? ? 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.
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. ? ? ?
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