CA9 knockdown enhanced ionizing radiation-induced ferroptosis and radiosensitivity of hypoxic glioma cells

Abstract

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.

Keywords:

• Glioma
• ferroptosis
• hypoxia
• radiosensitivity
• DAMPs

Acknowledgments

The authors thank Yanyan Li for technical assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (No. 32171234, 31870844 and 81874080), the State Key Laboratory of Radiation Medicine and Protection (GZK1202225) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Notes on contributors

Wenpeng Huang

Wenpeng Huang is a Master student at the Department of Radiation Biology, Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China.

Yuping He

Yuping He is a Master student at the Department of Radiation Biology, Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China.

Shuangyu Yang

Shuangyu Yang is a Master student at the Department of Radiation Biology, Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China.

Xuefei Xue

Xuefei Xue is a Master student at the Department of Radiation Biology, Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China.

Hualong Qin

Hualong Qin is a Senior Scientist of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, China.

Ting Sun

Ting Sun, PhD, is a Senior Scientist of Neuro-Oncology, The First Affiliated Hospital of Soochow University, Suzhou, China.

Wei Yang

Wei Yang, PhD, is a Professor of Radiation Biology, Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China.