Synergistic Sensitization of Pancreatic Cancer Cells by Nanosecond Pulsed Electric Fields and Cold Atmospheric Plasma via Amplifying ROS and Apoptotic Signaling

Pancreatic cancer remains a highly lethal malignancy, with standard therapies offering limited benefits in advanced stages; thus, novel strategies that exploit specific cancer cell vulnerabilities are urgently needed. Building on our previous findings that nanosecond pulsed electric fields (nsPEF) combined with cold atmospheric plasma (CAP) produce enhanced cytotoxicity, this study investigates the mo-lecular mechanisms underlying this synergy. Pan02 pancreatic cancer cells were subjected to nsPEF, CAP, or a combination of both. We assessed cell viability, reactive oxygen species (ROS) production, and mitochondrial integrity using metabolic assays, flow cytometry, and fluorescence microscopy. Apoptotic markers were evaluated via Western blotting and caspase activity assays. Combined nsPEF–CAP treatment significantly outperformed either modality alone in inducing cell death. Mechanistically, dual treatment triggered a surge in intracellular ROS, particularly mitochondrial superoxide, indicating severe oxidative stress. Distinct mitochondrial responses were observed: nsPEF reduced mitochondrial membrane potential (ΔΨm), whereas CAP alone caused a slight elevation. Notably, while CAP induced apoptosis (evidenced by increased cleaved caspase-3 and caspase-3/7 activity), lethal nsPEF (100 pulses) caused cell death without triggering apoptotic signaling. However, mild nsPEF (20 pulses) significantly potentiated CAP-induced apoptosis. These findings suggest that nsPEF sensitizes cells to CAP treat-ment by amplifying oxidative stress and mitochondrial dysfunction. This synergistic combination repre-sents a promising therapeutic approach for targeting pancreatic cancer cells resistant to conventional therapies.