Significant inhibition by the flavonoid antioxidant silymarin against 12-O-tetradecanoylphorbol 13-acetate-caused modulation of antioxidant and inflammatory enzymes, and cyclooxygenase 2 and interleukin-1alpha expression in SENCAR mouse epidermis: implications in the prevention of stage I tumor promotion.

The flavonoid antioxidant silymarin is used clinically in Europe and Asia for the treatment of liver diseases and is sold in the United States and Europe as a dietary supplement. Recently we showed that silymarin possesses exceptionally high cancer-preventive effects in different mouse skin carcinogenesis models and affords strong anticancer effects in human skin, cervical, prostate, and breast carcinoma cells. More recently, we showed that the anti-tumor-promoting effect of silymarin is primarily targeted against stage I tumor promotion in mouse skin (Cancer Res 1999;59:622-632). Based on this recent study, in this report, further investigations were made to identify and define the biochemical and molecular mechanisms of silymarin's effect during stage I tumor promotion in mouse skin. A single topical application of silymarin at 3-, 6-, and 9-mg doses onto SENCAR mouse skin followed 30 min later with 12-O-tetradecanoylphorbol 13-acetate (TPA) at a 3-microg dose resulted in a 76-95% inhibition (P < 0.001) of TPA-caused skin edema. Similarly, these doses of silymarin also showed 39-90%, 29-85%, and 15-67% protection (P < 0.05 or 0.001), against TPA-caused depletion of epidermal superoxide dismutase, catalase, and glutathione peroxidase activity, respectively. Pretreatment of mice with silymarin also produced highly significant inhibition of TPA-caused induction of epidermal lipid peroxidation (47-66% inhibition, P < 0.001) and myeloperoxidase activity (56-100% inhibition, P < 0.001). In additional studies assessing the effect of silymarin on arachidonic acid metabolism pathways involving lipoxygenase and cyclooxygenase (COX), similar doses of silymarin showed highly significant inhibition of TPA-caused induction of epidermal lipoxygenase (49-77% inhibition, P < 0.001) and COX (35-64% inhibition, P < 0.01 or 0.001) activity. Western immunoblot analysis showed that the observed effect of silymarin on COX activity was due to inhibition of TPA-inducible COX-2 with no change in constitutive COX-1 protein levels. In other studies, silymarin also showed dose-dependent inhibition of TPA-caused induction of epidermal interleukin 1alpha (IL-1alpha) protein (39-72% inhibition, P < 0.005 or 0.001) and mRNA expression. Taken together, the results from these biochemical and molecular studies further substantiate our recent observation of silymarin's anti-tumor-promoting effects primarily at stage I tumor promotion. Furthermore, the observed inhibitory effects of silymarin on COX-2 and IL-1alpha should be further explored to develop preventive strategies against those cancers in which these molecular targets play one of the causative roles, such as non-melanoma skin, colon, and breast cancers in humans.