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

Abstract

Thiol-containing drugs such as WR1065, the free thiol form of amifostine, have been shown to induce a delayed radioprotective effect in both malignant and non-malignant cells. In mammalian cells exposed to a dose as low as 40 μM WR1065, the redox-sensitive nuclear transcription factor κB (NFκB) is activated, leading to an elevation in the expression of the antioxidant gene manganese superoxide dismutase (SOD2) and a concomitant increase in active SOD2 enzyme levels that peaks 24 to 32 h later. Exposure of cells to ionizing radiation during the period of elevated SOD2 enzymatic activity results in an enhanced radiation resistance. This is seen as an increase in surviving fraction as determined by standard colony formation assays. To determine whether this delayed radioprotection can be maintained over a prolonged period in cells of either malignant or nonmalignant origin, both human microvascular endothelial cells (HMEC) and SA-NH mouse sarcoma cells were grown to confluence and exposed to 40 μM WR1065 using three administration protocols: (1) daily drug exposure for 10 days followed each day by irradiation with 2 Gy; (2) drug exposure once every 48 h followed by irradiation with 2 Gy 48 h later for 14 days; and (3) drug exposure every 72 h followed by irradiation with 2 Gy 72 h later for 12 days. As a function of each experimental condition, cell numbers and associated SOD2 enzymatic activities were measured at the time of each irradiation. None of the treatment conditions were toxic to either HMEC or SA-NH cells. SOD2 activity was elevated 5.3-and 1.8-fold over background on average for HMEC exposed to 40 μM WR1065 every 24 or 48 h, respectively. Likewise, SOD2 activity was elevated in SA-NH mouse sarcoma cells 7.8-and 4.9-fold after daily exposure to WR1065 or exposure to WR1065 once every 48 h, respectively. Both HMEC and SA-NH cells exhibited enhanced radiation resistance that correlated with the increase in SOD2 activity. The average respective increases in cell survival were 1.33 ± 0.01 (SEM), 1.23 ± 0.01 and 1.04 ± 0.01 for HMEC exposed to WR1065 every 24, 48 and 72 h, respectively, and 1.27 ± 0.01, 1.18 ± 0.02 and 1.02 ± 0.02 for SA-NH cells exposed to WR1065 every 24, 48 and 72 h, respectively. Both the elevation in WR1065-induced SOD2 enzymatic activity and the corresponding increase in radiation resistance were completely inhibited in HMEC and SA-NH cells transfected with human or mouse SOD2 siRNA oligomers and irradiated 24 h later. These data demonstrate that a delayed radioprotective effect can be induced and maintained over a prolonged period in both non-malignant and malignant cells exposed to thiol-containing drugs such as WR1065. For non-malignant cells this represents a novel paradigm for radiation protection. The ability of WR1065 to induce a persistent elevated radiation resistance in malignant cells, however,

SOD2-Mediated Effects Induced by WR1065 and Low-Dose Ionizing Radiation on Micronucleus Formation in RKO Human Colon Carcinoma Cells

“…We have extensively characterized a thiol-induced and SOD2-mediated delayed radioprotective effect using cell survival as an end point that in many ways parallels a phenomenon referred to as the radiation-induced adaptive response (17–20, 28–30). In the current study we have expanded our investigation not only to include a marker for chromosome damage, e.g., the micronucleus assay, but also to contrast the relative magnitudes of the thiol- and radiation-induced “adaptive” responses with each other and to determine the role of SOD2 in the mediation of each of these phenomena.…”

“…Increased intracellular SOD2 protein levels were accompanied by an associated increase in enzymatic activity that correlated with a 20 to 40% increase in cell survival when cells were exposed at that time to a 2-Gy challenge dose (17–19). Elevated levels of SOD2 and associated increased radiation resistance could be prolonged by continued treatment with WR1065 (20). Treatment of cells with NFκB inhibitors or SOD2 siRNA prior to exposure to WR1065 completely inhibited the thiol-induced increases in intracellular SOD2 levels and activities in mouse SA-NH tumor cells, human microvascular endothelial cells (HMEC), and RKO human carcinoma cells.…”

“…Treatment of cells with NFκB inhibitors or SOD2 siRNA prior to exposure to WR1065 completely inhibited the thiol-induced increases in intracellular SOD2 levels and activities in mouse SA-NH tumor cells, human microvascular endothelial cells (HMEC), and RKO human carcinoma cells. The specific inhibition of thiol-induced elevation of SOD2 activity was directly correlated with subsequent decreases in radiation resistance as shown by decreased cell survival (17–20). …”

SOD2-mediated adaptive responses induced by low-dose ionizing radiation via TNF signaling and amifostine

“…The elevation of SOD2 enzymatic activity induced in this manner reaches maximal levels between 20 and 24 h later, at which time if cells are challenged with a second high dose of ionizing radiation their relative resistance to cell killing can increase by about 40% [17]. The importance of both NFκB signaling and elevated SOD2 activity in the expression of this radio-induced adaptive response has been further demonstrated through the use of NFκB inhibitors [18, 19] and/or SOD2 siRNA transfection of cells [20, 21], both of which result in the abolishment of elevated SOD2 activity and concomitant enhanced cell survival or reduced micronuclei formation, a marker of genomic instability.…”

Manganese superoxide dismutase interacts with a large scale of cellular and mitochondrial proteins in low-dose radiation-induced adaptive radioprotection

“…MnSOD helps to eliminate free radicals in the mitochondria by converting superoxide anions to H 2 O 2 , which can then be detoxified by the catalase activity of peroxidase [13]. Loss of MnSOD expression or its enzymatic activity leads to a high risk of tumorigenesis [14, 15] and high sensitivity of cells to genotoxic condition including ionizing radiation [16]. In addition, mice with MnSOD haploinsufficiency have abnormal mitochondrial function correlated with increased oxidative damage [17].…”