Hydrogen Sulfide as a Radiosensitizer for Glioblastoma

摘要 多形性胶质母细胞瘤(GBM)是成人最常见的恶性中枢神经系统肿瘤。 常规治疗包括肿瘤手术切除后光子照射联合 辅助化疗的中位生存期为14.6个月。治疗方面的进展是 受限于GBM的极端耐化疗和抗辐射能力。光子和质子的结合疗法已经被 发现可增加存活时间，但因放射性坏死引起的并发症阻碍了实施 这种治疗方法。添加辐射增敏剂可以使用较低的辐射剂量来实现 肿瘤细胞毒性，同时保留正常组织。本申请旨在调查硫化氢的使用情况。 (H_2S)作为一种治疗GBM的新型放射增敏剂。硫化氢已被确定为第三个 实验中使用内源性气体递质和硫化钠(Na2S)、DATS和AP39来增加 细胞内的硫化氢。我们的初步研究表明，急性、高剂量的Na2S显著诱导DNA氧化 损伤并能杀死培养中的GBM细胞，而不损伤正常的脑血管内皮细胞。Na2S也发生了变化 GBM细胞系线粒体功能、蛋白质乙酰化增加、氧化谷胱甘肽增加以及 放射增敏的GBM细胞对质子或光子电离辐射。我们的假设是硫化氢增强了电离 通过增加GBM细胞产生的活性氧物种(ROS)来辐射选择性杀伤GBM细胞 线粒体，增加DNA损伤，导致聚(ADP-核糖)聚合酶过度激活 (PARP)。我们进一步假设，PARP的过度激活降低了细胞内NAD+的水平，抑制了 HDAC/sirtuins，由于抑制DNA修复和更高的DNA损伤水平而导致细胞死亡。 这一假设将在两个目标上进行验证，使用两个GBM细胞系，一个是星形胶质细胞系，一个是神经细胞系 和脑微血管细胞系。目标1将测试Na2S、DATS和AP39是否只增强GBM质子 或者光子电离辐射细胞的杀灭。我们将通过检测ROS，DNA损伤来探讨其机制 生产，和线粒体功能障碍。线粒体抗氧化剂MitoTEMPOL和两个GBM Rho 0 氧化磷酸化缺陷的细胞系也将被用来确定ROS是否在 线粒体。AIM 2将测试Na2S诱导的放射增敏是否通过PARP和 减少了DNA修复。在这项工作完成后，我们将理解为什么硫化氢使GBM细胞和 了解哪种硫化氢释放化合物是GBM细胞最好的放射增敏剂。这可能会打开一条 用于开发释放硫化氢的化合物作为辐射增敏剂。本建议书中的工作将由 研究生、本科生和高中生。这些学生将学习辐射生物学、氧化 应激和DNA修复，将对放射治疗和Proteus®One铅笔束的使用有新的见解。 这项建议符合R15关于加强什里夫波特学生教育的目标。资金将大幅增加 提高专业人员进行这些研究的能力，并将加强院校研究生课程。

ABSTRACT Glioblastoma multiforme (GBM) is the most common malignant central nervous system cancer in adults. Conventional treatment consisting of tumor surgical resection followed by photon radiation in combination with adjuvant chemotherapy results in a median survival of 14.6 months. Therapeutic advancements have been limited by the extreme chemo- and radio-resistance of GBM. Combination photon and proton therapy has been found to increase survival time, but complications due to radionecrosis have prevented the implementation of this therapy regimen. Addition of a radiosensitizing agent could allow the use of lower radiation doses to achieve tumor cytotoxicity while sparing normal tissue. This application aims to investigate the use of hydrogen sulfide (H2S) as a novel radiosensitizing agent for the treatment of GBM. H2S has been identified as the third endogenous gasotransmitter and sodium sulfide (Na2S), DATS and AP39 are used experimentally to increase cellular H2S. Our preliminary studies suggest acute, high doses of Na2S significantly induce oxidative DNA damage and can kill GBM cell lines in culture, while sparing normal cerebral endothelial cells. Na2S also altered GBM mitochondrial function, increased protein acetylation, increased oxidized glutathione in GBM cell lines and radiosensitized GBM cells to proton or photon ionizing radiation. Our hypothesis is that H2S enhances ionizing radiation-selective killing of GBM cells by increasing reactive oxygen species (ROS) production from the mitochondria, enhancing DNA damage and leading to hyper-activation of poly(ADP-ribose) polymerase (PARP). We further hypothesize that hyper-activation of PARP decreases cellular NAD+ levels, inhibiting HDAC/ sirtuins, resulting in cell death due to inhibition of DNA repair and higher DNA damage levels. This hypothesis will be tested in two aims using two GBM cell lines, an astrocyte cell line, a neuronal cell line and a cerebral microvascular cell line. Aim 1 will test whether Na2S, DATS and AP39 enhance only GBM proton or photon ionizing radiation cell killing. We will probe the mechanism by examining ROS, DNA damage production, and mitochondrial dysfunction. MitoTEMPOL, a mitochondrial antioxidant, and two GBM rho zero cell lines deficient in oxidative phosphorylation will also be used to determine if the ROS is generated in the mitochondria. Aim 2 will test whether Na2S-induced radiosensitization is through hyper-activation of PARP and reduced DNA repair. Upon completion of this work, we will understand why H2S radiosensitizes GBM cells and know which H2S-releasing compound is the best radiosensitizer of GBM cells. This will potentially open avenues for development of H2S-releasing compounds as radiosensitizers. Work in this proposal will be performed by graduate, undergraduate and high school students. These students will learn about radiation biology, oxidative stress and DNA repair and will gain novel insight into radiotherapy and the use of the Proteus®ONE pencil beam. This proposal meets the R15 goals of enhancing education of students in Shreveport. Funding will significantly improve the ability of the PI to perform these studies, and will enhance the institutional graduate program.