Dose-Dependent Ferroptosis Induction in Osteosarcoma via NRF2/GPX4 Axis Modulation by Plasma-Activated Liquid Therapy

Plasma-activated liquid (PAL), an indirect application form of cold-atmospheric plasma (CAP)-an ionized gas generating reactive oxygen and nitrogen species, has been proposed as an innovative therapeutic approach for various cancer types. Despite accumulating evidence suggesting that PAL induces cell death through multiple mechanisms, the involvement of ferroptosis, a form of cell death driven by iron and lipid peroxidation, in osteosarcoma (OS) remains predominantly unknown.

CAP was used to activate the liquid for various durations, resulting in different doses of PAL. The antitumor efficacy of PAL was directly correlated with both the dosage and duration of treatment and was achieved by increasing the level of intracellular reactive oxygen species. Through screening three effective PAL doses, we discovered that PAL significantly influenced the migration and invasion capabilities of OS cells. Proteomic sequencing revealed increases in several ferroptosis-related antioxidant proteins in the PAL-treated group. Subsequent findings revealed that PAL modulated nuclear factor erythroid 2-related factor 2 (NRF2) and its downstream ferroptosis-related genes, predominantly resulting in the induction of ferroptosis by depleting glutathione peroxidase 4 (GPX4) in human OS cells. Finally, utilizing an OS xenograft model, we found that PAL effectively suppressed tumor growth in vivo via ferroptosis.

Our study highlights the importance of the NRF2/GPX4 axis as a pivotal pathway in PAL-induced ferroptosis. In vivo experiments provided compelling evidence supporting the potential of PAL as a potent therapeutic strategy for OS treatment.

High-dose PAL-induced sustained oxidative stress by simultaneously targeting NRF2 inactivation and GPX4 degradation, establishing redox imbalance as a critical ferroptotic checkpoint in OS therapy. Antioxid. Redox Signal. 44, 292-310.