Natural source inhibitors of carcinogenesis are usually non-toxic or markedly less toxic then synthetic chemopreventive agents. The studies on the mechanism(s) of anti-tumor initiating and anti-tumor promoting properties of a variety of naturally occurring phytochemicals suggest that they are very important for the prevention of and other epithelial cancers in humans. The purpose of our study was to investigate the antioxidant and anti-carcinogenic properties of the selected natural antioxidants, i.e., grape seed extract (GSE, a major antioxidant proanthocyanidin B-2-gallate), resveratrol (RES), and ursolic acid (URA) as well as ellagic acid (ELA), in order to assess the mechanism(s) by which these antioxidants inhibit murine skin carcinogenesis. The following in vitro test tube assays were used: linoleic acid autooxidation assay, cytochrome c reduction assay and 2-deoxyribose assay, to measure quenching of peroxyl, superoxide and hydroxyl radicals, respectively. We have found that GSE, ELA and RES are potent scavengers of peroxyl radicals and superoxide anion while URA inhibits these radicals to a lesser extent. Hydroxyl anions were efficiently quenched by ELA (IC50 10 µM), and at higher concentrations by GSE. We also used ATP-bioluminescence, Caspase-Glo 3/7 and P450-Glo (CYP 1A1 and 1B1) assays to study antiproliferative, proapoptotic and CYP450 inhibiting effects of the above inhibitors. Three murine keratinocyte cell lines, non-tumorigenic (3PC), papilloma (MT1/2), and squamous cell carcinoma (Ca3/7), representing various stages of malignant transformation were used. Apoptosis induced by these compounds was associated with the increased activities of caspases 3 and 7 but the statistically significant effect was apparent only during GSP and URA treatments. The treatment of carcinoma cells with 10 µM URA and 50 µM GSE for 24 h increased the level of caspase 3 and 7 activity 2 and 6 times, respectively. On the other hand, URA was the only compound with no impact on activity of CYP 1A1 and 1B1, while all the other test compounds showed strong inhibitory effects for both cytochromes. IC50 for GSE was 4.9 µM for CYP1A1 and 2.6 µM for CYP 1B1 while for ELA IC50 was 32.7 and 2.1 respectively. RES showed the strongest effect - 0.05 and 0.34 µM. In conclusion, all selected natural source antioxidants are potent scavengers of free radicals in the tests described above. Also, significant activation of caspases 3 and 7 was observed in the different stages of epidermal tumor development. These results may have important implications for the development of new strategies for treating skin cancer. Supported by NIH grants CA 102747 and P30 CA 54174-16S1.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA