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

• Published in: Radiotherapy and oncology 2009-09, Vol.92 (3), p.334-338
• Main Authors: Florczak, Urszula, Toulany, Mahmoud, Kehlbach, Rainer, Peter Rodemann, H
• Format: Article
• Language: English
• Subjects: 2-ME; Adenocarcinoma - pathology; Adenocarcinoma - radiotherapy; Akt; Apoptosis - drug effects; Apoptosis - radiation effects; Blotting, Western; Cell Line, Tumor - metabolism; Cell Line, Tumor - radiation effects; DNA Breaks, Double-Stranded - drug effects; DNA Breaks, Double-Stranded - radiation effects; DNA Damage - drug effects; DNA Damage - radiation effects; DNA-Activated Protein Kinase - antagonists & inhibitors; DNA-Activated Protein Kinase - metabolism; DNA-PKcs; Enzyme Activation; Estradiol - analogs & derivatives; Estradiol - pharmacology; Hematology, Oncology and Palliative Medicine; Humans; Ionizing radiation; Lung Neoplasms - pathology; Lung Neoplasms - radiotherapy; Phosphorylation - drug effects; Phosphorylation - radiation effects; Proto-Oncogene Proteins c-akt - antagonists & inhibitors; Proto-Oncogene Proteins c-akt - metabolism; Radiation Dosage; Radiation Tolerance - drug effects; Radiation, Ionizing; Sensitivity and Specificity; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; Superoxide dismutases
• ISSN: 0167-8140; 1879-0887
• DOI: 10.1016/j.radonc.2009.06.005

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.