Differential effects of several phytochemicals and their derivatives on murine keratinocytes in vitro and in vivo: implications for skin cancer prevention.

The purpose of our study was to investigate in vitro the potential cancer preventive properties of several phytochemicals, i.e. grape seed extract (GSE), resveratrol (RES), ursolic acid (URA), ellagic acid (ELA), lycopene and N-acetyl-L-cysteine (NAC) to define the mechanisms by which these compounds may inhibit murine skin carcinogenesis. We measured quenching of peroxyl, superoxide and hydroxyl radicals by these phytochemicals. We also used adenosine triphosphate (ATP) bioluminescence, Caspase-Glo 3/7 and P450-Glo (CYP1A1 and CYP1B1) assays to study antiproliferative, proapoptotic and CYP-inhibiting effects of the phytochemicals. We next determined their effects on a 4 week inflammatory hyperplasia assay using 7,12-dimethylbenz[a]anthracene-induced murine skin carcinogenesis model to further understand their mechanism of action. Three murine keratinocyte cell lines, i.e. non-tumorigenic (3PC), papilloma-derived (MT1/2) and squamous cell carcinoma-derived (Ca3/7) cell lines, were used in in vitro assays. We have found that GSE, ELA and RES are potent scavengers of peroxyl and superoxide radicals. Statistically significant effects on activities of caspase-3 and -7 were observed only after GSE and URA treatments. All tested compounds protected cells from hydrogen peroxide-induced DNA damage. Using a short-term complete carcinogenesis assay, we have found that all selected compounds caused marked decreases of epidermal thickness and (except RES) reduced percentages of mice with mutation in codon 61 of Ha-ras oncogene. In conclusion, differential effects of tested phytochemicals on events and processes critical for the growth inhibition of keratinocytes in vitro and in vivo indicate that combinations of tested compounds may, in the future, better counteract both tumor initiation and tumor promotion/progression.