Chemoprevention of Colon Cancer, HDAC Inhibition, and Histone Status

项目3研究食物成分作为组蛋白去乙酰化酶（HDAC）抑制剂。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“去抑制”表观遗传学沉默的基因，如P21/WAF 1，导致癌细胞的生长停滞/凋亡。PI支持这样的观点，即饮食HDAC抑制剂在治疗环境中可能起类似的作用，但由于它们在食物中的摄入，它们也通过正常细胞中基因表达的表观遗传“引发”起化学预防作用。 中心假设是萝卜硫素（SFN）和吲哚-3-甲醇（I3 C）以及它们所来源的十字花科蔬菜是结肠中有效的化学预防剂，因为除了它们在起始阶段的阻断活性之外，它们还抑制HDAC活性并改变组蛋白修饰的模式在癌细胞中，通过抑制癌细胞中的乙酰化、甲基化、磷酸化，从而去抑制表观遗传学上沉默的基因，如调节细胞周期和凋亡的P21。 目的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，最后是人类转化工作（结肠镜筛查）。 目标1.在用SFN和I3 C处理的人结肠癌细胞中，定义HDAC、组蛋白状态（乙酰化/甲基化/磷酸化）和P21启动子区域上的Sp1/Sp3转录因子结合的变化，以及HDAC抑制机制。研究P21启动子的DNA甲基化状态和非组蛋白靶点如p53的变化。该目的使用免疫印迹、ChIP/re-ChIP、co-IP和qRT-PCR来检查可逆的组蛋白修饰、p21去阻遏和HDAC抑制/转换。 目标2.在2-氨基-1-甲基-6-苯基咪唑[4，5-B]吡啶（PhIP）大鼠结肠癌发生模型中，使用全食物（西兰花芽、布鲁塞尔芽）和单个植物化学物质（SFN、I3 C）检查结肠癌的化学预防，将Aim 1中的表观遗传生物标志物作为结肠肿瘤结局的预测因子。通过检查其他组织（肺、胸腺、前列腺）的表观遗传生物标志物与项目1和2相结合，这些生物标志物可能应用于临床环境，例如在结肠镜检查筛查期间。 目标3.在进行结肠镜筛查的患者中，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。测量SFN代谢物作为十字花科蔬菜摄入量的生物标志物，使用LC/MS/MS在表观遗传/翻译生物标志物核心，以及结肠活检中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.