Effects of resveratrol on 12-O-tetradecanoylphorbol-13-acetate-induced oxidative events and gene expression in mouse skin

doi:10.1016/S0304-3835(98)00250-X

Cancer Letters

Volume 134, Issue 1, 11 December 1998, Pages 81-89

https://doi.org/10.1016/S0304-3835(98)00250-X Get rights and content

Abstract

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a natural product shown to inhibit carcinogen-induced pre-neoplastic lesions in mouse mammary organ culture and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted mouse skin tumors. Application of TPA to mouse skin induces oxidative stress, as evidenced by numerous biochemical responses, including significant generation of H₂O₂ and enhanced levels of myeloperoxidase and oxidized glutathione reductase activities and decreases in glutathione levels and superoxide dismutase activity. TPA treatment also elevates the expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), c-myc, c-fos, c-jun, transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α). As currently reported, pre-treatment of mouse skin with resveratrol negated several of these TPA-induced effects in a dose-dependent manner. H₂O₂ and glutathione levels were restored to control levels, as were myeloperoxidase, oxidized glutathione reductase and superoxide dismutase activities. As judged by reverse transcriptase-polymerase chain reaction (RT-PCR), TPA-induced increases in the expression of c-fos and TGF-β1 were selectively inhibited. These data suggest that resveratrol inhibits tumorigenesis in mouse skin through interference with pathways of reactive oxidants and possibly by modulating the expression of c-fos and TGF-β1.

Introduction

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a naturally occurring stilbene found in various plant species. Of particular note, significant quantities of the compound are present in grape skins and red wines as a result of being generated by Vitis vinifera L. (Vitaceae) in response to fungal infection [1]. Cancer chemopreventive potential has been established since resveratrol inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced pre-neoplastic lesion formation in mouse mammary organ culture and reduces the incidence and multiplicity of DMBA/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papillomas in the two-stage mouse skin model [2]. However, the mechanism by which resveratrol inhibits carcinogenesis has not been elucidated.

DMBA/TPA-induced responses have been extensively studied in mouse skin, which is commonly used as a model for the process of carcinogenesis. It is well-known, for example, that protein kinase C activity, expression and intracellular distribution are modulated by treatment with TPA [3], [4]. In addition, the tumor promoting activity mediated by TPA has been associated with oxidative stress, as exemplified by increased production of superoxide anion radicals, H₂O₂ and lipid hydroperoxides [5]. Furthermore, topical treatment of murine epidermis with tumor promoters modulates cellular antioxidant defense systems, as exemplified by a reduction of superoxide dismutase activity, which is able to detoxify superoxide anion radicals and interference with reduced glutathione (GSH) metabolism, a key intracellular component capable of protecting cellular constituents from attack by peroxide and free radicals [6]. Although the mechanisms by which cellular pro-oxidant states facilitate tumor promotion are poorly understood, the sequential formation of reactive oxygen species (ROS) in TPA-stimulated mouse skin may readily initiate free radical chain reactions, triggering or amplifying some essential events in the process of tumorigenesis.

The application of TPA to mouse skin or certain cells in culture results in a host of biological and biochemical alterations, including stimulation of arachidonic acid metabolism, accumulation of neutrophils and inflammatory responses [7]. TPA-induced oxidative stress may produce selective growth and altered gene expression through modification of regulatory molecules. For example, TPA effectively enhances expression of the proto-oncogenes c-myc, c-fos and c-jun in mouse skin [8]. This type of altered gene expression may be critical in transmitting tumor-promoting signals from the extracellular environment to the nuclear transcriptional machinery.

In the current investigation, utilizing CD-1 mice, we have evaluated the potential of resveratrol to modulate TPA-induced oxidative stress by assessing H₂O₂ production, myeloperoxidase (MPO) activity, oxidized glutathione (GSSG) reductase activity, GSH levels and SOD activity and the expression of several genes, including cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), c-myc, c-fos, c-jun, transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α). The results of these studies help to define the mechanism by which resveratrol functions as a chemopreventive agent.

Section snippets

Treatment of animals

Female CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA) and used at 5–6 weeks of age. For topical treatment with tumor promoter and resveratrol, the backs of mice were shaved with electric clippers 2 days prior to treatment. Animals showing hair regrowth were not used. Utilizing nine groups of three mice, 10 nmol TPA in 0.2 ml acetone, or acetone alone, was applied topically to the dorsal skin of each mouse. The mice were sacrificed by CO₂ anesthesia followed by cervical

TPA-induced oxidative events in mouse skin

Treatment of mouse skin with TPA caused overproduction of cellular oxidants and modulation of the antioxidant defense system. As observed during a time course study (Fig. 1), a single application of 10 nmol TPA to mouse skin led to modulation of several parameters of oxidative stress, relative to control animals receiving acetone only. For example, a marked elevation of H₂O₂ production (Fig. 1A) was observed at 4 and 6 h post-treatment and a considerable increase in MPO activity (Fig. 1B) was

Discussion

As demonstrated previously, resveratrol facilitates a number of responses relevant to cancer chemoprevention and inhibits TPA-induced tumor promotion in mouse skin [2]. Mechanisms by which TPA acts directly on the plasma membrane and triggers a host of biochemical and cellular responses have been extensively studied [14]. In the current report, we elected to investigate the effect of resveratrol on early responses induced by the treatment of a single application of TPA. In particular, in the

Acknowledgements

We thank D.D. Lantvit for technical assistance. This work was supported by grant P01 CA48112 from the National Cancer Institute.

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Effects of resveratrol on 12-O-tetradecanoylphorbol-13-acetate-induced oxidative events and gene expression in mouse skin

Abstract

Introduction

Section snippets

Treatment of animals

TPA-induced oxidative events in mouse skin

Discussion

Acknowledgements

Physiol. Plant Pathol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Adv. Cancer Res.

J. Biol. Chem.

Free Rad. Biol. Med.

Food Chem. Toxicol.

Cell