Role of redox state in ovarian cancer response to cisplatin

氧化还原状态在卵巢癌顺铂反应中的作用

• 批准号: 7767362
• 负责人: LARRY W DANIEL
• 金额: $ 30.2万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-11 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7767362
• 关键词: Abbreviations; Acetates; Acetylcysteine; Affect; Alkaline Phosphatase; Antioxidants; Apoptosis; Apoptosis Inhibitor; Apoptotic; Ascites; Avidin; BIRC4 gene; Biotin; Cell Membrane Permeability; Cell Proliferation; Cell physiology; Cells; Cessation of life; Characteristics; Chemicals; Chemotherapy-Oncologic Procedure; Chloride Ion; Chlorides; Chromosomes, Human, Pair 10; Cisplatin; Complex; Cysteine; Data; Development; Drug usage; Embryo; Esters; Genetic Transcription; Goals; Growth; Growth Factor; Human; Inflammatory; Inhibition of Apoptosis; Label; Lead; Life; Link; Liquid substance; Lysophosphatidic Acid Receptors; Lysophospholipids; MAPK8 gene; MEKs; Malignant neoplasm of ovary; Manganese; Mass Spectrum Analysis; Methods; Modification; Molecular; Molecular Conformation; Molecular Target; Mono-S; NADPH Oxidase; NF-kappa B; Nuclear Translocation; Oxidation-Reduction; PTEN gene; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Phosphoric Acids; Phosphorylation; Play; Poly(ADP-ribose) Polymerases; Precipitation; Production; Proliferating; Propionic Acids; Protein Tyrosine Phosphatase; Proteins; Reactive Oxygen Species; Reagent; Regulation; Research; Resistance; Role; Second Messenger Systems; Serum; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Sulfenic Acids; Taxane Compound; Tetradecanoylphorbol Acetate; Transcriptional Activation; Transcriptional Regulation; Tumor Necrosis Factor-alpha; Western Blotting; autocrine; base; cancer cell; cell growth; chemotherapeutic agent; chemotherapy; cysteinesulfenic acid; cytokine; dimedone; diphenyliodonium; disulfide bond; docetaxel; enzyme activity; human BIRC4 protein; human CYBA protein; inhibitor/antagonist; insight; kidney cell; lipid mediator; lysophosphatidic acid; mitochondrial membrane; neoplastic; novel; oxidation; p65; phorbol-12-myristate; public health relevance; pyridine; research study; response; second messenger; taxane; tetrakis(4-benzoic acid)porphyrin; transcription factor

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are implicated in the signaling pathways that control the growth of ovarian cancer cells and their resistance to chemotherapy. However, the molecular mechanisms by which ROS control cell function remain elusive. We have shown that lysophosphatidic acid (LPA) stimulates ROS production in ovarian cancer cells and that disrupting this signaling results in growth inhibition and apoptosis. The Raf-MEK-ERK pathway and NF-B appear to be two major targets of regulation by ROS. NF-B dependent signaling is major determinant of resistance to chemotherapy. Thus, higher levels of ROS, which are a characteristic of cancer cells, may stimulate NF-B dependent synthesis of anti-apoptotic proteins. The two main chemotherapeutic drugs for the treatment of ovarian cancer are cisplatin and taxanes. Both of these drugs stimulate ROS production and their anti-neoplastic effects are altered by antioxidants. Thus, ROS signaling is a critical determinant of cellular control of growth and apoptosis. Our goal is to identify the targets of ROS that control these fates. We have developed novel methods for analyzing the cysteine sulfenic acid modification that can be used to identify proteins modified in response to ROS. We will use this approach to determine the ROS-responsive signaling pathways in ovarian cancer cells. We will analyze the response to growth and apoptotic signals, which we have determined involve ROS signaling. Lysophosphatidic acid (LPA) is a lipid mediator of proliferative signaling and cisplatin and taxanes are chemotherapeutic agents which stimulate apoptosis by a ROS-dependent mechanism. The goal of this project is to determine how ROS- dependent signaling in response to LPA, cisplatin and taxanes can lead to either proliferation or apoptosis in ovarian cancer cells. The overall strategy is to use treatment conditions which lead to a growth response or apoptosis and determine what proteins are oxidized in a functionally critical way. How do these oxidations lead to changes in NF-kB activity and how does NF-kB-dependent transcription affect the cells fate? The specific aims are: 1. To determine the role of redox state in the control growth and apoptosis. 2. To determine the role of ROS in the control of NF-kB dependent transcriptional regulation and resistance to chemotherapy. 3. To determine the downstream targets of ROS-signaling in resistance to apoptosis. We will use our newly developed reagents to identify and characterize proteins in the pathways that lead to apoptosis or resistance to apoptosis. This approach involves labeling modified proteins with biotin derivatives of dimedone, followed by avidin pull-down and analysis by western blotting and mass spectrometry. We will use this approach to determine the ROS-responsive signaling pathways in ovarian cancer cells. In summary, the experiments outlined here will contribute to our understanding of how apoptosis and cell growth are controlled by ROS in ovarian cancer cells. This will allow for a better understanding of how ROS play a role in the normal growth control and in resistance to chemotherapy. PUBLIC HEALTH RELEVANCE: Reactive oxygen species (ROS) are second messengers in growth factor signaling pathways and in the response of ovarian cancer cells to chemotherapy. However, the molecular targets for ROS are not known. The goal of this research is to identify which proteins are oxidized in response to growth factors and chemotherapy and determine how these alterations control cell growth or apoptosis. We will use our newly developed reagents that react specifically with oxidized proteins and act as a tag to allow identification of the protein targets for oxidation.

描述（由申请人提供）：活性氧（ROS）与控制卵巢癌细胞生长及其对化学疗法的耐药性的信号通路有关。但是，ROS控制细胞功能的分子机制仍然难以捉摸。我们已经表明，溶物磷脂酸（LPA）刺激了卵巢癌细胞中的ROS产生，并且破坏了这种信号导致生长抑制和凋亡。 RAF-Mek-ERK途径和NF-B似乎是ROS调节的两个主要目标。 NF-B依赖性信号传导是对化学疗法耐药性的主要决定因素。因此，较高水平的ROS是癌细胞的特征，可能刺激抗凋亡蛋白的NF-B依赖性合成。治疗卵巢癌的两种主要化学治疗药物是顺铂和紫杉烷。这两种药物都刺激ROS的产生及其抗塑性作用都会因抗氧化剂而改变。因此，ROS信号传导是细胞控制生长和凋亡的关键决定因素。我们的目标是确定控制这些命运的ROS的目标。我们开发了用于分析半胱氨酸硫酸修饰的新方法，该方法可用于鉴定响应ROS修饰的蛋白质。我们将使用这种方法来确定卵巢癌细胞中的ROS响应信号通路。我们将分析对生长和凋亡信号的响应，我们确定的涉及ROS信号。溶物磷脂酸（LPA）是增生信号传导和顺铂和紫杉烷的脂质介质，是化学治疗剂，通过依赖ROS的机制刺激凋亡。该项目的目的是确定对LPA，顺铂和紫杉烷的反应信号传导如何导致卵巢癌细胞的增殖或凋亡。总体策略是使用导致生长反应或凋亡的治疗条件，并以功能关键的方式确定哪些蛋白质被氧化。这些氧化如何导致NF-KB活性的变化？NF-KB依赖性转录如何影响细胞的命运？具体目的是：1。确定氧化还原状态在控制生长和凋亡中的作用。 2。确定ROS在控制NF-KB依赖性转录调控和对化学疗法的抗性中的作用。 3。确定抗凋亡抗性的ROS信号的下游靶标。我们将使用新开发的试剂来识别和表征导致凋亡或抗凋亡抗性的途径中的蛋白质。这种方法涉及将修饰的蛋白用二聚体的生物素衍生物标记，然后进行抗生物素下拉和通过蛋白质印迹和质谱法分析。我们将使用这种方法来确定卵巢癌细胞中的ROS响应信号通路。总而言之，此处概述的实验将有助于我们理解卵巢癌细胞中ROS如何控制凋亡和细胞生长。这将使人们更好地了解ROS如何在正常生长控制和对化疗的抗性中发挥作用。 公共卫生相关性：活性氧（ROS）是生长因子信号通路和卵巢癌细胞对化学疗法的反应中的第二信使。但是，ROS的分子靶标尚不清楚。这项研究的目的是确定哪些蛋白质是响应生长因子和化学疗法氧化的，并确定这些改变如何控制细胞生长或凋亡。我们将使用新开发的试剂，该试剂与氧化蛋白特别反应，并充当标签，以识别蛋白质靶标的氧化靶标。