OXIDANTS AND CELL DEATH

氧化剂和细胞死亡

• 批准号: 6293782
• 负责人: EMILY B SHACTER
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6293782
• 关键词: ADP ribosylation; adenosine triphosphate; antineoplastics; apoptosis; cell death; flow cytometry; fluorescence microscopy; gel electrophoresis; hydrogen peroxide; inflammation; necrosis; neoplastic cell; oligomycins; oxidative stress

The goal of our work is to elucidate the consequences of exposure of biological substrates to oxidative stress. In these studies, we investigate how oxidants may affect tumor progression, focusing on the two following questions: (1) how are tumor cells killed by oxidative stress? and (2) how can oxidants modulate tumor cell killing by anti-neoplastic drugs? Inherent in these studies is an investigation of cell death pathways. Cell death can occur through several different mechanisms which are distinguished by unique morphological and biochemical traits. The two most widely described forms of cell death are necrosis and apoptosis. Cells dying by apoptosis fragment into subcellular "apoptotic bodies" while cells dying by necrosis swell and then lyse. It is thought that death by apoptosis is physiologically advantageous because apoptotic cells can be phagocytosed by nearby cells such that the contents are degraded intracellularly. In contrast, death by necrosis is thought to promote an inflammatory response caused by the release of the intracellular contents. Most chemotherapeutic agents kill tumor cells by inducing apoptosis. Oxidants such as superoxide, hydrogen peroxide (H2O2), and the hydroxyl radical are generated under a variety of conditions in vivo such as during acute and chronic inflammation. Solid tumors are often infiltrated by inflammatory phagocytes which can generate oxidative stress within the tumor tissue. Treatment of cells in vitro with H2O2 causes DNA strand breaks, oxidation of lipids and proteins, activation of poly(ADP)-ribosylation, and depletion of cellular energy stores. We have found that in the presence of H2O2, human B lymphoma cells are unable to undergo apoptosis. This was established using the chemotherapy drug VP-16 to induce apoptosis in Burkitt's lymphoma cell lines and measuring markers of apoptotic death, including cell morphology (fluorescence microscopy), annexin V binding (FACS analysis), induction of an oligonucleosomal endonuclease (agarose gel electrophoresis), and caspase activation (enzyme assays and Western blot immunoassays). When cells are treated with VP-16 in the presence of 75-100 ?M H2O2, all of these biochemical hallmarks of apoptosis are inhibited and the cells undergo non-apoptotic cell death, similar to the death observed when they are treated with H2O2 alone. The mechanism whereby H2O2 inhibits apoptosis is by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which preserve cellular ATP levels, and can be mimicked by agents such as oligomycin which inhibit ATP synthesis. Overall, our data show that H2O2 can manipulate cell death pathways, diverting the cell away from apoptosis. The main physiological significance of these findings will be found in whether oxidative stress interferes with chemotherapy-induced tumor cell death and clearance in vivo. Experiments are currently underway to address this question.

我们工作的目的是阐明 暴露于生物底物对氧化应激的暴露。 在 这些研究，我们研究了氧化剂如何影响肿瘤 进步，重点介绍以下两个问题：（1） 肿瘤细胞是否被氧化应激杀死？ （2）如何 氧化剂调节抗塑性药物杀死肿瘤细胞？ 这些研究固有的是对细胞死亡的研究 途径。 细胞死亡可以通过几种不同 以独特的形态学和 生化特征。 两种最广泛描述的形式 细胞死亡是坏死和凋亡。 细胞死亡 细胞凋亡碎片成亚细胞“凋亡人士” 坏死死亡的细胞肿胀，然后裂解。 这是思想的 凋亡死亡在生理上是有利的 因为凋亡细胞可以被附近细胞吞噬 使内容物在细胞内降解。 在 对比，死亡死亡被认为促进 由释放引起的炎症反应 细胞内含量。 大多数化学治疗剂杀死 肿瘤细胞通过诱导凋亡。 氧化剂，例如超氧化物，过氧化氢（H2O2）和 羟基自由基在多种 体内的条件，例如急性和慢性期间 炎。 实体瘤通常被 炎症性吞噬细胞可以产生氧化应激 在肿瘤组织中。 在体外治疗细胞 H2O2引起DNA链断裂，脂质的氧化和 蛋白质，聚（ADP） - 脱骨基化的激活和耗竭 细胞能量存储。 我们发现在场 H2O2，人B淋巴瘤细胞无法接受 凋亡。 这是使用化学疗法药物建立的 VP-16在伯基特淋巴瘤细胞系中诱导凋亡和 测量凋亡死亡的标记物，包括细胞 形态学（荧光显微镜），膜联蛋白V结合（FACS 分析），诱导寡核合体内核酸酶 （琼脂糖凝胶电泳）和胱天蛋白酶激活（酶 测定和蛋白质印迹免疫测定）。 当细胞处理时 在75-100？m H2O2的存在下，所有这些 抑制凋亡的生化标志，细胞被抑制 经历非凋亡细胞死亡，类似于死亡 单独使用H2O2处理时观察到。 机制 H2O2抑制细胞凋亡是通过耗尽细胞 ATP。 H2O2的影响可以通过抑制剂的抑制剂来克服 聚（ADP） - 核糖基化，保留细胞ATP水平， 并且可以被诸如寡霉素之类的药物模仿 ATP合成。 总体而言，我们的数据表明H2O2可以 操纵细胞死亡路径，将细胞从 凋亡。 这些的主要生理意义 在氧化应激是否干扰时会发现发现 与化学疗法诱导的肿瘤细胞死亡和清除 体内。 目前正在进行实验以解决此问题 问题。