2-Methoxyestradiol-induced radiosensitization is independent of SOD but depends on inhibition of Akt and DNA-PKcs activities.
OBJECTIVE
2-Methoxyestradiol (2-ME) is described as an inhibitor of the superoxide dismutase (SOD) enzyme activity. However, it attenuates PI3K/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.


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 gammaH2AX 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 4Gy stimulated SOD enzyme activity as early as 1min after radiation exposure. Expression of Cu/Zn-SOD (SOD1) as well as Mn-SOD (SOD2) was increased by single doses of 1-4Gy within 24-36h. 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.


CONCLUSIONS
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.
Florczak
Urszula
U
Department of Radiation Oncology, University of Tuebingen, Germany.
Toulany
Mahmoud
M
Kehlbach
Rainer
R
Peter Rodemann
H
H
D000077584:2-Methoxyestradiol; D000230:Adenocarcinoma / Q000473:pathology / Q000532:radiotherapy; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D015153:Blotting, Western; D045744:Cell Line, Tumor / Q000378:metabolism* / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology / Q000532:radiotherapy; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000187:drug effects; D011839:Radiation, Ionizing; D012680:Sensitivity and Specificity; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*
D004958:Estradiol; D000077584:2-Methoxyestradiol; D013482:Superoxide Dismutase; D051747:DNA-Activated Protein Kinase; D051057:Proto-Oncogene Proteins c-akt