The purpose of our study was to determine the effect of combined topical and dietary treatment with several natural compounds on the expression of intermediate biomarkers related to early stages of skin tumorgenesis, i.e., initiation and promotion. We tested ellagic acid (ELA) and calcium D-glucarate (CG) given in the diet, while resveratrol (RES) was applied topically; grape seed extract (GSE) was applied topically or given in the diet. The 4-week inflammatory-hyperplasia assay, based on the 7,12-dimethylbenz[a]anthracene (DMBA)-induced complete skin carcinogenesis model in SENCAR mice was used. Inhibitors were applied either topically 20 min prior to DMBA or were given in the AIN-93G diet beginning 2 weeks prior to the first dose of DMBA. We analyzed by RT-PCR and immunochistochemical analyses the expression of biomarkers of cell proliferation, apoptosis and inflammation. Topical application of DMBA resulted in epidermal hyperplasia characterized by significant increase in the mRNA levels of PCNA, p21, Bcl-2 and AP-1 components with simultaneous increased expression of the inflammation marker COX-2 and of the Phase I CYP1B1 enzyme compared to untreated controls. Skin tissue of mice receiving combinations of diets with topical treatments showed significant alterations in DMBA-induced effects: decreased cell proliferation (PCNA) and Bcl2 expression; decreased p21, negative regulator of the cell cycle; decreased inflammation (COX-2). Combinations of the phytochemicals seemed to also exert influence on the modulation of Phase I drug metabolizing enzymes. All the selected combinations caused a marked decrease of the CYP1B1 level compared to DMBA-treated group and also to groups treated with a single compound and DMBA. All combinations also diminished the DMBA-induced mRNA expression of proto-oncogenes c-jun and c-fos, components of the transcription factor AP-1, which can regulate the induction of COX-2, an enzyme associated with inflammation and tumorigenesis. In fact, COX-2 expression was also reduced follow treatment with combined inhibitors. Resveratrol showed a weak effect when used alone, as shown in our previous study of its effect on epithelial thickness, BrdU index of proliferation, percentage of mice with mutation in 61 codon of Ha-ras. Unexpectedly, however, combinations of RES with dietary ELA and GSE were the most effective compared to DMBA-treated group and also to groups treated with the single compounds and DMBA. In conclusion, resveratrol combinations with ELA, GSE and other phytochemicals are very potent inhibitors of skin tumorgenesis based on the suppression of epidermal hyperplasia as well as on modulation of intermediate biomarkers of cell proliferation, cell survival, inflammation, oncogene mutation and apoptosis. Supported by NIH grants CA 102747 and P30 CA 54174-16S1.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4789.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO