Inhibition of glutamine metabolism increases sensitivity to plasma-activated medium-induced cytotoxicity

Non-thermal atmospheric pressure plasma (NTP), an ionized gas containing electrons, ions, radicals, and photons, has various biological effects, including wound healing and anticancer effects. Plasma-activated medium (PAM), which is prepared by irradiating medium with NTP, preferentially kills cancer cells. Large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) included in PAM are closely related to its anticancer effects. The precise mechanism of PAM-induced cytotoxicity is not fully understood; however, PAM exposure has been reported to reduce cellular energy metabolism. Glutamine (Gln) is an important amino acid as an energy source in cancer cells. Gln is converted to glutamate by glutaminase (GLS), and is utilized to synthesize ATP and glutathione (GSH). Expression levels of GLS have been shown to be higher in certain types of cancers. In this study, we examined the effects of GLS inhibition on PAM cytotoxicity using breast cancer MDA-MB-231 cells. Pretreatment with BPTES, a glutaminase 1 (GLS1) inhibitor, dose-dependently enhanced PAM-induced cell death. PAM-induced ROS production and γ-H2AX formation, a DNA damage marker, were increased in cells pretreated with BPTES compared with PAM alone. BPTES pretreatment enhanced a PAM-induced decrease in intracellular GSH, indicating the possibility that BPTES reduces the antioxidant capacity of MDA-MB-231 cells. In addition, BPTES pretreatment enhanced PAM-induced loss of the mitochondrial membrane potential and reduction of ATP production. Moreover, GLS1 knockdown promoted PAM-induced cell death. Taken together, the combination of GLS1 inhibitors such as BPTES is considered to be useful for enhancing the cytotoxic effects of PAM against cancer cells.