Eugenol restricts DMBA croton oil induced skin carcinogenesis in mice: Downregulation of c-Myc and H-ras, and activation of p53 dependent apoptotic pathway

Abstract

Background

Eugenol is the active component of essential oil isolated from clove (Syzigium aromaticum). Eugenol has antimutagenic, antigenotoxic, anti-inflammatory properties. The anticarcinogenic effect of eugenol was evident in different types of cell lines. However, its anticarcinogenic effect in in vivo has not yet been fully explored.

Objective

The aim of this study is to evaluate the chemopreventive potential of eugenol in an experimental skin carcinogenesis mice model system.

Method

Skin tumor was induced by topical application of DMBA croton oil in Swiss mice. To assess the chemopreventive potential of eugenol, it was orally administered 15 days prior carcinogen treatment. The development of skin carcinogenesis was confirmed by histopathological analysis. Cellular proliferation and apoptosis in the skin tumor were analyzed by in situ cellular proliferation and in situ cell death assay. Expression of some proliferation and apoptosis associated genes was analyzed by RT-PCR and protein expression was analyzed by Western blot.

Results

Reduction in incidence and sizes of skin tumors along with overall increase in survival of mice were seen due to eugenol treatment. Restriction of skin carcinogenesis at the dysplastic stage along with reduced rate of cellular proliferation and increase in apoptosis were evident in eugenol treated skin tumors. Eugenol treatment led to the downregulation of c-Myc, H-ras and Bcl2 expression along with upregulation of P53, Bax and active Caspase-3 expression in the skin lesions.

Conclusion

Restriction of skin carcinogenesis at dysplastic stage by eugenol was due to attenuation of c-Myc, H-ras and modification of some p53 associated gene expression.

Introduction

Eugenol (4-allyl-2-methoxyphenol) is the active component of clove Syzigium aromaticum and it is also present in number of other aromatic plants like basil, cinnamon and bay leaves [1]. Eugenol is widely used as a general antiseptic in medical and dental practice on account of its potent fungicidal, bactericidal, antioxidant and anti-inflammatory properties [2], [3]. Antigenotoxic activity of eugenol was evident in several studies [4], [5], [6], [7], [8]. Eugenol was found to suppress the mutagenicity of furylfuramide, 4NQO, aflatoxin B in Salmonella typhimurium [4]. Eugenol was found to modulate detoxification enzyme [5] and in MCF-7 cell line, DMBA induced DNA damage was inhibited due to induction of detoxification enzymes [6]. Oral feeding of eugenol along with trans-anethol exerts antigenotoxic property against cyclophosphamide, procarbazine, N-methyl-N′-nitro-N-nitrosoguanidine and urethane in mice as assessed by bone marrow micronucleus test [7]. However, there is some ambiguity about the antigenotoxic property of eugenol. Rompelberg et al. [8] showed oral feeding of eugenol in rats did not reduce the genotoxicity of DMBA and aflatoxin B1.

The anticarcinogenic activity of eugenol has not yet been fully explored. Pro-apoptotic activity of eugenol was reported in HL-60 [9], melanoma cell line (G361) [10] and human osteosarcoma (HOS) cells [11]. In in vivo system mouse skin tumor induced by DMBA croton oil was inhibited by radical scavenging activity of eugenol [12]. B(a)P induced skin carcinogenesis was also partially inhibited by topical application of eugenol in Swiss mice [13]. Similarly, Kaur et al. [14] showed pretreatment of eugenol (topical) could inhibit the formation of DMBA-TPA induced skin carcinogenesis. However, Azuine et al. [15] showed topical application of eugenol was less effective in reducing DMBA induced skin carcinogenesis in Swiss mice. The ambiguities of eugenol action in different studies might be due to the differences in the application procedure, as topical application of eugenol takes time to be in the circulation. This problem can be avoided by oral administration of eugenol.

Two stage skin carcinogenesis model is useful for analysis of the chemopreventive mechanism of different compounds. Overexpression of c-Myc in skin epidermis is associated with epidermal hyperproliferation, delayed differentiation and altered programmed cell death [16]. Similarly, mutation and overexpression of H-ras have been suggested to be one of the initiating event in the skin carcinogenesis [17], [18]. Deletion of H-Ras could reduce the skin tumor formation suggesting the importance of this gene in skin tumor formation [19]. The evidence that, c-Myc deficient skin epidermis is resistant to H-Ras-induced tumorigenesis, suggests the cooperation of these two genes in skin tumor development [20]. Inactivation of tumor suppressor gene p53 due to mutation was reported to be associated with the conversion of skin papilloma to carcinoma [21], [22]. Differential application of eugenol in DMBA-TPA induced skin carcinogenesis enhanced the p53 expression along with increase in apoptotic process [14]. The skin is a highly regulated organ maintaining proper balance between cellular proliferation and programmed cell death. It was evident that different chemopreventive agents could modulate these cellular processes in restriction of the carcinogenesis [23], [24] though the molecular events associated with these processes are not known clearly.

In the present study attempts have been made to understand the chemopreventive mechanism of eugenol in DMBA croton oil induced skin carcinogenesis mice model system. Two weeks before carcinogen exposure, eugenol was treated orally and continued through out the experiment. Effect of eugenol on cellular proliferation and apoptosis was analyzed. Our study indicated the restriction of skin carcinogenesis by eugenol due to induction of some apoptosis associated genes and inhibition of some proliferation associated genes.