Effect of Autophagy Inhibitors on Radiosensitivity in DNA Repair-Proficient and -Deficient Glioma Cells

Abstract

Background and Objectives: The development of radioresistance is a fundamental barrier to successful glioblastoma therapy. Autophagy is thought to play a role in facilitating the DNA repair of DNA damage foci in radiation-exposed tumor cells, thus, potentially contributing to their restoration of proliferative capacity and development of resistance in vitro. However, the effect of autophagy inhibitors on DNA damage repair is not fully clear and requires further investigation. Materials and Methods: In this work, we utilized M059K (DNA-PKcs proficient) and M059J (DNA-PKcs deficient) glioma cell lines to investigate the role of autophagy inhibitors in the DNA repair of radiation-induced DNA damage. Cell viability following radiation was determined by trypan blue exclusion in both cell lines. Cell death and autophagy assays were performed to evaluate radiation-induced cell stress responses. DNA damage was measured as based on the intensity of phosphorylated γ-H2AX, a DNA double-stranded breaks (DSBs) marker, in the presence or absence of autophagy inhibitors. Results: The cell viability assay showed that M059J cells were more sensitive to the same dose of radiation (4 Gy) than M059K cells. This observation was accompanied by an elevation in γ-H2AX formation in M059J but not in M059K cells. In addition, the DAPI/TUNEL and Senescence-associated β-galactosidase (SA-β-gal) staining assays did not reveal significant differences in apoptosis and/or senescence induction in response to radiation, respectively, in either cell line. However, acridine orange staining demonstrated clear promotion of acidic vesicular organelles (AVOs) in both cell lines in response to 4 Gy radiation. Moreover, DNA damage marker levels were found to be elevated 72 h post-radiation when autophagy was inhibited by the lysosomotropic agent bafilomycin A1 (BafA1) or the PI3K inhibitor 3-methyl adenine (3-MA) in M059K cells. Conclusions: The extent of the DNA damage response remained high in the DNA-PKcs deficient cells following exposure to radiation, indicating their inability to repair the newly formed DNA-DSBs. On the other hand, radioresistant M059K cells showed more DNA damage response only when autophagy inhibitors were used with radiation, suggesting that the combination of autophagy inhibitors with radiation may interfere with DNA repair efficiency.

Keywords

• DNA damage
• autophagy
• senescence
• glioblastoma
• DNA-PKcs
• radiation