 (5Aza C) 来处理前列腺癌和正常的细胞系。BTG3的 信使核糖核酸 (mRNA) 的表达通过在组织和细胞系中的定量实时聚合酶链反应测定。硫酸氢钠改性的聚合酶链反应，克隆和测序被用于检查肿瘤样本和细胞株中的启动子甲基化。通过酶活性/抑制作用的测定结果来检查染料木素和 5Aza-C 对 DNA 甲基转移酶的影响。进行芯片检测来分析金雀异黄素治疗造成的染色质改性。

结果︰ BTG3的 mRNA 表达在癌组织和细胞中明显下调。染料木素和 5Aza-C 诱导所有 PC 细胞系 中的BTG3 mRNA 表达。在肿瘤样本和肿瘤细胞株中观察到BTG3 启动子完整的甲基化。染料木素和 5Aza-C 治疗明显降低了启动子甲基化，重新激活 了BTG3的表达。染料木素和5Aza-C提高了乙酰化组蛋白 3，4，组蛋白3在赖氨酸 4上的二甲基化、 组蛋白 3 在赖氨酸4和 RNA 聚合酶 II上的三甲基化，减少了 DNA 甲基转移酶和甲基结合结构域蛋白 2 的活性，并且增加了组蛋白乙酰转移酶 （HAT）的 活性。

结论︰ 这是第一份表明了BTG3 沉寂在前列腺癌中并且可以被染料木黄酮诱导的启动子去甲基化和活跃组蛋白修饰激活的报告。染料木黄酮表现出与5Aza-C类似的效果。5Aza-C作为一种治疗前列腺癌的手段，目前正在进行第二阶段临床试验。因为染料木黄酮是一种天然、无毒的膳食异黄酮，这些结果表明，染料木黄酮是一种新颖的、 有利治疗前列腺癌的治疗剂。