The effect of 2-N-hydroxyamino-1-methyl-6-phenylimidazo [4,5-b]pyridine (NOH-PhIP) and 5α-dihydrotestosterone (DHT) individually and in combination on proteomic profiles in the rat prostate gland.

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In the USA prostate cancer is the second leading cause of cancer-related death in men; nonetheless, the etiology and molecular mechanisms underlying prostate cancer remain fully undefined. Epidemiological and preclinical studies have revealed that exposure to excessive amount of androgens and increased intake of meat products that contain carcinogenic heterocyclic aromatic amines (HAA) are possible causes of the disease. We hypothesized that the combination of testosterone and HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is more potent in the development of prostate cancer than the individual agents alone because (a) the ultimate carcinogenic form of PhIP, NOH-PhIP can induce both covalent and oxidative DNA damages, (b) metabolic activation of testosterone to 5α-dihydrotestosterone (DHT) will provide an environment of enhanced oxidative stress and cell proliferation, and (c) oxidative stress exerted by DHT on NOH-PhIP-induced DNA damaged cells can inhibit p53 and DNA repair leading to persistence of DNA adducts, mutations and subsequently carcinogenesis. To test our hypotheses, 24 male F344 rats were sacrificed; the prostate glands were rapidly excised under sterile conditions, separated into anterior, ventral, and dorsolateral lobes, and placed in ice-cold basal medium. The prostate tissue was minced with razor blade into approximately 1 mm³ pieces, and cultured at 37 °C in an atmosphere of 5% CO₂/air for 10 days; the medium was changed every other day. The cultures were treated, in duplicate, with DMSO control, and DMSO solutions of 10^-7 M DHT and 10^-3 M NOH-PhIP, individually and in combination. After 24h treatment, the prostate lobes were harvested and stored at -80°C until analysis. Proteins were isolated using the ToPI-DIGE protein isolation kit. Fifty micrograms of “treated” and “control” proteins were labeled with Cy3 and Cy5 respectively, and analyzed by 2D-DIGE using pH3-10 linear immobiline drystrips and 12.5% SDS-PAGE gels. Gel images were captured with a digital imager and candidate protein spots that displayed ≥ 2.0 - fold difference in abundance between the “treated” and “control” samples were detected using the Biological Variation Analysis module of DeCyder (Version 6.5). Protein spots were picked, in-gel digested and identified by LC-MS/MS. Proteins that showed differential expression (DE) as a function of treatment ranged from 2.7% - 4% (anterior), 2.9% - 8% (dorsolateral) and 3.7% - 8.6% (ventral). Treatment with DHT, NOH-PhIP and DHT+NOH-PhIP repressed majority of the differentially expressed proteins in the anterior (81%) and ventral (75%) lobes and activated majority of the differentially expressed proteins in the dorsolateral lobe (52%). The biological roles of the candidate proteins are being evaluated. Support: Penn State Cancer Institute