d-Limonene modulates inflammation, oxidative stress and Ras-ERK pathway to inhibit murine skin tumorigenesis

d-Limonene, a common monoterepene has been shown to have antiproliferative, apoptosis-inducing and chemopreventive effects. In the present study, we have investigated the effects of d-limonene on the growth of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor development. We found that d-limonene (50 and 100 mg/kg body weight) treatments to the mouse skin significantly reduced the TPA-induced (a) edema and hyperplasia (p < 0.001); (b) expression of cyclooxygenase-2; (c) ornithine decarboxylase activity (p < 0.001); and (d) [3H] thymidine incorporation into DNA (p < 0.001). In addition, treatment of d-limonene effectively restored the level of reduced glutathione, glutathione peroxidase, glutathione reductase, glutathione S-transferase, catalase and malondialdehyde production in TPA-treated mouse skin. In a two-stage skin tumorigenesis study, d-limonene significantly reduced the tumor burden (p < 0.005) and tumor incidence as compared to DMBA/TPA-treated mice. d-Limonene treatment also extended the latency period of tumor development from 4 to 9 weeks. d-Limonene treatment decreased the expression level of Ras, Raf and phosphorylation of extracellular signal-regulated protein kinase 1/2 in DMBA/TPA-induced tumors. A decrease in the expression of Bcl-2 and an increase in Bax expression were also observed in tumor tissues of mice treated with d-limonene. Taken together, our findings suggest that d-limonene may exert its chemopreventive activity through the inhibition of inflammation, oxidative stress and Ras-signaling as well as the induction of pro-apoptotic state during TPA-mediated promotion of DMBA-induced skin cancer in mouse model.