2001

Delayed radioprotection is defined as an enhancement in the radiation resistance of cells 24 h after their exposure to thiol-containing drugs such as WR1065. The proposed underlying mechanism of action for this effect involves thiol-induced activation of NFκB that leads to enhanced expression of Sod2. To further characterize the effects of WR1065 on NFκB and Sod2 enzyme levels as they relate to the delayed radioprotective effect, human microvascular endothelial cells (HMEC) were treated with helenalin, a sesquiterpene lactone that selectively and irreversibly alkylates the p65 subunit of NFκB, thereby blocking DNA binding. HMEC were exposed to helenalin concentrations ranging from 1 to 20 μM for 2.5 h, and toxicity determined by clonogenic assay. No effect on survival was observed for cells treated with 1 μM helenalin, while the 20 μM dose resulted in no survivors. Surviving fractions of 0.71 and 0.05 were obtained for cells exposed to 5 μM or 10 μM helenalin, respectively. The 1 μM and 5 μM concentrations were chosen for examination of their ability to inhibit WR1065-mediated NFκB activation. As previously observed, treatment of HMEC with 40 μM or 4 mM WR1065 for 30 min induced NFκB DNA binding 30 min after drug treatment. A slight inhibition of NFκB DNA binding was observed in cells exposed to 1 μM helenalin for 2 h prior to WR1065 treatment, while exposure to 5 μM helenalin completely inhibited NFκB. To assess the effects of WR1065 treatment on Sod2 levels, HMEC were exposed to 5 μM helenalin for 2 h and then treated with either 40 μM or 4 mM WR1065 for 30 min. The drugs were then removed and the cells were cultured in drug-free medium for 24 h. At that time, relative Sod2 protein levels were determined by Western blotting. Treatment of HMEC with WR1065 resulted in a 10- to 20-fold enhancement in the Sod2 levels relative to sham-treated cells. This elevation was inhibited in the cells exposed to helenalin prior to WR1065 treatment. Delayed radioprotection was examined in HMEC treated with 40 μM or 4 mM WR1065 for 30 min, washed, and incubated in drug-free medium for 24 h before being irradiated with 2 Gy, or exposed to 5 μM helenalin for 2 h prior to WR1065 treatment. Protection against radiation-induced cell killing was observed for the cells treated with both 40 μM and 4 mM WR1065. In contrast, no cytoprotection was observed for HMEC irradiated 24 h after helenalin exposure, the time frame during which the maximum elevation in Sod2 is observed in cells treated with WR1065. These data support our hypothesis that the delayed protection observed in cells 24 h following exposure to WR1065 is the result of its ability to induce NFκB DNA binding activity and subsequently enhance Sod2 enzyme levels. This work was supported by NIH/NCI grants R01-CA37435 and CA99005 (D.J.G.).

[Proc Amer Assoc Cancer Res, Volume 46, 2005]