Metabolic Oxidative Stress and TRAIL Cytotoxicity

描述（由申请人提供）：拟议研究项目的长期目标是了解哺乳动物细胞对各种应激反应的分子机制。在下一个授权期间，我们将研究代谢氧化应激对TRAIL（肿瘤坏死因子相关凋亡诱导配体）诱导的凋亡性死亡的影响。我们推测，肿瘤微环境，特别是葡萄糖剥夺，通过促进肿瘤坏死因子依赖性途径和/或非依赖性途径增强TRAIL诱导的细胞毒性。低葡萄糖浓度诱导代谢性氧化应激，随后在TRAIL存在下促进细胞色素c释放。细胞色素c的释放促进caspase介导的信号转导途径。我们还假设代谢性氧化应激诱导神经酰胺升高，从而激活神经酰胺活化蛋白磷酸酶（CAPP）和/或灭活磷酸肌醇-3激酶[PI（3）K]。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-κ B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）低葡萄糖浓度对TRAIL诱导的细胞毒性的影响，（2）低葡萄糖浓度对TRAIL激活的凋亡途径的影响，（3）依赖于TRAIL的caspase途径和凋亡相关蛋白调节的caspase途径之间的交叉作用，（4）代谢性氧化应激-神经酰胺-PI（3）K/CAPP-Akt-NF-κ B-FLIP途径在TRAIL敏感性中的作用，（5）肿瘤微环境的多种其它特征性特征对TRAIL细胞毒性的影响。拟议的研究（目的1）将使用生存测定法来检查TRAIL诱导的细胞毒性在不同浓度的葡萄糖。第二个目标将集中在使用生物化学方法来研究低葡萄糖浓度如何促进TRAIL激活的caspase信号转导通路。Aim 3的研究将使用分子和生物化学方法来阐明线粒体依赖性caspase途径和细胞凋亡相关蛋白调节的caspase途径之间的串扰。我们将采用分子遗传学和生物化学技术来阐明PI（3）K/CAPP-Akt-FLIP通路在低葡萄糖增强的TRAIL细胞毒性中的作用（目的4）。最后，我们将整合信号通路的不同方面。我们相信，本研究的成功结果将支持TRAIL治疗人类癌症的开发和临床应用。

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.