Chemoprevention of Colon Cancer, HDAC Inhibition, and Histone Status

项目3研究作为组蛋白脱乙酰酶(HDAC)抑制剂的食物成分。HDAC抑制剂通过相应基因启动子中的Sp1/Sp3转录因子结合位点，对表观遗传沉默的基因(如P21/WAF1)进行‘去抑制’，导致癌细胞生长停滞/凋亡。PI支持这样的观点，即饮食中的HDAC抑制剂在治疗环境中可能起到类似的作用，但由于它们在食物中的摄取，它们还通过表观遗传学对正常细胞中的基因表达起到化学预防作用。 中心假设是萝卜硫醚(SFN)和吲哚-3-甲醇(I3C)及其衍生的十字花科蔬菜在结肠中是有效的化学预防药物，因为除了它们在起始阶段的阻断活性外，它们还抑制HDAC活性并改变癌细胞中组蛋白修饰的模式(乙酰化、甲基化、磷酸化)，从而抑制表观遗传沉默的基因，如调节细胞周期和细胞凋亡的P21。 目标1-3从人类结肠癌细胞的机制研究开始，然后是大鼠结肠癌模型的临床前剂量-反应实验，最后是人类翻译工作(结肠镜筛查)。 目的：1.研究SFN和I3C对人结肠癌细胞株P21启动子区域HDACs、组蛋白状态(乙酰化/甲基化/磷酸化)和Sp1/SP3转录因子结合的影响，以及HDAC抑制的机制。研究P21启动子的DNA甲基化状态和非组蛋白靶标如P53的变化。本研究的目的是利用免疫印迹、ChlP/Re-ChIP、co-IP和qRT-PCR来检测可逆组蛋白修饰、p21抑制和HDAC抑制/翻转。 目的2.在2-amino-1-methyl-6-phenylimidazo[4，5-b]pyridine(PhIP)大鼠结肠癌模型中，研究使用整体食物(西兰花芽、甘蓝芽)和单独的植物化学物质(SFN、I3C)对结肠癌的化学预防，验证AIM 1的表观遗传生物标记物作为结肠癌预后预测因子的有效性。通过检查其他组织(肺、胸腺、前列腺)中可能在临床环境中应用的表观遗传生物标记物，如结肠镜检查，与项目1和2相结合。 目的3.在进行结肠镜检查的患者中，招募低、中、高十字花科蔬菜消费者，评估组织活检标本和循环血细胞的HDAC活性和组蛋白状态。利用表观遗传/翻译生物标记物核心中的LC/MS/MS检测SFN代谢物作为十字花科蔬菜摄入量的生物标记物，以及结肠活检组织中K-ras的突变状态。

Project 3 studies food components as histone deacetylase (HDAC) inhibitors. HDAC inhibitors 'de-repress' epigenetically silenced genes, such as P21/WAF1, through Sp1/Sp3 transcription factor binding sites in the corresponding gene promoters, causing growth arrest/apoptosis in cancer cells. The PI has championed the view that dietary HDAC inhibitors might act similarly in the therapeutic setting, but because of their ingestion in foods they also serve a chemopreventive role via epigenetic 'priming' of gene expression in normal cells. The CENTRAL HYPOTHESIS is that sulforaphane (SFN) and indole-3-carbinol (I3C), and the cruciferous vegetables from which they derive, are effective chemopreventive agents in the colon because, in addition to their blocking activities during the initiation phase, they inhibit HDAC activity and alter the pattern of histone modifications (acetylation, methylation, phosphorylation) in cancer cells, thereby de-repressing epigenetically silenced genes such as P21 that regulate the cell cycle and apoptosis. Aims 1-3 start with mechanistic studies in human colon cancer cells, followed by preclinical dose-response experiments in a rat colon cancer model, ending with human translational work (colonoscopy screening). Aim 1. In human colon cancer cells treated with SFN and I3C, define the changes HDACs, histone status (acetylation/methylation/phosphorylation), and Sp1/Sp3 transcription factor binding on the promoter region of P21, and mechanisms of HDAC inhibition. Study the DNA methylation status of the P21 promoter and changes in non-histone targets such as p53. This aim uses immunoblotting, ChlP/re-ChIP, co-IP and qRT-PCR to examine reversible histone modifications, p21 de-repression, and HDAC inhibition/turnover. Aim 2. In the 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) rat colon carcinogenesis model, examine chemoprevention of colon cancer using whole foods (broccoli sprouts, Brussels sprouts) and individual phytochemicals (SFN, I3C), Validate epigenetic biomarkers from Aim 1 as predictors of colon tumor outcome. Integrate with Projects 1 and 2 by examining other tissues (lung, thymus, prostate) for epigenetic biomarkers that might be applied in the clinical setting, such as during colonoscopy screening. Aim 3. In patients presenting for screening colonoscopy, recruit low, mid, and high cruciferous vegetable consumers and assess tissue biopsy specimens and circulating blood cells for HDAC activity and histone status. Measure SFN metabolites as a biomarker of cruciferous vegetable intake, using LC/MS/MS in the Epigenetic/Translational Biomarkers core, and the mutation status of K-ras in colon biopsies.