The main goal of this proposal is to investigate whether genistein inhibits prostate cancer growth through epigenetic pathways. The rationale for this project is that Western men have a 5-6 fold higher incidence of prostate cancer than Asians. One reason for this discrepancy may be the high soy diet (genistein) consumed by Asians. We hypothesize that genistein inhibits prostate cancer growth through epigenetic pathways.
Specific Aim # 1. To investigate whether genistein can suppress prostate cancer growth through cell cycle arrest, cyclin genes and apoptosis. Based on the published literature and preliminary data, we lypothesize that genistein will inhibit prostate cancer cell growth in vitro. We will investigate whether genistein alone or in combination with demethylating agent (5-'-aza-2-deoxycytidine (5-AZA-C) and histone deacetylase inhibitor (trichostatin (TSA) can inhibit prostate cancer growth in vitro. A panel of human prostate cell lines will be studies for cell proliferation, cell cycle, cyclin genes and apoptosis.
Specific Aim # 2. To investigate the molecular mechanisms of genistein action through epigenetic pathways. Based on our preliminary data, we hypothesize that genistein inhibits prostate cancer growth through modulation of epigenetic events, such as DNA methylation and histone acetylation. To test this hypothesis, we will analyze: 1) Global methylation and enzymatic activity of DNA methyltransferase (DNMT) using radiolabeled S- adenosylmethionine (SAM) assays. 2) Promoter methylation of candidate genes using bisulfite modification of DNA based techniques. 3) Alterations in gene-specific histone acetylation using ChIP assay, and enzymatic activities of histone acetyl transferases (HATs). 4) Histone deacetylase (HDACs) analysis using biochemical assays. 5). Alterations in mRNA expression of genes that encode enzymes participating in DNA methylation and histone acetylation including DNMTs, demethylases, HATs, HDACs using RT-PCR and northern blot assays. 6) To analyze transcription factors which mediate the effects of genistein on prostate cancer.
Specific Aim # 3. To investigate whether genistein has anti-tumor effects through reversal of epigenetic pathways in prostate cancer using an in vivo system. Based on the published literature, genistein or demethylating agents have anti-proliferative effects on various cancers. We hypothesize that genistein will inhibit prostate cancer growth through reversal of epigenetic pathways. Studies will be conducted in athymic nude mice implanted with androgen-responsive LNCaP and androgen nonresponsive DuPro prostate cancer cells. Mice will be fed a diet supplemented with genistein, or a combination of genistein with 5-AZA-C or TSA. The following experiments will be conducted: 1) Determine the in vivo anti-tumor effects of genistein and in combination with 5-AZA-C or TSA. 2) To evaluate expression of steroid receptor genes and tumor suppressor genes at the mRNA and protein levels using Northern blot, and Western blotting analysi respectively. 3) To analyze whether genistein treatment can modulate global and candidate gene methylation in tumor tissue using a radiolabeled SAM assay and bisulfite modified genomic sequencing techniques respectively. 4) To analyze, in tumor tissue, gene-specific histone acetylation using ChIP assay and the enzymatic activities of HATs and HDACs using biochemical assays. Accomplishment of these experiments will demonstrate whether epigenetic events are modulated by genistein and provide rationale for a clinical trial of genistein alone or in combination with other epigenetic modulators for the treatment or prevention of prostate cancer. ? ? ?
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