Onconase Induces Apoptosis in Dabrafenib-Resistant Melanoma Cell Lines Through Dysregulation of ROS Homeostasis, Antioxidant Protein Expression, and Mitochondrial Dynamics.

Advanced melanoma remains difficult to treat due to its intrinsic resistance to conventional therapies and the frequent development of acquired resistance to targeted agents, such as BRAF inhibitors. Onconase (ONC), an amphibian ribonuclease with established antitumor activity, had been previously shown to have selective cytotoxicity toward melanoma cells. In this study, we investigated the molecular mechanisms underlying ONC-induced cytotoxicity in BRAF-mutated melanoma cell lines that are either sensitive or resistant to the BRAF inhibitor dabrafenib. We focused on oxidative stress regulation, mitochondrial dynamics, and cell death-related signaling pathways. ONC treatment resulted in a marked increase in reactive oxygen species (ROS) levels, concomitant with a pronounced downregulation of NRF2 and multiple NRF2-dependent antioxidant proteins. These effects were particularly evident in dabrafenib-resistant melanoma cells. In parallel, ONC impaired mitochondrial plasticity by inhibiting mitochondrial biogenesis and fission, as evidenced by reduced PGC1α, DRP1, and FIS1 expression. Confocal analysis confirmed the presence of more enlarged mitochondria in ONC-treated cells. Mitophagy and autophagy are hindered by ONC due to the downregulation of PINK1, beclin1, ATG3 expression, as well as the lack of LC3B activation. These mitochondrial defects were associated with mitochondrial-dependent apoptosis, characterized by caspase-9 activation and strong downregulation of the antiapoptotic protein survivin. Lipid peroxidation was also induced by ONC, especially in the A375 cell line. Additionally, ONC inhibited key proliferation-related signaling pathways, including STAT3 and NF-κB, and reduced cyclin-dependent kinase 1, 2, and 4 activities. Collectively, these findings demonstrate that ONC disrupts redox homeostasis, mitochondrial function, and survival signaling in melanoma cells, exerting particularly potent effects in BRAF inhibitor-resistant populations. This study provides mechanistic insight into the anti-melanoma activity of ONC and supports its potential therapeutic application in drug-resistant melanoma.