Therapeutic potential of no-ozone cold plasma-activated saline in osteosarcoma through mitochondrial and MAPK pathway modulation

Plasma has the unique ability to trigger potent chemical reactions, making it a promising tool in biomedical applications. Recent advancements focus on low-temperature plasmas, particularly no-ozone cold plasma (NCP), which produces RONS at body-compatible temperatures. NCP has shown notable potential in inducing apoptosis in various cancer cells. This study investigated the efficacy of no-ozone cold plasma-activated saline in human osteosarcoma cell lines, and elucidated the underlying molecular mechanisms. Cells were indirectly exposed to plasma-activated media (PAM) and PAS. Cell viability was assessed using the SRB assay, while wound healing and oxidative stress were evaluated through migration, H₂O₂, and ROS assays. Immunofluorescence and Western blotting were used to analyze apoptotic markers and signaling pathways. PAS treatment significantly reduced osteosarcoma cell viability in a cell selective manner, impaired MG-63 cell migration and increased ROS levels in a time-dependent manner. Apoptotic indicators such as cytochrome c and AIF translocation, caspase-3 and PARP cleavage, Bax upregulation, and Bcl-2 downregulation were observed. Moreover, PAS modulated MAPK signaling by enhancing p38 phosphorylation and reducing ERK activity. These findings highlight PAS as a selective and noninvasive potential therapy for osteosarcoma as its selectively targets osteosarcoma cells while sparing normal cells, and triggers apoptosis through both mitochondrial dysfunction and MAPK pathway modulation.