Inhibition of DNA‑PK activity sensitizes A549 cells to X‑ray irradiation by inducing the ATM‑dependent DNA damage response.

Abstract

Non-small cell lung cancer (NSCLC) is radioresistant to X-rays due to powerful cellular DNA damage repair mechanisms. DNA-dependent protein kinase (DNA-PK) is a key enzyme involved in DNA damage repair and the phenomenon and molecular mechanism of NSCLC radionsensitivity were investigated following inhibition of DNA-PK activity. In the present study A549 cells were treated with the DNA-PK inhibitor NU7026 and/or siRNA directed against ataxia telangiectasia mutated (ATM), followed by exposure to 4 Gy X-ray irradiation. Radiosensitivity, DNA damage, apoptosis and protein expression were measured by colony formation assay, γH2AX foci immunofluorescence, Annexin V/PI staining and western blotting, respectively. A Balb/c-nu/nu xenograft mouse model was established by subcutaneous injection of A549 cells and was used to examine the effect of administering NU7026 via intraperitoneal injection prior to 4 Gy X-ray exposure. The xenograft tumors were weighed and observed by hematoxylin and eosin staining after irradiation. NU7026 treatment followed by X-ray irradiation significantly decreased the colony formation ratio of A549 cells, and increased γH2AX foci and cell apoptosis. Furthermore, the combined treatment of NU7026 and X-rays resulted in growth inhibition and cell apoptosis in A549 xenograft tumors. Consequently, apoptosis regulators full-length transactivating (TA) p73 and an N-terminally truncated (DN) p73 were upregulated and downregulated respectively, leading to activation of glucosyltransferases and Rab-like GTPase activators and myotubularins domain-containing 4 (GRAMD4) protein to reduce the Bcl-2/Bax protein ratio. In addition, ATM siRNA efficiently prevented γH2AX foci formation, and enhanced NU7026-induced inhibition of survival and promoted apoptosis. In conclusion, inhibition of DNA-PK activity increased the radiosensitivity of A549 cells to X-ray irradiation. NU7026 treatment activated the ATM-dependent DNA damage response and induced p73 apoptosis pathway. DNA-PK inhibitor may be an effective constituent of radiosensitization products. DNA damage repair pathway could be a potential target for radiosensitization.