Delayed radioprotection by nuclear transcription factor κB -mediated induction of manganese superoxide dismutase in human microvascular endothelial cells after exposure to the free radical scavenger WR1065

Abstract

The free radical scavenger WR1065 (SH) is the active thiol form of the clinically approved cytoprotector amifostine. At doses of 40 μM and 4 mM it can activate the redox-sensitive nuclear transcription factor κB (NFκB) and elevate the expression of the antioxidant gene manganese superoxide dismutase (MnSOD) in human microvascular endothelial cells (HMEC). MnSOD contains binding motifs for a number of transcription factors other than NFκB and codes for a potent antioxidant enzyme localized in the mitochondria that is known to confer enhanced radiation resistance to cells. The purpose of this study was to determine the effect of WR1065 exposure on the various transcription factors known to affect MnSOD expression along with the subsequent kinetics of intracellular elevation of MnSOD protein levels and associated change in sensitivity to ionizing radiation in HMEC. Cells were grown to confluence and exposed to WR1065 for 30 min. Affects on the transcription factors AP1, AP2, CREB, NFκB, and Sp1 were monitored as a function of time ranging from 30 min to 4 h after drug exposure using a gel-shift assay. Only NFκB exhibited a marked activation and that occurred 30 min following the cessation of drug exposure. MnSOD protein levels, as determined by Western blot analysis, increased up to 16-fold over background control levels by 16 h following drug treatment, and remained at 10-fold or higher levels for an additional 32 h. MnSOD activity was evaluated using a gel-based assay and was found to be active throughout this time period. HMEC were irradiated with X-rays either in the presence of 40 μM or 4 mM WR1065 or 24 h after its removal when MnSOD levels were most elevated. No protection was observed for cells irradiated in the presence of 40 μM WR1065. In contrast, a 4 mM dose of WR1065 afforded an increase in cell survival by a factor of 2. A "delayed radioprotective" effect was, however, observed when cells were irradiated 24 h later, regardless of the concentration of WR1065 used. This effect is characterized as an increase in survival at the 2 Gy dose point, i.e., a 40% increase in survival, and an increase in the initial slope of the survival curve by a factor of 2. The anti-inflammatory sesquiterpene lactone, Helenalin, is an effective inhibitor of NFκB activation. HMEC were exposed to Helenalin for 2 h at a nontoxic concentration of 5 μM prior to exposure to WR1065. This treatment not only inhibited activation of NFκB by WR1065, but also inhibited the subsequent elevation of MnSOD and the delayed radioprotective effect. A persistent marked elevation of MnSOD in cells following their exposure to a thiol-containing reducing agent such as WR1065 can result in an elevated resistance to the cytotoxic effects of ionizing radiation and represents a novel radioprotection paradigm.

Doxorubicin-Induced Oxidative Injury of Cardiomyocytes - Do We Have Right Strategies for Prevention?

“…Namely, MnSOD is nuclear-encoded mitochondrial enzyme that scavenges O 2  . -in mitochondrial matrix, and has been shown to be highly protective against radiation-induced ROS (Murley et al, 2006). Based on the current data, we speculate that succesful amifostine protection of DOX-induced damage of heart capillaries, whose endothelium, as a rich source of oxidants, contributes a lot to the oxidant-rich environment at that locus in this model (Dobric et al, 1998;Dragojevic-Simic et al, 2007;Dragojevic-Simic et al, 2011a), may be mediated by its antioxidant properties resulting in downregulation of oxidative stress and redox-sensitive signalling cascades.…”

Maintenance of Manganese Superoxide Dismutase (SOD2)-Mediated Delayed Radioprotection Induced by Repeated Administration of the Free Thiol Form of Amifostine

“…Additional reports followed indicating that the thiol-containing drugs oltipraz, captopril, mesna and WR1065, the active free thiol form of amifostine, could also elevate SOD2 gene expression through their ability to activate NFκB in exposed malignant and nonmalignant cells of human and mouse origin (27,28). After exposure of cells to these thiols, activation of NFκB occurred within 30 min and was followed by elevated SOD2 gene expression and a 10-to 20-fold buildup of active SOD2 enzyme that peaked about 24 to 30 h later (22)(23)(24)(25)(26)(27)(28). Cells irradiated at times of maximum SOD2 buildup after thiol exposure exhibited a 20 to 40% increase in resistance to ionizing radiation: hence the term delayed radioprotective effect (22)(23)(24)(25).…”

“…We earlier identified a novel radiation protection paradigm known as the thiol-induced SOD2-mediated "delayed radioprotective effect" (22)(23)(24)(25). This radioprotection paradigm is based on earlier observations that the thiol reducing agents N-acetylcysteine, dithiothreitol and 2-mercaptoethanol had the ability to activate NFκB and subsequently elevate SOD2 gene expression (26).…”

“…After exposure of cells to these thiols, activation of NFκB occurred within 30 min and was followed by elevated SOD2 gene expression and a 10-to 20-fold buildup of active SOD2 enzyme that peaked about 24 to 30 h later (22)(23)(24)(25)(26)(27)(28). Cells irradiated at times of maximum SOD2 buildup after thiol exposure exhibited a 20 to 40% increase in resistance to ionizing radiation: hence the term delayed radioprotective effect (22)(23)(24)(25). This effect could be inhibited by treating cells with the NFκB inhibitors BAY11-7082 or Helenalin, by stably transfecting them with a mutant IκBα gene under the control of a CMV promoter to prevent NFκB activation after thiol exposure (23,24), or by transfecting cells with SOD2 siRNA to prevent thiol-induced elevation of SOD2 enzyme levels and activity (25).…”

“…Cells irradiated at times of maximum SOD2 buildup after thiol exposure exhibited a 20 to 40% increase in resistance to ionizing radiation: hence the term delayed radioprotective effect (22)(23)(24)(25). This effect could be inhibited by treating cells with the NFκB inhibitors BAY11-7082 or Helenalin, by stably transfecting them with a mutant IκBα gene under the control of a CMV promoter to prevent NFκB activation after thiol exposure (23,24), or by transfecting cells with SOD2 siRNA to prevent thiol-induced elevation of SOD2 enzyme levels and activity (25). The proposed mechanism of action underlying the delayed radioprotective effect is that reducing agents having an active thiol moiety can reduce cysteine residues on the p50 and p65 subunits of NFκB that results in activation and enhanced binding of this transcription factor to the NFκB binding site(s) of SOD2 (29).…”