Nitrogen-oxygen plasma induces selective cancer cell death via apoptosis with lipid-peroxidation-associated mechanisms across multiple cancer cell models

Plasma-based approaches are attracting interest as potential anticancer strategies due to their ability to generate reactive species that damage tumor cells. However, clinical translation remains limited by challenges in biocompatibility, reproducibility, and chemical tunability. In this study, we utilized a nitrogen-oxygen gas mixture to generate a controllable spectrum of reactive species and evaluated its effects in endometrial (HEC1A), cervical (HeLa), melanoma (B16F10), and lung (H1299) cancer models. Nitrogen-oxygen plasma treatment preferentially reduced cancer cell viability compared to normal fibroblasts in vitro. Mechanistically, plasma exposure increased intracellular oxidative stress, and co-treatment with GSH attenuated cytotoxicity, indicating a central role for reactive species. Apoptosis was supported by Annexin V/PI staining, caspase-3 and PARP cleavage, mitochondrial depolarization, and increased γH2AX, phospho-p53, BiP, and CHOP. Transcriptomic profiling revealed activation of apoptotic pathways alongside modulation of ferroptosis-associated genes. Ferrostatin-1 partially rescued cell viability, linking cytotoxicity to lipid peroxidation, whereas iron chelation did not reduce lipid peroxidation. These findings indicate a contribution of lipid peroxidation without canonical iron-dependent ferroptosis. In vivo, treatment significantly suppressed tumor growth in a B16F10 melanoma allograft model following intratumoral administration. Together, these results demonstrate that nitrogen-oxygen plasma induces apoptosis with additional oxidative lipid damage, supporting its therapeutic potential.