Epigenetic modifications by dietary PEITC in prostate cancer

描述(申请人提供)：背景和假设：前列腺癌(PCA)仍然是美国男性癌症死亡的第二大原因。在之前的资助期间，我们发现苯乙基异硫氰酸酯(PEITC)和姜黄素(Cur)单独或联合通过诱导促凋亡和抗增殖的信号通路抑制裸鼠PC-3移植瘤和TRAMP小鼠的前列腺癌的发生和发展。R01继续应用的目的是研究PEITC对前列腺癌的表观遗传效应。本申请中提出的研究的基本原理来自我们已发表和未发表的初步研究，这些研究表明：(I)随着TraMP PCa的进展，抗氧化应激转录因子Nrf2和Nrf2靶向基因II期解毒和抗氧化酶逐渐丧失；(Ii)PEITC治疗导致Nrf2和Nrf2靶向基因UGT1A1在TRAMP前列腺癌中重新表达，与肿瘤抑制相关；(Iii)NRF2基因在TRAMP肿瘤和TRAMP C1细胞系中通过启动子CpG高甲基化而表观调控；(Iv)芯片检测显示Mbd2和三甲基-组蛋白H3(Lys9)蛋白与这些CpG位点的结合增加；(V)用DNMT抑制剂5-aza和HDACi TSA处理后，Nrf2的表达恢复；(Vi)PEITC利用DNA甲基化PCR阵列逆转了LNCaP细胞中96个基因启动子中某些基因的甲基化状态；(Vii)PEITC通过MSP检测和qPCR检测恢复了14-3-3基因的表达。尽管有这些有希望的结果，但我们对PEITC的表观遗传机制(S)的理解仍存在重大差距。根据我们的初步研究结果，我们想要验证我们的假设，即表观遗传事件将推动流浪汉前列腺癌的发生和进展，而PEITC修改这些改变，从而产生其PCA化学预防作用。在体内和体外，将详细研究TRAMP中Nrf2和Nrf2靶基因的表观遗传学事件以及LNCaP中的促凋亡/抗增殖基因。具体目的：本项目的具体目的是：(1)研究TRAMP小鼠前列腺癌发生发展过程中的表观遗传学改变，以及PEITC通过表观遗传学修饰的化学预防作用。(2)研究膳食PEITC诱导的促凋亡/抗增殖基因对SCID小鼠LNCaP原位移植瘤生长抑制的表观遗传修饰作用。(3)探讨PEITC对体内AIMS 1和2基因调控的体外表观遗传学机制。建议研究的意义：通过饮食或化学干预进行PCA的癌症化学预防是合乎逻辑的，将为美国数十万男性带来巨大的临床好处。这项拟议研究的成功将进一步推动PEITC的临床试验，以确定其化学预防表观遗传机制、生物标志物和对抗人类前列腺癌的有效性。

DESCRIPTION (provided by applicant): Background and Hypothesis: Prostate cancer (PCa) remains the second leading cause of cancer death in men in the United States. During the previous funding period, we have found that phenethyl isothiocyanate (PEITC) and curcumin (CUR) alone and in combination inhibit the development and progression of PCa in nude mice PC-3 xenograft and TRAMP mice via induction of pro-apoptotic and anti-proliferative signaling pathways. The objective of this R01 continuation application is to investigate the epigenetic effects of PEITC against PCa. Rationale for the studies proposed in this application is derived from our published and unpublished preliminary studies showing that: (i) As TRAMP PCa progresses, there is a progressive loss of the anti-oxidative stress transcription factor Nrf2 and Nrf2-target genes phase II detoxifying and antioxidant enzymes; (ii) Treatment with PEITC resulted in the re-expression of Nrf2 and Nrf2-target gene UGT1A1 in the TRAMP prostate tumor correlating with tumor suppression; (iii) Nrf2 gene is epigenetically regulated in TRAMP tumor and TRAMP C1 cell line through promoter CpG hypermethylation; (iv) ChIP assays revealed increased binding of the MBD2 and trimethyl-histone H3 (Lys9) proteins to these CpG sites; (v) treatment with DNMT inhibitor 5-aza and HDACi TSA restores the expression of Nrf2; (vi) PEITC reverses the methylation status of certain genes in LNCaP cells from a panel of 96 gene promoters using DNA methylation PCR Array; (vii) PEITC restores expression of 14-3-3 gene by MSP assay and qPCR. Despite these promising results, significant gaps exist in our understanding of the epigenetic mechanism(s) of PEITC. Based on the results of our preliminary studies we would like to test our hypothesis that epigenetic events would drive the development and progression of PCa in TRAMP and PEITC modifies these alterations leading to its PCa chemopreventive effect. The epigenetic events of Nrf2 and Nrf2-target genes in TRAMP as well as pro- apoptotic/anti-proliferative genes in LNCaP will be investigated in detailed both in vivo and in vitro. Specific Aims: The specific aims of this project are to: (1) To investigate the epigenetic alterations during the development and progression of prostate tumorigenesis in TRAMP mice and the chemopreventive effects of PEITC through epigenetic modifications. (2) To determine the epigenetic modifications of pro-apoptotic/anti- proliferative genes elicited by dietary PEITC on the growth inhibition of LNCaP orthotopic xenograft tumors in SCID mice. (3) To elucidate the in vitro epigenetic mechanisms of regulation of the genes obtained from in vivo Aims 1 and 2 by PEITC in TRAMP C1, C3 and LNCaP cell lines. Significance of the Proposed Research: Cancer chemoprevention of PCa through dietary or chemical intervention would be logical and would be tremendous clinical benefit to hundreds of thousands of men in the United States. The success of the proposed study will further drive clinical trials of PEITC to determine its chemopreventive epigenetic mechanisms, biomarkers and effectiveness against human PCa.