Abstract
INTRODUCTION
Cold atmospheric plasma (CAP) selectively induces reactive oxygen and nitrogen species (ROS/RNS) in many types of cancerous cells. ROS-mediated lipid peroxidation is thought to induce ferroptosis, apoptosis, and autophagy. We hypothesize that ferroptosis and apoptosis are key mechanisms of CAP-mediated cytotoxicity in high-grade glioma (HGG).
METHODS
B16, U87, GL261, EPD-210FHTC and human astrocyte NHA hTERT cells were treated with CAP for 10, 30, 60, 90, and 180 seconds. Proliferation and propidium iodide (PI)/annexin V flow cytometry assays were employed to quantify cytotoxicity, cell cycle phases and apoptosis. Mitochondrial superoxide concentration was measured using MitoSOX Red. Cells were pre-treated with ferroptosis inhibitors Ferrostatin-1 and Deferoxamine (DFO) in rescue assays.
RESULTS
Survival of GL261 and U87 cells after 90 seconds of CAP treatment was 3.7% and 7%, respectively, compared to 62% in NHA cells. A CAP dose-dependent increase in mitochondrial superoxide concentration was observed in GL261 and NHA (R2=0.88 and 0.99, respectively). A shift of EPD and NHA cells into G0 phase was noted after 180 seconds of treatment, compared to baseline (55.4% versus 1.2%, 100% vs. 27.5% respectively). Early apoptosis was more prominent in NHA cells (79% of dead cells), and late apoptosis in EPD cells after 60 seconds of treatment (86% of dead cells). DFO pre-treatment significantly reduced CAP cytotoxicity in GL261 (93% vs. 58% after 10 seconds) and U87 cells (85% vs. 13% after 60 seconds). DFO pre-treatment had no effect on NHA response to CAP.
CONCLUSION
CAP treatment induces dose-dependent increases in ROS and apoptosis in HGG lines tested more significantly than in NHA cells. CAP induces G1-phase cell cycle arrest in treated HGG cells and G0 arrest in non-cancerous cells. CAP-mediated cytotoxicity was significantly mitigated with DFO pre-treatment in HGG cells, suggesting that ferroptosis plays a critical role in the mechanism of CAP treatment in HGG.
Dose-Dependent Cortical Thinning After Partial Brain Irradiation in High-Grade Glioma
