Murley Jeffrey S, Kataoka Yasushi, Weydert Christine J, Oberley Larry W, Grdina David J
Department of Radiation and Cellular Oncology, The University of Chicago, IL 60637, USA.
Free Radic Biol Med. 2006 Mar 15;40(6):1004-16. doi: 10.1016/j.freeradbiomed.2005.10.060. Epub 2005 Nov 21.
The free radical scavenger WR1065 (SH) is the active thiol form of the clinically approved cytoprotector amifostine. At doses of 40 microM and 4 mM it can activate the redox-sensitive nuclear transcription factor kappaB (NFkappaB) 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 NFkappaB 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, NFkappaB, 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 NFkappaB 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 microM 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 microM 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 NFkappaB activation. HMEC were exposed to Helenalin for 2 h at a nontoxic concentration of 5 microM prior to exposure to WR1065. This treatment not only inhibited activation of NFkappaB 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.
自由基清除剂WR1065（SH）是临床批准的细胞保护剂氨磷汀的活性硫醇形式。在40微摩尔和4毫摩尔的剂量下，它可以激活对氧化还原敏感的核转录因子κB（NFκB），并提高人微血管内皮细胞（HMEC）中抗氧化基因锰超氧化物歧化酶（MnSOD）的表达。MnSOD除了含有NFκB的结合基序外，还含有许多其他转录因子的结合基序，并且编码一种定位于线粒体的强效抗氧化酶，已知该酶能赋予细胞增强的抗辐射能力。本研究的目的是确定暴露于WR1065对已知影响MnSOD表达的各种转录因子的影响，以及随后HMEC中MnSOD蛋白水平的细胞内升高动力学和对电离辐射敏感性的相关变化。细胞生长至汇合后，暴露于WR1065 30分钟。使用凝胶迁移分析监测药物暴露后30分钟至4小时内对转录因子AP1、AP2、CREB、NFκB和Sp1的影响。只有NFκB表现出明显的激活，并且在药物暴露停止后30分钟出现。通过蛋白质印迹分析确定，药物处理后16小时，MnSOD蛋白水平比背景对照水平增加了16倍，并在另外32小时内保持在10倍或更高水平。使用基于凝胶的分析评估MnSOD活性，发现其在此期间一直具有活性。在存在40微摩尔或4毫摩尔WR1065的情况下或在去除WR1065 24小时后（此时MnSOD水平最高），用X射线照射HMEC。在存在40微摩尔WR1065的情况下照射的细胞未观察到保护作用。相比之下，4毫摩尔剂量的WR1065使细胞存活率提高了2倍。然而，当细胞在24小时后照射时，无论使用的WR1065浓度如何，都观察到了“延迟辐射保护”效应。这种效应的特征是在2 Gy剂量点存活率增加，即存活率增加40%，并且存活曲线的初始斜率增加2倍。抗炎倍半萜内酯海伦alin是NFκB激活的有效抑制剂。在暴露于WR1065之前，将HMEC以5微摩尔的无毒浓度暴露于海伦alin 2小时。这种处理不仅抑制了WR1065对NFκB的激活，还抑制了随后MnSOD的升高和延迟辐射保护效应。细胞暴露于含硫醇的还原剂如WR1065后，MnSOD持续显著升高，可导致对电离辐射细胞毒性作用的抗性增加，这代表了一种新的辐射保护模式。
