Osteosarcoma cells promote intracellular iron detoxification to mitigate GPX4-mediated ferroptosis

Osteosarcoma is an aggressive and highly metastatic cancer that arises in bones. Ferroptosis, an iron-dependent form of cell death, is critically controlled by glutathione peroxidase 4 (GPX4). In this study, we characterized GPX4 and its upstream regulator xCT across five osteosarcoma cell lines (U2OS, MG-63, HOS, Saos2, and 143B). We also demonstrated that chemical inhibition of these proteins using RSL3 (GPX4 inhibitor) and erastin (xCT inhibitor) significantly suppressed osteosarcoma cell growth. In U2OS and MG-63 cells, GPX4 inhibition triggered lipid peroxidation, NRF2 activation, and upregulated antioxidant genes including, AKR1C1, HO-1, and NQO1, indicating a compensatory response to oxidative stress. Furthermore, GPX4 inhibition, through RSL3 treatment or transient GPX4 knockdown, increased ferritin heavy chain 1 and ferroportin expression, promoting intracellular iron depletion and conferring resistance to ferroptosis. RSL3 exposure also elevated post-translational regulators such as HIF-1α, c-MET and MSPR/RON, associated with cell survival pathways. Pharmacological inhibition of these signaling molecules synergistically enhanced RSL3-induced cytotoxicity. Collectively, our findings reveal that GPX4 inhibition initiates ferroptosis while simultaneously activating NRF2-driven antioxidant defenses, iron homeostasis mechanisms, and adaptive cell survival signaling. The results highlight potential therapeutic strategies that combine GPX4 inhibition with targeted disruption of compensatory pathways to overcome ferroptosis resistance in osteosarcoma.