Abstract
Studies have demonstrated that reactive oxygen species (ROS) generated by NADPH oxidase are essential for melanoma proliferation and survival. However, the mechanisms by which NADPH oxidase regulates these effects are still unclear. In this work, we investigate the role of NADPH oxidase-derived ROS in the signaling events that coordinate melanoma cell survival. Using the highly metastatic human melanoma cell line MV3, we observed that pharmacological NADPH oxidase inhibition reduced melanoma viability and induced dramatic cellular shape changes. These effects were accompanied by actin cytoskeleton rearrangement, diminished FAKY397 phosphorylation, and decrease of FAK-actin and FAK-cSrc association, indicating disassembly of focal adhesion processes, a phenomenon that often results in anoikis. Accordingly, NADPH oxidase inhibition also enhanced hypodiploid DNA content, and caspase-3 activation, suggesting activation of the apoptotic machinery. NOX4 is likely to be involved in these effects, since silencing of NOX4 significantly inhibited basal ROS production, reduced FAKY397 phosphorylation and decreased tumor cell viability. Altogether, the results suggest that intracellular ROS generated by the NADPH oxidase, most likely NOX4, transmits cell survival signals on melanoma cells through the FAK pathway, maintaining adhesion contacts and cell viability.
Highlights
Melanoma arises from the malignant transformation of pigment-producing cells, and its incidence has increased in many countries, being a prominent worldwide public health challenge [1,2,3].Development of skin cancer is a multistage process mediated by different cellular, biochemical and molecular changes, involving the activation of several anti-apoptotic and pro-survival signaling pathways
Reagents HEPES, ethylenediaminetetraacetate (EDTA), bovine serum albumin (BSA), penicillin, streptomycin, dihydrorhodamine 123 (DHR), phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin, and soybean trypsin inhibitor (SBTI), sodium orthovanadate (Na3VO4), apocynin (4-acetovanillone), 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenol tetrazolium bromide (MTT), pyrrolidine dithiocarbamate (PDTC), diphenyleneiodonium (DPI), TRITC-labelled phalloidin, cycloheximide (CHX), ribonuclease A (RNase A), propidium iodide (PI), diethylenetriamine pentaacetic acid (DTPA), RPMI-1640 medium, superoxide dismutase conjugated to polyethylene glycol (PEG-SOD), catalase conjugated to PEG (PEG-CAT) and sulforhodamine B were from Sigma-Aldrich
It has already been described that some melanoma cell lines can produce intracellular reactive oxygen species (ROS) in a NADPH oxidase-dependent manner [26]
Summary
Melanoma arises from the malignant transformation of pigment-producing cells (melanocytes), and its incidence has increased in many countries, being a prominent worldwide public health challenge [1,2,3].Development of skin cancer is a multistage process mediated by different cellular, biochemical and molecular changes, involving the activation of several anti-apoptotic and pro-survival signaling pathways. The metastatic ability of these cells is closely related to a rearrangement on the integrin-coordinated signaling hierarchy [4,5]. Epidemiological studies have demonstrated that the major risk factors for melanoma relate to both environmental exposure and genetic alterations. Melanoma incidence in white populations has revealed an inverse correlation with latitude and positive correlation with ultraviolet radiation (UVR) index [6,7]. Skin exposure to UVR generates ROS in excessive quantities [8]. Rather than this occurring as a direct effect of UVR, it has been shown that the observed ROS accumulation relies on ROS generated by highly specialized enzymatic systems [9]
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