Metabolic Oxidative Stress and TRAIL Cytotoxicity

描述(申请人提供)：拟议研究项目的长期目标是了解哺乳动物细胞对各种压力做出反应的分子机制。在下一次授予期间，我们将研究代谢氧化应激在TRAIL(肿瘤坏死因子相关凋亡诱导配体)诱导的凋亡死亡中的作用。我们推测，肿瘤微环境，尤其是葡萄糖缺乏，通过促进线粒体依赖途径和/或非依赖途径，增强了TRAIL诱导的细胞毒性。在TRAIL存在的情况下，低糖浓度诱导代谢氧化应激，进而促进细胞色素c的释放。细胞色素c的释放促进了线粒体介导的caspase信号转导途径。我们还假设代谢性氧化应激导致神经酰胺的升高，神经酰胺激活的蛋白磷酸酶(CAPP)和/或磷脂酰肌醇-3激酶[PI(3)K]失活。CAPP的激活和/或PI(3)K的失活通过去磷酸化使Akt失活，从而通过抑制NF-kappaB信号转导途径下调抗凋亡蛋白FliP的表达。本项目的具体目的是检测(1)低糖浓度对TRAIL诱导的细胞毒性的影响，(2)低糖浓度对TRAIL激活的细胞凋亡途径的影响，(3)线粒体依赖的半胱氨酸天冬氨酸酶途径和凋亡相关蛋白调节的半胱氨酸天冬氨酸酶途径之间的相互作用，(4)代谢氧化stress-ceramide-PI(3)K/CAPP-Akt-NF-kappaB-FLIP途径在TRAIL敏感性中的作用，(5)肿瘤微环境的其他特征对TRAIL细胞毒性的影响。拟议的研究(目标1)将使用生存决定试验来检查TRAIL在不同浓度的葡萄糖中诱导的细胞毒性。第二个目标将集中在使用生化方法来研究低糖浓度如何促进TRAIL激活的caspase信号转导途径。Aim 3的研究将使用分子和生化方法来阐明线粒体依赖的caspase途径和凋亡相关蛋白调节的caspase途径之间的相互作用。我们将利用分子遗传学和生化技术来阐明PI(3)K/CAPP-Akt-Flip通路在低糖增强TRAIL细胞毒作用中的作用(目标4)。最后，我们将整合信号通路的不同方面。我们相信，这项研究的成功将为TRAIL用于治疗人类癌症的开发和临床应用提供支持。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research project is to understand the molecular mechanisms of cellular responses to a variety of stresses in mammalian cells. During the next granting period, we will investigate the effect of metabolic oxidative stress on TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptotic death. We postulate that tumor microenvironment, in particular glucose deprivation, enhances TRAIL-induced cytotoxicity by facilitating the mitochondria-dependent pathway and/or -independent pathway. Low glucose concentrations induce metabolic oxidative stress and subsequently promote cytochrome c release in the presence of TRAIL. Cytochrome c release promotes the mitochondria-mediated caspase signal transduction pathway. We also hypothesize that metabolic oxidative stress-induces an elevation of ceramide which activates ceramide-activated protein phosphatase (CAPP) and/or inactivates phosphoinositide-3 kinase [PI(3)K]. The activation of CAPP and/or inactivation of PI(3)K inactivates Akt through dephosphorylation and consequently down-regulates the expression of FLIP, an antiapoptotic protein, by inhibiting NF-kappaB signal transduction pathway. The specific aims of this project are to examine (1) the effect of low glucose concentrations on TRAIL-induced cytotoxicity, (2) the effect of low glucose concentrations on the TRAIL-activated apoptotic pathway, (3) cross-talk between the mitochondria-dependent caspase pathway and the apoptosis associated protein-regulated caspase pathway, (4) the role of the metabolic oxidative stress-ceramide-PI(3)K/CAPP-Akt-NF-kappaB-FLIP pathway in TRAIL sensitivity, (5) the effect of a variety of other characteristic features of the tumor microenvironment on TRAIL cytotoxicity. The proposed studies (Aim 1) will use survival determination assays to examine TRAIL-induced cytotoxicity in various concentrations of glucose. The second aim will focus on the use of biochemical approaches to investigate how low glucose concentrations promote the TRAIL-activated caspase signal transduction pathway. The studies for Aim 3 will use molecular and biochemical approaches to elucidate cross-talk between the mitochondria-dependent caspase pathway and apoptosis associated protein-regulated caspase pathway. We will employ molecular genetics and biochemical techniques to elucidate the role of the PI(3)K/CAPP-Akt-FLIP pathway in the low glucose-enhanced TRAIL cytotoxicity (Aim 4). Finally we will integrate different aspects of the signaling pathways. We believe that the successful outcome of this study will support the development and clinical application of TRAIL for the treatment of human cancer.