Others and we have shown that fish oil vs corn oil, is protective against experimentally-induced colon cancer. Recently we reported that this protective effect is due to a higher steady state level of apoptosis with fish oil supplementation. In addition, we show a synergistic protective effect of the combination of fish oil and pectin (a highly fermentable fiber) on both tumor incidence and enhancement of spontaneous apoptosis. We now show data that the combination of fish oil and pectin also enhances targeted apoptosis within the first 12 hours after administration of the carcinogen azoxymethane (AOM). The overall goal of this proposal is to understand, at a mechanistic level, how fish oil, high in n-3 fatty acids, induces apoptosis in colonocytes and thus protects against experimentally-induced colon tumorigenesis. A secondary goal is to determine how supplementing fish oil-containing diets with pectin compared to cellulose synergistically enhances the protective effect of fish oil. Hypotheses: The observed enhancement of apoptosis with fish oil supplementation is due to (1) down regulation of Cox-2 expression providing a permissive environment for butyrate-induced apoptosis; and/or (2) alterations in mitochondrial function which are permissive for butyrate-induced apoptosis. To test these hypotheses we have three specific aims: 1. Determine in vivo expression of Cox-2, apoptosis, p21waf1/CipI p27kip1, Cyclin Dl, cell proliferation and differentiation as a function of dietary lipid, butyrate, and carcinogen administration, during the stage of promotion. Using a 3 x 2 x 2 factorial design (fish oil, fish oil ethyl esters, or corn oil supplementation; plus or minus butyrate; saline or AOM), we will use quantitative immunohistochemistry on serial sections of rat colon to detect patterns of expression of Cox-2, apoptosis, p21 WAFI/CIPI and Cyclin Dl; and p27Kip1 differentiation, and apoptosis in the same cells. 2. Determine in vivo expression of the same markers as in specific aim #1, as a function of dietary lipid, butyrate, and AOM, but during the initiation stage of colon cancer. 3. Determine in an ex vivo system if fish oil alters mitochondrial function thus creating a permissive environment for butyrate-induced apoptosis. Using a 3 x 2 factorial design (fish oil; fish oil ethyl esters or corn oil; saline or AOM), during the stage of promotion we will determine the effect on known indicators of mitochondrial function, reactive oxygen species, cardiolipin fatty acid composition and butyrate-induced apoptosis. An understanding of how diet affects colon tumor incidence has important consequences for the design of clinical trials and for future dietary recommendations.
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