Role of redox state in ovarian cancer response to cisplatin

描述（由申请人提供）：活性氧（ROS）参与控制卵巢癌细胞生长及其对化疗耐药性的信号通路。然而，ROS控制细胞功能的分子机制仍然难以捉摸。我们已经表明，溶血磷脂酸（LPA）刺激卵巢癌细胞中的ROS产生，破坏这种信号传导导致生长抑制和凋亡。Raf-MEK-ERK途径和NF-B似乎是ROS调节的两个主要靶点。NF-B依赖性信号传导是化疗耐药的主要决定因素。因此，作为癌细胞特征的较高水平的ROS可刺激抗凋亡蛋白的NF-B依赖性合成。治疗卵巢癌的两种主要化疗药物是顺铂和紫杉烷。这两种药物都刺激ROS的产生，它们的抗肿瘤作用被抗氧化剂改变。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。我们已经开发了分析半胱氨酸次磺酸修饰的新方法，该方法可用于鉴定响应于ROS的修饰蛋白。我们将使用这种方法来确定卵巢癌细胞中的ROS反应信号通路。我们将分析对生长和凋亡信号的反应，我们已经确定这些信号涉及活性氧信号传导。溶血磷脂酸（LPA）是增殖信号传导的脂质介质，顺铂和紫杉烷是通过ROS依赖性机制刺激细胞凋亡的化疗剂。该项目的目标是确定响应于LPA、顺铂和紫杉烷的ROS依赖性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处理条件，并确定哪些蛋白质以功能关键的方式被氧化。这些氧化是如何导致NF-κ B活性的变化的？NF-κ B依赖性转录是如何影响细胞命运的？具体目标是：1.探讨氧化还原状态在调控细胞生长和凋亡中的作用。2.确定ROS在控制NF-κ B依赖的转录调节和化疗抗性中的作用。3.确定抗凋亡ROS信号的下游靶点。我们将使用我们新开发的试剂来鉴定和表征导致细胞凋亡或抗细胞凋亡的途径中的蛋白质。这种方法包括用双甲酮的生物素衍生物标记修饰的蛋白质，然后用亲和素下拉并通过蛋白质印迹和质谱法进行分析。我们将使用这种方法来确定卵巢癌细胞中的ROS反应信号通路。总之，这里概述的实验将有助于我们理解卵巢癌细胞中ROS如何控制细胞凋亡和细胞生长。这将有助于更好地了解ROS如何在正常生长控制和耐化疗中发挥作用。 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疗反应的第二信使。然而，ROS的分子靶点尚不清楚。这项研究的目的是确定哪些蛋白质在响应生长因子和化疗时被氧化，并确定这些改变如何控制细胞生长或凋亡。我们将使用我们新开发的试剂，这些试剂与氧化蛋白质特异性反应，并作为标签，以识别氧化的蛋白质靶点。

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.