Hepatocyte Nox proteins in the pathogenesis of hepatocellular carcinoma

肝细胞Nox蛋白在肝细胞癌发病机制中的作用

• 批准号: 8238851
• 负责人: JINAH CHOI
• 金额: $ 31.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-24 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8238851
• 关键词: 3-nitrotyrosine; Ablation; Cell Culture System; Cell Nucleus; Cells; Chronic; Chronic Hepatitis; Chronic Hepatitis C; Cirrhosis; DNA; DNA Damage; DNA Double Strand Break; Development; Electron Transport; Enzymes; Family; Fibrosis; Generations; Genes; Genetic; Genotype; Goals; HCV Animal Models; Hepatic; Hepatitis C; Hepatitis C virus; Hepatocarcinogenesis; Hepatocyte; Human; Hydrogen Peroxide; Hydroxyl Radical; Individual; Infectious hepatitides; Inflammation; Inflammatory; Knockout Mice; Laboratories; Link; Liver; Liver diseases; Location; Malignant Neoplasms; Modification; Molecular; Mutation; Nitric Oxide; Nox enzyme; Nuclear; Oncogenic; Oxidases; Oxidative Stress; Pathogenesis; Patients; Peroxonitrite; Primary carcinoma of the liver cells; Probability; Production; Proteins; Public Health; Reaction; Reactive Oxygen Species; Role; Secondary to; Signal Transduction; Source; Stress; Transgenic Mice; Transgenic Organisms; Tumor Necrosis Factor-alpha; Virus Diseases; cancer initiation; carcinogenesis; cytokine; design; fibrogenesis; human TGFB1 protein; in vivo; nitrosative stress; oxidative DNA damage; prevent; response; therapy development; toll-like receptor 4; tumor; virus core

DESCRIPTION (provided by applicant): Hepatitis C virus is a major etiologic agent of hepatocellular carcinoma (HCC). HCV-induced HCC is typically preceded by chronic hepatitis and cirrhosis. Studies suggest that HCV has a direct oncogenic potential and increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage that can be mutagenic in the development of cancer. However, the source of reactive oxygen species (ROS) and how HCV induces HCC through oxidative stress, secondary to chronic inflammation/cirrhosis, are still largely unresolved. Recently, our laboratory discovered that hepatocyte NAD(P)H oxidases 1 and 4 (Nox1 and Nox4) are prominent sources of ROS in hepatocytes producing infectious HCV as well as in the HCV-infected human liver. Furthermore, Nox4 was prominent in the nucleus of these cells, particularly in the presence of HCV, and increased the generation of ROS and nitrotyrosine in the nucleus which is likely to increase the probability of reactive species damaging the host nuclear DNA in carcinogenesis. HCV elevated Nox4 through a fibrogenic cytokine, transforming growth factor beta 1. In addition, HCV infection is characterized by chronic, low level of inflammation. Toll-like receptor 4 (TLR4) and tumor necrosis factor alpha (TNF?) are elevated in hepatitis C patients. Nox4 is required for TLR4 signaling, and TNF? can induce Nox4. Thus, hepatocyte Nox4 may serve as a link between inflammation, fibrosis, and cancer during HCV infection. Importantly, the ability to regulate Nox enzymes makes them potential targets for therapy. Nevertheless, the pathogenic consequence of persistent Nox4 induction by HCV and combined oncogenic effects of HCV plus inflammation have not been clearly delineated. Therefore, the goal of this study is to determine the role of Nox enzymes in the HCV- and inflammation-induced DNA damage in hepatocarcinogenesis, focusing on hepatocyte Nox4. The study will employ infectious HCV-producing cell culture systems and an HCV animal model. We hypothesize that hepatocyte Nox4, together with inflammation-driven Noxes and other sources of ROS, increases mutations to the host DNA to facilitate the development of HCC during chronic HCV infection. The specific aims are: 1) to determine the role of hepatocyte Nox1 and Nox4 in the HCV-induced oxidative modification/mutations to the host DNA in hepatocarcinogenesis. Subcellular localization of Nox1 and Nox4 and molecular determinants of perinuclear/nuclear location of Nox4 will continue to be characterized as they relate to the probability of reactive species damaging the host DNA; 2) to determine the role of Nox4 in the spontaneous, HCV-induced hepatic tumors in vivo. HCV transgenic/Nox4 knockout mice will be generated by crossing HCV core transgenic mice with Nox4 knockout mice, which are available, to examine whether the rate of DNA damage and hepatic tumors decreases in the HCV transgenic mice with genetic ablation of nox4 gene; 3) to examine the combined effects of HCV and inflammation on the hepatocyte Nox enzymes, DNA damage, and hepatocarcinogenesis. Results from this study will increase our understanding of hepatocyte Nox4 and the role of inflammation in cancer, for the development of therapy. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a growing public health concern and is estimated to have infected nearly four to five million individuals in the U.S. alone. One of the most devastating consequences of HCV infection is hepatocellular carcinoma, which is still on the rise. This study examines newly described hepatocyte NAD(P)H oxidases (Nox proteins) as a persistent and robust, intra/peri-nuclear source of reactive species, which would promote the DNA damage in response to fibrogenic and pro-inflammatory cytokines increased by HCV, thereby linking chronic hepatitis/fibrogenesis to hepatocarcinogenesis during HCV infection. Results from this study will increase our understanding of HCV, Nox proteins, and the relationship between oxidative stress and HCV-induced liver disease for the development of therapy directed at the source of the reactive species.

描述（由申请人提供）：丙型肝炎病毒是肝细胞癌（HCC）的主要病原体。 HCV 诱发的 HCC 通常先发生慢性肝炎和肝硬化。研究表明，HCV 具有直接致癌潜力，会增加细胞核附近羟基自由基和过氧亚硝酸盐的产生，造成 DNA 损伤，从而在癌症的发展过程中产生突变。然而，活性氧 (ROS) 的来源以及 HCV 如何通过继发于慢性炎症/肝硬化的氧化应激诱发 HCC，在很大程度上仍未得到解决。最近，我们的实验室发现，肝细胞NAD(P)H氧化酶1和4（Nox1和Nox4）是产生感染性HCV的肝细胞以及HCV感染的人类肝脏中ROS的重要来源。此外，Nox4 在这些细胞的细胞核中很突出，特别是在存在 HCV 的情况下，并且增加了细胞核中 ROS 和硝基酪氨酸的生成，这可能会增加活性物质在致癌过程中破坏宿主核 DNA 的可能性。 HCV 通过纤维化细胞因子、转化生长因子 β1 升高 Nox4。此外，HCV 感染的特点是慢性、低水平炎症。丙型肝炎患者中 Toll 样受体 4 (TLR4) 和肿瘤坏死因子 α (TNF?) 升高。 TLR4 信号传导需要 Nox4，而 TNF？可以诱导Nox4。因此，肝细胞Nox4可能是HCV感染过程中炎症、纤维化和癌症之间的联系。重要的是，调节 Nox 酶的能力使它们成为潜在的治疗目标。然而，HCV 持续诱导 Nox4 的致病后果以及 HCV 加炎症的综合致癌作用尚未明确。因此，本研究的目的是确定Nox酶在肝癌发生过程中HCV和炎症诱导的DNA损伤中的作用，重点关注肝细胞Nox4。该研究将采用感染性 HCV 产生细胞培养系统和 HCV 动物模型。我们假设肝细胞 Nox4 与炎症驱动的 Noxes 和其他 ROS 来源一起，增加宿主 DNA 的突变，从而促进慢性 HCV 感染期间 HCC 的发展。具体目标是：1）确定肝细胞Nox1和Nox4在HCV诱导的肝癌发生过程中宿主DNA氧化修饰/突变中的作用。 Nox1 和 Nox4 的亚细胞定位以及 Nox4 核周/核位置的分子决定因素将继续得到表征，因为它们与活性物质破坏宿主 DNA 的概率有关； 2)确定Nox4在体内HCV诱导的自发性肝肿瘤中的作用。将HCV核心转基因小鼠与现有的Nox4敲除小鼠杂交，产生HCV转基因/Nox4敲除小鼠，以检测Nox4基因基因敲除的HCV转基因小鼠的DNA损伤和肝肿瘤发生率是否降低； 3) 检查HCV和炎症对肝细胞Nox酶、DNA损伤和肝癌发生的综合影响。这项研究的结果将增加我们对肝细胞 Nox4 以及炎症在癌症中的作用的了解，以促进治疗的发展。 公共卫生相关性：丙型肝炎病毒 (HCV) 是一个日益严重的公共卫生问题，估计仅在美国就感染了近四到五百万人。 HCV 感染最具破坏性的后果之一是肝细胞癌，其发病率仍在上升。这项研究检查了新描述的肝细胞 NAD(P)H 氧化酶（Nox 蛋白）作为一种持久而强大的核内/核周活性物质来源，它会促进 DNA 损伤，以响应 HCV 增加的纤维形成和促炎细胞因子，从而将慢性肝炎/纤维形成与 HCV 感染期间的肝癌发生联系起来。这项研究的结果将加深我们对 HCV、Nox 蛋白以及氧化应激与 HCV 诱导的肝病之间关系的了解，从而开发针对活性物质来源的治疗方法。