Phytocompounds from essential oil of Mentha aquatica L. cv. Lime prevent vemurafenib-promoted skin carcinogenesis via inhibiting HRASQ61L keratinocytes and reprogramming macrophage activities.

Abstract

Twenty to thirty percent of patients taking BRAF inhibitors such as vemurafenib (PLX4032) for melanoma develop cutaneous squamous cell carcinomas. This study aimed to elucidate the chemopreventive effect of essential oil from Mentha aquatica L. cv. Lime (EO) and its major constituents, limonene and carvone (L+C) that made up 45.68% of the EO, against PLX4032-induced cutaneous side effects. PLX4032 accelerated skin papilloma formation and keratinocyte HRAS mutation in 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage skin carcinogenesis mouse model was used to evaluate the in vivo bioefficacy of EO and L+C. The effects and molecular mechanisms of EO and L+C on deregulating mouse PDVHRASQ61L keratinocyte activities were demonstrated using a spectrum of bioactivity assays, western blotting, immunochemistry, and keratinocyte-macrophage co-culture assay. Treatment with EO suppressed colony formation ability, cell migration, invasion, and induced G2/M cell-cycle arrest and apoptosis in PDVHRASQ61L keratinocytes, and L+C treatment inhibited colony formation, cell migration and invasion of PDV cells. In mouse skin irritated with DMBA/TPA (DT group) or DMBA/TPA with PLX4032 (DTP group), topical application of EO and L+C significantly delayed papilloma appearance and reduced papilloma incidence compared to DT or DTP controls. Histopathology results showed that EO and L+C treatment attenuated K14+ keratinocyte proliferation and paradoxical MAPK activation, and shifted the macrophage population from M2 (CD163+) to M1 (iNOS+) in the mouse skin microenvironment. The conditioned medium of EO or L+C pre-treated PDV keratinocytes promoted M0 macrophages to differentiate from THP-1 cells into M1-like macrophages. This study demonstrates that EO and L+C in combination prevent PLX4032-induced cutaneous side-effects and skin carcinogenesis in mice through reprogramming the macrophage cell population and inhibiting keratinocyte activity. Both mint EO and the natural products L+C can be considered to be effective chemopreventive agents that might be useful in reducing cutaneous lesions in human patients administrated with BRAF inhibitors.