Characteristics of plasma-activated water and its effect on apoptosis of malignant melanoma cells

Plasma-activated water (PAW) has emerged as a promising indirect cold atmospheric plasma modality for cancer therapy due to its rich reactive oxygen and nitrogen species (RONS) content, yet its effects on malignant melanoma and the underlying mechanisms require further elucidation. In this study, deionized water was irradiated with a helium atmospheric pressure plasma jet (APPJ) for 1, 2, 3, 4, and 5 min to generate five PAWs (PAW1, PAW2, PAW3, PAW4, PAW5). Physicochemical analysis of PAWs revealed progressive increases in conductivity, decreases in pH, and accumulation of hydrogen peroxide (H₂O₂), nitrite (NO₂^-), and nitrate (NO₃^-) with prolonged APPJ irradiation. Compared to control group, PAWs treatment reduced malignant melanoma cell (B16) viability in a time-dependent manner, concomitant with increased apoptosis and elevated lactate dehydrogenase release. Oxidative stress markers demonstrated an obvious increase in malondialdehyde (MDA) content and a progressive decline in superoxide dismutase (SOD) activity. PAW5 treatment exerted the strongest cytotoxic effect on B16 cells. Western blotting analysis revealed significant downregulation of anti-apoptotic Bcl-2, accompanied by a sustained upregulation of pro-apoptotic Bax. This was followed by significant activation of cleaved caspase-9 and cleaved caspase-3, indicating the induction of the mitochondrial apoptotic pathway. These findings demonstrated that PAW-induced oxidative stress triggered the mitochondrial apoptotic pathway in melanoma cells, supporting the potential of PAW as a novel therapeutic strategy for malignant melanoma.