Delayed Radioprotection by NFκB-Mediated Induction of Sod2 (MnSOD) in SA-NH Tumor Cells after Exposure to Clinically Used Thiol-Containing Drugs

Abstract

The ability of thiol-containing reducing agents to activate transcription factors leading to changes in gene expression and enzyme activities provides an additional mechanism to potentially protect against radiation-induced cell killing. Manganese superoxide dismutase (Sod2) is one such gene whose expression levels have been shown to be elevated after exposure to the thiol compounds WR-1065 and N-acetyl-L-cysteine (NAC), resulting in an increase in radiation resistance. To further characterize this effect, SA-NH sarcoma cells, both wild-type and a clone stably transfected with a plasmid containing an IkappaBalpha gene mutated at serines 32 and 36, which prevents the inducible phosphorylation of these residues and the subsequent activation of NFkappaB (SA-NH+mIkappaBalpha1), were grown to confluence and then exposed to amifostine's free thiol WR-1065 at a concentration of 4 mM for 30 min. Effects of thiol exposure on NFKB activation in SA-NH+mIkappaBalpha1 cells were determined by a gel shift assay, and changes in Sod2 protein levels in these cells 24 h after exposure to 40 microM or 4 mM WR-1065 were measured by Western blot analysis and compared with wild-type cells exposed to the NFkappaB inhibitor BAY 11-7082. Changes in radiation response, measured immediately after thiol exposure or 24 h later, were determined using a colony-forming assay and were correlated with NFKB activation and Sod2 protein levels. The effects of captopril, mesna and NAC, each at a dose of 4 mM, on radiation response were also determined and contrasted with those of WR-1065. Only WR-1065 and captopril protected SA-NH cells when present during irradiation, i.e. 1.57 and 1.31 times increase in survival at 2 Gy, respectively. All four thiols were protective if irradiation with 2 Gy occurred 24 h later; i.e. increases in survival of 1.40, 1.22, 1.35, and 1.25 times were found for WR-1065, captopril, mesna and NAC, respectively. This delayed radioprotective effect correlated with elevated Sod2 protein levels in wild-type SA-NH tumor cells but was not observed in SA-NH+mIkappaBalpha1 cells, indicating that interference with thiol-induced NFKB activation abrogates this delayed radioprotective effect. Because the delayed radioprotective effect is readily demonstrable at a radiation dose of 2 Gy 24 h after exposure to clinically approved thiol-containing drugs such as amifostine, captopril, mesna and NAC, it suggests a new potential concern regarding the issue of tumor protection and the use of these agents in cancer therapy.