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

Abstract

Background: Twenty to thirty percent of patients taking BRAF inhibitors such as vemurafenib (PLX4032) for melanoma develop cutaneous squamous cell carcinomas.

Purpose: 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.

Methods: 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 PDV^HRASQ61L keratinocyte activities were demonstrated using a spectrum of bioactivity assays, western blotting, immunochemistry, and keratinocyte-macrophage co-culture assay.

Results: Treatment with EO suppressed colony formation ability, cell migration, invasion, and induced G₂/M cell-cycle arrest and apoptosis in PDV^HRASQ61L 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.

Conclusion: 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.

Keywords: BRAF inhibitor; Chemoprevention; Macrophage reprogramming; Mentha aquatica L. cv. Lime; Skin carcinogenesis.