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Marta Osrodek, 1 Michal Rozanski, 1, 2 Malgorzata Czyz 1 1Department of Molecular Biology of Cancer, Medical University of Lodz, Lodz, Poland; 2Laboratory of Transcriptional Regulation, Institute of Medical Biology, Polish Academy of Sciences, Lodz, PolandCorrespondence: Malgorzata CzyzDepartment of Molecular Biology of Cancer, Medical University of Lodz, 6/8 Mazowiecka Street, 92-215 Lodz, PolandTel +48 42 272 57 02Email malgorzata.czyz@umed.lodz.plBackground: Despite the progress made in the clinical management of metastatic melanoma, a patient’s response to treatment cannot be fully predicted, and intrinsic or acquired resistance that is developed in most melanoma patients warrants further research efforts. In addition to genetic factors, microenvironmental input should be considered to explain the diversity of response to treatment among melanoma patients. In this study, we evaluated the impact of insulin on patient-derived BRAFV600E melanoma cells, either untreated or treated with vemurafenib or trametinib, inhibitors of BRAFV600 and MEK1/2, respectively.Methods: Cells were cultured in serum-free conditions, either with or without insulin. The activity of the MAPK/ERK and PI3K/AKT pathways was assessed by Western blotting, cell viability, and percentages of Ki-67- and NGFR-positive cells by flow cytometry. Transcript levels were analyzed using qRT-PCR, and γ-H2AX levels by immunoblotting and confocal microscopy. A luminescence-based assay was used to measure glutathione content.Results: While insulin did not influence the MAPK/ERK pathway activity, it had a strong influence on melanoma cells, in which this pathway was suppressed by either vemurafenib or trametinib. In the presence of insulin, both drugs were much less efficient in 1) inhibiting proliferation and reducing the percentage of Ki-67-positive cells, and 2) inducing apoptosis and phosphorylation of histone H2AX in melanoma cells. Changes induced by vemurafenib and trametinib in glutathione homeostasis and DNA repair gene expression were also ... |
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