Dietary HDAC Inhibitors in Colon Cancer Prevention

描述(由申请人提供)： 该应用程序响应PA-06-412“饮食、表观遗传事件和癌症预防”，专注于作为组蛋白脱乙酰酶(HDAC)抑制剂的食品成分。随着人们意识到组蛋白乙酰化在许多癌症中发生改变，HDAC抑制剂作为癌症治疗药物正受到越来越多的关注。强效的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，使P21/WAF1等基因在表观上沉默，导致癌细胞生长停滞/凋亡。PI提出，饮食中的HDAC抑制剂在治疗环境中可能起到类似的作用，但由于它们经常在食物中摄入，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。中心假设是西兰花中的萝卜硫素和大蒜中的有机硫化合物是有效的化学预防药物，因为母体化合物(S)激活了Nrf2/Are信号，而代谢产物抑制了HDAC活性，从而抑制了调控细胞周期和细胞凋亡的P21等基因。通过作用于基因启动子中的Sp1/Sp3位点，膳食制剂的HDAC抑制效应可以独立于Nrf2/ARE途径发生。目标1-3是连续的，从单独的饮食化合物和人类结肠癌细胞中的HDAC抑制机制开始(目标1)，然后是体内的结肠癌抑制研究(目标2)，以及体内的联合药物(目标3A，B)，最后是人体翻译研究(目标3C)。目的1.在人结肠癌细胞中，确定与P21基因启动子区域相关的特定的HDAC，以及饮食中的HDAC抑制剂处理后这些HDAC的变化。利用免疫印迹、芯片/再芯片、qRT-PCR和siRNA敲除，系统地研究了与P21启动子区域相关的HDAC，它们对饮食中HDAC抑制剂的反应，以及特定的转录因子(Sp1，SP3)在用SFN(目标1A)或大蒜化合物(目标1B)处理的HT29细胞中去抑制p21的作用。目的2.在1，2-二甲基肼(DMH)处理的小鼠中，验证饮食HDAC抑制剂在启动后增加组蛋白乙酰化和去抑制p21，从而抑制结肠癌形成的假设。结果表明，在给予SFN(目标2A)或大蒜有机硫化合物(目标2B)的小鼠中，肿瘤抑制与HDAC抑制、乙酰化组蛋白的诱导和p21的抑制有关。目的3.研究SFN和大蒜化合物作为饮食HDAC抑制剂在小鼠中的协同作用，并将研究结果转化为人类志愿者，采用结合富含SFN的西兰花芽和大蒜油补充剂的全食方法。结果表明，SFN和大蒜化合物能协同抑制DMH诱导的小鼠结肠肿瘤(AIM 3A)，并在小鼠正常细胞(AIM 3B)，即外周血单核细胞(PBMC)中引起HDAC抑制和组蛋白超乙酰化。在人类志愿者中，西兰花芽和大蒜油补充剂会导致外周血单核细胞中HDAC抑制和组蛋白超乙酰化的假设(AIM 3C)。与癌症相关的遗传变化不同，表观遗传变化是潜在的可修改的；因此，饮食制剂的HDAC抑制作用值得研究，这对癌症的预防和治疗具有重要意义。与公共卫生相关：癌症的发展通常被认为是DNA序列中的遗传变化(即突变)，但最近的研究表明，所谓的“表观遗传”变化。后者涉及与DNA相关的蛋白质的可逆变化，并影响基因的开启或关闭方式。这个项目研究了西兰花和大蒜中的化学保护因子如何通过表观遗传机制，启动肿瘤抑制基因，从而导致结肠癌细胞停止生长和/或迅速经历细胞自杀。

DESCRIPTION (provided by applicant): This application responds to PA-06-412 "Diet, epigenetic events & cancer prevention" by focusing on food components that act as histone deacetylase (HDAC) inhibitors. HDAC inhibitors are receiving increasing interest as cancer therapeutic agents, with the realization that histone acetylation is altered in many cancers. Potent 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 proposed that dietary HDAC inhibitors might act similarly in the therapeutic setting, but because of their regular 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) from broccoli and organosulfur compounds from garlic are effective chemopreventive agents because the parent compound(s) activate Nrf2/ARE signaling whereas the metabolites inhibit HDAC activity, thereby de-repressing genes such as P21 that regulate the cell cycle and apoptosis. By acting through Sp1/Sp3 sites in gene promoters, HDAC inhibitory effects of dietary agents can occur independently of the Nrf2/ARE pathway. Aims 1-3 are sequential, starting with individual dietary compounds and HDAC inhibitor mechanisms in human colon cancer cells (Aim 1), followed by colon tumor suppression studies in vivo (Aim 2), and combined agents in vivo (Aim 3A,B), ending with translational studies in humans (Aim 3C). Aim 1. In human colon cancer cells, define the specific HDACs that associate with the promoter region of the P21 gene, and the changes in these HDACs following treatment with dietary HDAC inhibitors. Using immunoblotting, ChIP/re-ChIP, qRT-PCR and siRNA knockdown, systematically examine HDACs associated with the promoter region of P21, their de-recruitment in response to dietary HDAC inhibitors, and the role of specific transcription factors (Sp1, Sp3) in de-repressing p21, in HT29 cells treated with SFN (Aim 1A) or garlic compounds (Aim 1B). Aim 2. Test the hypothesis that, post-initiation, dietary HDAC inhibitors increase histone acetylation and de- repress p21, thereby suppressing colon tumor formation in 1,2-dimethylhydrazine (DMH)-treated mice. Show that tumor suppression is correlated with HDAC inhibition, induction of acetylated histones, and de-repression of p21 in mice given SFN (Aim 2A) or garlic organosulfur compounds (Aim 2B). Aim 3. Examine the cooperative effects of SFN and garlic compounds as dietary HDAC inhibitors in mice, and translate the findings into human volunteers, using a `whole food' approach that combines SFN-rich broccoli sprouts and garlic oil supplements. Show that SFN and garlic compounds cooperate to suppress DMH- induced colon tumors in mice (Aim 3A), and cause HDAC inhibition and histone hyperacetylation in normal cells of the mouse (Aim 3B), i.e. in peripheral blood mononuclear cells (PBMCs). Test the hypothesis that, in human volunteers, broccoli sprouts and garlic oil supplements cause HDAC inhibition and histone hyperacetylation in PBMCs (Aim 3C). Unlike genetic changes associated with cancer, epigenetic changes are potentially modifiable; thus, HDAC inhibitory effects of dietary agents are worthy of study, with implications for cancer prevention and treatment. PUBLIC HEALTH RELEVANCE: Cancer development is often considered in terms of genetic alterations in the DNA sequence (i.e. mutations), but recent work has implicated so-called `epigenetic' changes. The latter involve reversible alterations in the proteins that associate with DNA, and affect how genes are turned `on' or `off'. This project examines how chemoprotective factors in broccoli and garlic, working through epigenetic mechanisms, can turn on tumor suppressor genes, thereby causing colon cancer cells to halt their growth and/or rapidly undergo cell suicide.