Photosensitization of human skin fibroblasts by vemurafenib promotes pleiotropic effects on membrane-enclosed organelles and apoptosis

© 2022 Elsevier B.V. All rights reserved.Vemurafenib (VB), a BRAF inhibitor and a first-line treatment for unresectable or metastatic melanoma, is strongly phototoxic towards normal skin cells. Herein, we show that in cultured HS 68 human diploid dermal fibroblasts, low concentrations of VB suffice to promote photosensitization to low doses of UVA (∼ 5 J/cm2), as evidenced by a significant decrease in cell viability. In contrast to data obtained in chemico our results support a role for ROS (reactive oxygen species). Indeed, peroxidation of cellular lipids was observed which could be alleviated by the lipophilic antioxidant BHT (2,6-di-tert-butyl-4-methylphenol). Using in vivo confocal laser scanning microscopy and vital fluorescent probes it was shown at the single cell level that the plasma membrane and lipid-rich organelles, namely mitochondria, endoplasmic reticulum, and lysosomes, as well as actin filaments, were severely damaged by the UVA-induced VB-photosensitization. Finally, we showed that mitochondrial impairment was concurrent with caspase 3/7 activation and cell death by apoptosis.This work was supported by AbbVie, Lda., donation FPJ937, and Fundação para a Ciência e Tecnologia/FCT, Portugal (FCT PTDC/MEC-DER/30198/2017 FARM ID).info:eu-repo/semantics/publishedVersio

Interplay Between Membrane Lipid Peroxidation and Photoproduct Formation in the Ultraviolet A-Induced Phototoxicity of Vemurafenib in Skin Keratinocytes

International audienceAccording to some authors, the phototoxic response to ultraviolet A (UVA) of patients treated with vemurafenib (VB) may involve VB metabolites. However, the production of singlet oxygen and free radicals and photoproduct formation upon UVA light absorption by the lipophilic VB have been demonstrated. This work is aimed at determining the contribution of reactive oxygen species (ROS), lipid photoperoxidation, and VB photochemistry in the UVA-induced photocytotoxicity in NCTC 2544 keratinocytes. The potent membrane lipid peroxidation effectiveness of VB-photosensitization has been proved by the observation of an effective photohemolysis accompanied by thiobarbituric reactive substances (TBARS) formation in 2% red blood cell (RBC) suspensions. Photohemolysis is inhibited by human serum albumin (HSA) that binds VB and by the antioxidants 2,6-di-tert-butyl-4-methylphenol and Trolox. These data on RBC suggest that VB is readily incorporated in cell membranes and provide clues for understanding the UVA-induced VB-photosensitization of keratinocytes. In keratinocytes, ROS and TBARS formation with 10 mM VB is inhibited by approximately 40% and 50% by 30 mM Trolox and 50 mM vitamin E, respectively, but the light dose-dependent cell survival is unaffected. Whereas cell photokilling depends on the VB concentration, much smaller changes in the lethal doses (LD) than theoretically expected are observed for 25% or 50% cell photokilling when changing absorbed UVA doses and irradiation wavelengths. The lack of antioxidant effect on cell survival and the unexpectedly small LD dependence on absorbed UVA light doses and on irradiation wavelengths strongly suggest that, instead of metabolites, membrane photosensitization and photoproduct formation contribute to the cell photocytotoxicity