Roles of intracellular and extracellular ROS formation in apoptosis induced by cold atmospheric helium plasma and X-irradiation in the presence of sulfasalazine

Sulfasalazine (SSZ) is a well-known anti-inflammatory drug and also an inhibitor of the cystine-glutamate antiporter that is known to reduce intracellular glutathione (GSH) level and increase cellular oxidative stress, indicating its anti-tumor potential. However, the combination of SSZ with other physical modalities remains unexplored. Here, the effects of SSZ on cold atmospheric helium plasma (He-CAP), which produces approximately 24 x higher concentration of hydroxyl radicals (. OH) compared to X-irradiation (IR) in aqueous solution, and on IR-induced apoptosis in human leukemia Molt-4 cells were studied to elucidate the mechanism of apoptosis enhancement. Both the Annexin V-FITC/PI and DNA fragmentation assay revealed that pre-treatment of cells with SSZ significantly enhanced He-CAP and IR-induced apoptosis. Similar enhancement was observed during the loss of mitochondrial membrane potential, intracellular Ca2+ ions, and mitochondria-and endoplasmic reticulum-related proteins. The concentration of intracellular reactive oxygen species (ROS) was much higher in He-CAP treated cells than in X-irradiated cells. On the other hand, strong enhancement of Fas expression and caspase-8 and -3 activities were only observed in X-irradiated cells. It might be possible that the higher concentration of intracellular and extracellular ROS suppressed caspase activities and Fas expression in He-CAP-treated cells. Notably, pretreating the cells with an antioxidant N-acetyl-L-cysteine (NAC) dramatically decreased apoptosis in cells treated by He-CAP, but not by IR. These results suggest that IR-induced apoptosis is due to specific and effective ROS distribution since intracellular ROS formation is marginal and the high production of ROS inside and outside of cells plays unique roles in He-CAP induced apoptosis. We conclude that our data provides efficacy and mechanistic insights for SSZ, which might be helpful for establishing SSZ as a future sensitizer in He-CAP or IR therapy for cancer.