Mechanism of induction of apoptosis in melanoma cancer cells by non-thermal plasma

Induction of apoptosis, or programmed cell death, is an important factor in cancer therapy as cancer cells frequently have acquired the ability to avoid apoptosis and continue to multiply in an unregulated manner; thereby becoming resistant to traditional chemotherapeutic drugs. Nonthermal atmospheric plasma discharge may provide a novel approach to induction of apoptosis in cancer cells. The purpose of this study was to evaluate the apoptotic effects of non-thermal plasma on melanoma cells and understand the mechanism of initiation of apoptosis in melanoma cancer cells by non-thermal plasma. Melanoma cancer cell line (ATCC A2058) was cultured in Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, and 10% fetal bovine serum. Cells were incubated at 37 C with 95% humidified air and 5% carbon dioxide. Melanoma cells were treated with increasing levels of non-thermal plasma by altering dose rate and were evaluated by Trypan blue exclusion test, TUNEL analysis, Caspase 3 cleavage and Annexin-PI staining to determine viability and apoptotic activity at various time points after plasma treatment. Trypan blue testing revealed that plasma treatment at low power for up to 15 seconds (1.5 J/cm²) did not significantly increase the number of dead cells immediately following treatment (time zero); however, at higher doses, the percent dead cells increased linearly with dose of plasma. TUNEL analysis of cells treated for 15 seconds at high power (15 J/cm²) demonstrated an increase in apoptosis at 24 and 48 hours post-treatment (p < 0.05). Annexin-V staining revealed a significant increase in apoptosis in plasma-treated cells at 24, 48, and 72 hours post-treatment (p < 0.05). Caspase-3 cleavage was observed at 48 hours post plasma treatment at a dose of 15 seconds (15 J/cm²). Pretreatment with N-acetyl cysteine (NAC), a free radical scavenger, significantly decreased apoptosis in plasma-treated cells. Plasma treatment induces apoptosis in melanoma cells through a pathway that appears to be dependent on production of reactive oxygen species by plasma in fluid. ROS penetrate the cells and may induce high levels of DNA damage resulting in the induction of apoptosis. Plasma may be a useful tool to induce directed cell death without inducing necrosis and inflammation.