Inhibition of NFκB DNA Binding by Helenalin Abolishes the Induced Radioprotective Effect by Amifostine’s Active Metabolite WR1065 in Human Microvascular Endothelial Cells.

David J. Grdina

Why this Project?

This project is designed to study induced radioprotection after exposure to thiol-containing drugs such as WR1065. The proposed underlying mechanism of action for this effect is supposed to involve thiol-induced activation of NFκB that leads to enhanced expression of Sod2 in human microvascular endothelial cells This hypothesis will be tested in this project.

Project Goals:

1. To further characterize the effects of WR1065 on NFκB and Sod2 enzyme levels as they relate to the induced radioprotective effect as a function of radiation dose.
   
   
2. This project will evaluate the relationships between the induced WR1065 protection and the induced NFκB DNA binding activity and subsequently enhanced Sod2 enzyme levels.

Experimental Approach:

1. Human microvascular endothelial cells will be used to test the effect of helenalin, a sesquiterpene lactone that selectively and irreversibly alkylates the p65 subunit of NFκB, thereby blocking DNA binding.
   
   
2. The human cells were exposed to helenalin concentrations ranging from 1 to 20 μM for 2.5 h and the toxicity was determined by clonogenic assay.
   
   
3. Induced radioprotection of 40 μM or 4 mM WR1065 following radiation with 2 Gy, or exposed to 5 μM helenalin for 2 h prior to WR1065 treatment will be characterized in endothelial cells.

Outcomes:

1. Treatment of human cells with WR1065 induced NFκB DNA binding and provided protection to radiation-induced cell killing.
   
   
2. 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 DNA binding.
   
   
3. Treatment of human microvascular epithelial cells with WR1065 resulted in a 10- to 20-fold enhancement in the Sod2 levels which was inhibited in the cells exposed to helenalin prior to WR1065 treatment.
   
   
4. In contrast, no cytoprotection was observed for cells irradiated 24 h after helenalin exposure, the time frame during which the maximum elevation in Sod2 is observed in cells treated with WR1065.