CA9 Knockdown Enhanced Ionizing Radiation-Induced Ferroptosis and Radiosensitivity of Hypoxic Glioma Cells.

PURPOSE:Ferroptosis is a type of regulatory cell death, caused by excessive lipid peroxidation This study aimed to explore whether ionizing radiation could induce ferroptosis in glioma cells and whether carbonic anhydrase 9 (CA9) knockdown could enhance the killing effect of ionizing radiation on hypoxic glioma cells through ferroptosis.MATERIALS AND METHODS:The protein levels of Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) were detected by Western blot in glioma cells irradiated by different doses of X-ray. The relative mRNA levels of ferroptosis markers and intracellular iron-associated proteins were detected by Real-time qPCR. Lipid peroxidation of glioma cells was detected by oxidation-sensitive probe C11-BODIPY581/591 staining. CCK-8 Assay was used to detect cell viability after X-ray irradiation. Cloning formation assay was used to assess the radiosensitivity of glioma cells. The exposure of cell surface calreticulin was measured by immunofluorescence staining.RESULTS:X-ray induced lipid peroxidation and ferroptosis markers expression in U251 and GL261 glioma cells. Knockdown of CA9 in hypoxic glioma cells significantly altered the expression of iron regulation-related proteins and enhanced X-ray-induced ferroptosis and radiosensitivity. The ferroptosis inhibitor significantly improved the survival of cells irradiated by X-ray, while ferroptosis inducers (FINs) enhanced the lethal effect of X-ray on cells. Enhancing ferroptosis in glioma cells promoted the exposure and release of damage-associated molecular patterns (DAMPs).CONCLUSIONS:Ionizing radiation can induce ferroptosis in glioma cells. CA9 knockdown can enhance the radiosensitivity of hypoxic glioma cells and overcome the resistance of ferroptosis under hypoxia. Enhancing ferroptosis will become a new idea to improve the efficacy of radiotherapy for glioma.