Hydrogen Sulfide as a Radiosensitizer for Glioblastoma

摘要 多形性胶质母细胞瘤（GBM）是成人中最常见的恶性中枢神经系统癌症。 常规治疗包括肿瘤手术切除，随后进行光子辐射， 辅助化疗的中位生存期为14.6个月。治疗的进步 受到GBM的极端化学和放射抗性的限制。联合光子和质子治疗已经 发现增加生存时间，但由于放射性坏死的并发症阻止了 这个治疗方案添加放射增敏剂可以允许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。本申请旨在研究硫化氢的使用 (H2S)作为治疗GBM的新型放射增敏剂。H2S已被确定为第三种 实验上使用内源性气体递质和硫化钠（Na_2S）、DATS和AP_（39）来增加 细胞内H2S我们的初步研究表明，急性，高剂量的Na 2S显着诱导氧化DNA 损伤并能杀死培养中的GBM细胞系，同时保留正常的脑内皮细胞。Na 2S也改变了 GBM线粒体功能，增加的蛋白乙酰化，GBM细胞系中增加的氧化型谷胱甘肽， 对质子或光子电离辐射放射敏感的GBM细胞。我们的假设是H2S增强了电离 放射选择性杀死GBM细胞，通过增加活性氧（ROS）的产生， 线粒体，增强DNA损伤并导致聚（ADP-核糖）聚合酶的过度活化 （PARP）。我们进一步假设PARP的过度激活降低了细胞NAD+水平，抑制了细胞内NAD+的表达。 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。 将使用两种GBM细胞系、星形胶质细胞细胞系和神经元细胞系在两个目标中测试该假设 和脑微血管细胞系。目标1将测试Na 2S、DATS和AP 39是否仅增强GBM质子 或光子电离辐射杀死细胞。我们将通过检测活性氧、DNA损伤 生产和线粒体功能障碍。线粒体抗氧化剂MitoTEMPOL和两种GBM rho zero 氧化磷酸化缺陷的细胞系也将用于确定ROS是否在细胞中产生。 线粒体目的2将测试Na 2S诱导的放射增敏是否是通过PARP的过度活化， 减少DNA修复。在完成这项工作后，我们将了解为什么H2S放射增敏GBM细胞， 知道哪种释放H2S的化合物是GBM细胞的最佳放射增敏剂。这可能会打开 用于开发释放H2S的化合物作为放射增敏剂。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 并将对放射治疗和Proteus®ONE笔形射束的使用获得新的见解。 这项建议符合R15的目标，加强教育的学生在什里夫波特。资金将大大 提高PI执行这些研究的能力，并将加强机构研究生课程。

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.