Role of redox state in ovarian cancer response to cisplatin

描述(由申请人提供)：活性氧物种(ROS)与控制卵巢癌细胞生长及其对化疗的耐药性的信号通路有关。然而，ROS控制细胞功能的分子机制仍然不清楚。我们已经证明，溶血磷脂酸(LPA)刺激卵巢癌细胞产生ROS，破坏这一信号转导会导致生长抑制和细胞凋亡。Raf-MEK-ERK通路和NF-B似乎是ROS调控的两个主要靶点。核因子-B依赖的信号转导是化疗耐药的主要决定因素。因此，更高水平的ROS，这是癌细胞的特征，可能会刺激依赖于核因子-B的抗凋亡蛋白的合成。治疗卵巢癌的两种主要化疗药物是顺铂和紫杉烷。这两种药物都能刺激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。我们开发了分析半胱氨酸磺酸修饰的新方法，可以用来识别响应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ROS反应的信号通路。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ROS信号。溶血磷脂酸(LPA)是增殖信号的脂质介质，顺铂和紫杉烷是通过ROS依赖机制刺激细胞凋亡的化疗药物。该项目的目标是确定LPA、顺铂和紫杉烷反应中依赖ROS的信号如何导致卵巢癌细胞的增殖或凋亡。总体策略是利用导致生长反应或凋亡的治疗条件，并确定哪些蛋白质在功能上是关键的氧化方式。这些氧化如何导致核因子-kB活性的变化，以及依赖核因子-kB的转录如何影响细胞的命运？其具体目的是：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在控制核因子-kB依赖的转录调控和化疗耐药中的作用。3.确定ROS信号在抗细胞凋亡中的下游靶点。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抵抗细胞凋亡的途径中的蛋白质。这种方法包括用生物素衍生物二美酮标记修饰的蛋白质，然后亲和素下拉并通过蛋白质印迹和质谱学进行分析。我们将使用这种方法来确定卵巢癌细胞中ROS反应的信号通路。总之，这里概述的实验将有助于我们理解ROS是如何控制卵巢癌细胞的凋亡和细胞生长的。这将有助于更好地了解ROS如何在正常生长控制和化疗耐药中发挥作用。

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.