2-Methoxyestradiol-induced radiosensitization is independent of SOD but depends on inhibition of Akt and DNA-PKcs activities

Abstract

Background and purpose 2-Methoxyestradiol (2-ME) is described as an inhibitor of the superoxide dismutase (SOD) enzyme activity. However, it attenuates PI3 K/Akt pathway and induces radiosensitization in human tumor cells as well. Since the activation of catalytic subunit of DNA-protein kinase (DNA-PKcs) is partially regulated by Akt activity, in the present study we investigated whether 2-ME-induced radiosensitization is dependent on inhibition of Akt and DNA-PKcs activities or on SOD targeting. Materials and methods This study was performed using the lung carcinoma cell line A549. Ionizing radiation-induced SOD activity was analyzed by superoxide dismutase activity assay. Applying Western blotting, the pattern of radiation-induced SOD expression and activation of Akt as well as DNA-PKcs was analyzed. Colony formation assay and γH2AX foci assay were performed to measure radiosensitization and DNA-double strand break (DNA-DSB) repair. To downregulate SOD expression small interfering RNA (siRNA) was used. Results Irradiation with 4 Gy stimulated SOD enzyme activity as early as 1 min after radiation exposure. Expression of Cu/Zn-SOD (SOD1) as well as Mn-SOD (SOD2) was increased by single doses of 1–4 Gy within 24–36 h. 2-ME blocked radiation-induced SOD enzyme activity but not protein expression and enhanced radiation sensitivity. Pretreatment with 2-ME blocked IR-induced Akt as well as DNA-PKcs phosphorylation and impaired the repair of DNA-DSB. SiRNA targeting of SOD1 and SOD2 affected neither DNA-PKcs phosphorylation nor post-irradiation survival while inhibition of Akt by specific inhibitor abrogated 2-ME-induced radiosensitization. Conclusion These results may indicate that 2-ME-induced radiosensitization is independent of SOD inhibition but mainly depends on inhibition of Akt and DNA-PKcs activities.