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. 2009 Jun;30(6):1008-15.
doi: 10.1093/carcin/bgp069. Epub 2009 Mar 27.

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

Magdalena C Kowalczyk  1 , Zbigniew Walaszek, Piotr Kowalczyk, Tatsuya Kinjo, Margaret Hanausek, Thomas J Slaga
Affiliations

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

Magdalena C Kowalczyk et al. Carcinogenesis. 2009 Jun.

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
(A) Superoxide scavenging (percentage inhibition of the cytochrome c reduction) by GSE (filled square), ELA (filled triangle), RES (filled diamond) and URA (filled circle) at 21°C. (B) Inhibition of auto-oxidation of linoleic acid to its conjugated diene hydroperoxide in the presence of GSE (filled square), ELA (filled triangle), RES (filled diamond) and NAC (open circle). (C) The viability of keratinocytes exposed to the compounds tested for 24 h in the ATP assay. Cells were treated with different concentrations of the compounds and the dose–response curves provided an IC50 values. (D) The growth inhibitory effect of the combined test compounds on MT1/2 papilloma cells. The concentrations 10 μM for GSE, 2 μM for RES, 10 μM for ELA, 100 μM for NAC and 10 μM for LYC were chosen. Interaction was examined after 24 h of treatment using the ATP assay. The cell viability is presented as a percent of control. P value (★) less than 0.05 was considered statistically significant.
Fig. 2.
Fig. 2.
(A) Effects of preincubation of the compounds tested on the rate of H2O2-mediated DNA damage in Ca3/7 cells, as determined by the comet assay. All compounds lowered the rate of H2O2-mediated DNA damage compared with the positive control. In each experiment, 50 cells were analyzed per sample and values are means ± SEM of the comet tail length. (B) To show the effect of GSE and URA on cellular caspase-3 and -7 activity, Ca3/7 cells were exposed to different concentrations of these compounds for 24 h. Activated caspase-3/-7 level was measured by conversion of aminoluciferin into a luminescent signal.
Fig. 3.
Fig. 3.
Inhibition of CYP1A1 and CYP1B1 activities by GSE (A), ELA (B), RES (C) and LYC (D).
Fig. 4.
Fig. 4.
(A) Effects of the test compounds on sustained epidermal hyperplasia after multiple treatments with each compound. The results are the averages of 20 measurements at various locations along the epidermis of the skin specimen from each treatment group. (B) Mutations in codon 61 of Ha-ras oncogene were analyzed in skin of SENCAR mice treated with the test compounds.
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