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.
In addition to genetic changes affecting DNA sequence information, we now realize that cancer development involves so-called epigenetic events, which represent a major new research priority area at NIH. One aspect of intense current interest concerns the histone (protein) modifications that silence tumor suppressor genes in cancer cells. We find that dietary agents can reverse such modifications, thereby re-expressing tumor suppressor genes and triggering cancer cells to arrest their growth and/or commit suicide via apoptosis.
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|Harper Jr, Tod A; MorrÃ©, Jeff; Lauer, Fredine T et al. (2015) Analysis of dibenzo[def,p]chrysene-deoxyadenosine adducts in wild-type and cytochrome P450 1b1 knockout mice using stable-isotope dilution UHPLC-MS/MS. Mutat Res Genet Toxicol Environ Mutagen 782:51-6|
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