Many epidemiological studies have supported that frequent consumption of cruciferous vegetables such as broccoli, cauliflower, cabbage, watercress and Brussels sprout is associated with a decreased risk in various type of human cancer including colon, lung, esophageal and stomach. Cruciferous vegetables contain high quantity of isothiocyanates (ITCs) including sulforaphane (SUL) and phenethylisothiocyanate (PEITC). These compounds have been shown to inhibit chemically induced carcinogenesis in various animal models. Our long-term goal is to elucidate the mechanisms of inhibition of carcinogenesis by ITCs. In this renewal application, we will test the hypothesis that ITC prevents carcinogenesis by inhibiting cellular proliferation and enhancing apoptosis in the intestine by examining the dose-response of the ITCs and the related pharmacokinetics, metabolism and tissue levels of the drugs, to examine the in vivo mechanism and to elucidate the related molecular mechanisms using in vitro cell culture models. The following specific aims are designed to address our hypothesis. 1. Investigate the dose-response of inhibition of carcinogenesis by ITCs in Min intestinal neoplasia mice. We will study the inhibition of cell proliferation and tumor progression by different doses of ITCs, SUL and PEITC, in short-term as well as long-term treatments. We will also examine the tumor and plasma levels of ITCs and their metabolites under different experimental conditions using LC/MS and characterize the relationship between drug levels and response. The tumor and plasma levels of these compounds will serve as potential references for evaluating the mechanisms of anti-carcinogenesis and for comparing results in animals and humans in the future. 2. Examine the in vivo mechanisms of inhibition of carcinogenesis by ITCs in Min intestinal neoplasia mice. We will study whether the inhibition of cell proliferation and tumor progression by ITCs, SUL and PEITC described in Aim 1 above, could be related to the activity of pertinent signal transduction pathways (such as MAPKcaspases- apoptosis and APC-beta-catenin-cell cycle pathways) in long-term and short-term animal experiments. DNA microarray analysis of tumor samples after ITC treatments will be conducted to complement and to further delineate the mechanisms of ITCs. 3. Elucidate in-depth mechanistic studies in colon cancer cell lines on the signal transduction pathways leading to growth inhibition and apoptosis induced by ITCs, including the role of APC-beta-catenin-cell cycle arrest pathway and MAPK-caspase-apoptosis pathway. Additional signaling pathways will be explored based on the DNA microarray analysis described above in Aim 2. These studies will complement the studies in Aim 2 and will provide the basic understanding of the chemopreventive actions of isothiocyanates in general.
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