Cold atmospheric plasma treated medium (PTM) induce antitumor effects in human ovarian cancer cells

Abstract Plasma treated solutions (PTS) have emerged as a promising anticancer strategy due to their ability to generate reactive oxygen and nitrogen species (RONS) that induce oxidative stress and disrupt multiple cellular pathways. Despite increasing interest in plasma-based cancer therapies, the mechanisms underlying their effects in ovarian cancer remain incompletely understood. This study aimed to investigate the biological effects of plasma treated medium (PTM) in ovarian cancer models, focusing on cell viability, caspase 3/7 activity, DNA damage, cell cycle progression, and mitochondrial metabolism. Methods: Three established ovarian cancer cell lines were treated with PTM to evaluate dose-dependent effects on cell viability using a resazurin assay. Apoptotic signaling was assessed by caspase 3/7 activity assays. DNA damage was analyzed by immunofluorescence staining of γH2AX, while cell cycle alterations were evaluated using propidium iodide staining and flow cytometry. Mitochondrial metabolism was investigated using fluorescence lifetime imaging microscopy together with NADH and FAD autofluorescence imaging. In addition, patient-derived high-grade serous ovarian cancer cells isolated from ascites were analyzed as independent biological replicates to assess biological relevance. Results: PTM induced dose-dependent inhibition of cell viability with IC 50 values ranging from 1:4 to 1:1.1 PTM dilutions across the tested cell lines. PTM treatment triggered γH2AX-associated DNA damage, caspase 3/7 activity, and dynamic alterations in cell cycle progression. Fluorescence lifetime imaging revealed significant reductions in free NADH and changes in protein-bound NADH and FAD, consistent with mitochondrial dysfunction. These metabolic alterations were accompanied by morphological changes from filamentous mitochondrial networks to punctate structures. Patient-derived ovarian cancer cells showed partial responses, primarily reflected in cell cycle modulation, highlighting both biological relevance and intercellular heterogeneity. Conclusions: PTM exerts multifactorial anticancer effects in ovarian cancer models by inducing DNA damage, caspase 3/7 activity, cell cycle alterations, and mitochondrial dysfunction. These findings provide further insight into the biological effects of plasma-based cancer therapies and support the potential of plasma treated solutions as a novel therapeutic strategy for ovarian cancer.