Reactive Oxygen Species–Driven Mitochondrial Apoptotic Signaling and ER Stress Responses in HNSCC Cells Treated with Cold Atmospheric Plasma

Abstract Objective: Head and neck squamous cell carcinoma (HNSCC) remains a challenging cancer with poor survival and significant treatment-related morbidity. Cold atmospheric plasma (CAP) has potential anticancer effects; however,its specific molecular mechanisms in HNSCC remain incompletely understood. This study examined the antitumor actions of CAP on HNSCC together with associated ROS-mediated mitochondrial pathways. Methods: Two HNSCC cell lines (FaDu and TU212) were treated with CAP for varying durations (0–240 s). Cell viability and migration were evaluated using CCK-8 and wound-healing assays, respectively, while apoptosis was examined using flow cytometry. Intracellular ROS levels were quantified. The activation of mitochondrial apoptotic pathway was evaluated by Western blotting for key markers (Bax,Blc-2 and cleaved caspase-3) and transmission electron microscopy. Furthermore, antitumor effects were verified using a FaDu xenograft mouse model, with tumor growth,proliferation (Ki-67),and apoptosis(C-Caspase-3) analyzed via immunofluorescence. Results: CAP administration led to marked time-dependent reductions in HNSCC cell viability and migration.Mechanistically, CAP persistently elevates intracellular ROS, triggering mitochondrial pathway-mediated apoptosis and ER stress, and marked ultrastructural changes including ER vacuolization and nuclear fragmentation. In mice, CAP administration markedly suppressed tumor growth without causing systemic toxicity or skin damage, correlating with reduced proliferation and enhanced apoptosis within the tumor microenvironment. Conclusion: CAP exerts potent antitumor effects against HNSCC by inducing a self-amplifying oxidative stress response that activates endogenous apoptosis and endoplasmic reticulum stress. These results indicate the potential of CAP in treating HNSCC and provide a mechanistic rationale for its future clinical translation.