Investigation of an anti-cancer phytochemical targeting Nrf2

针对 Nrf2 的抗癌植物化学物质的研究

• 批准号: 8181521
• 负责人: Donna D Zhang
• 金额: $ 30.28万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8181521
• 关键词: Adaptor Signaling Protein; Adjuvant; Antioxidants; Basic Science; Biological Factors; Biological Process; Cancer Etiology; Cancer Model; Cancer cell line; Cessation of life; Chemopreventive Agent; Cisplatin; Complex; Data; Defense Mechanisms; Development; Disease; Effectiveness; Enzymes; Exhibits; Genes; Goals; Health; Human; In Vitro; Investigation; Knockout Mice; Ligase; Luc Gene; Luciferases; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Methods; Molecular Target; Non-Small-Cell Lung Carcinoma; Organ; Oxidative Stress; Pathway interactions; Pharmaceutical Preparations; Phytochemical; Plant Extracts; Pre-Clinical Model; Property; Protein Subunits; Proteins; Regulation; Research; Resistance; Response Elements; Side; Signal Pathway; Somatic Mutation; Testing; Therapeutic; Toxic Environmental Substances; Ubiquitination; Xenobiotics; Xenograft procedure; base; biological adaptation to stress; cancer cell; cancer therapy; chemical carcinogen; combat; coping; genetic regulatory protein; in vivo; inhibitor/antagonist; novel; pre-clinical; stable cell line; transcription factor; tumor; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The transcription factor Nrf2 has emerged as a master regulator of a cellular protective mechanism by upregulating antioxidant response element (ARE)-bearing genes encoding antioxidant enzymes, detoxifying enzymes, xenobiotic transporters, and stress response proteins. Keap1, a substrate adaptor protein for a Cullin3 (Cul3)-based E3 ubiquitin ligase, tightly regulates the Nrf2-ARE signaling pathway. Under basal conditions, Nrf2 is constantly targeted for Keap1-mediated ubiquitination and subsequent proteasomal degradation to maintain a low constitutive level in al human organs. Upon activation of the pathway, the enzymatic activity of the Keap1-Cul3-Rbx1 E3 ligase is inhibited, resulting in stabilization of Nrf2 and activation of Nrf2 downstream genes. Since the discovery of the Nrf2-Keap1-ARE signaling pathway in 1999, Nrf2 has been viewed as a "good" transcription factor that protects us from oxidative stress-related diseases, including cancer. The chemopreventive property of Nrf2 has been well documented by the following two facts: (i) many of the well- studied chemopreventive compounds elicit their activities through activation of the Nrf2-ARE signaling pathway, and (ii) Nrf2-null mice are highly susceptible to chemical carcinogens and are no longer protected by chemopreventive compounds. Paradoxically, the "dark side" of Nrf2 has recently been revealed. For instance, somatic mutations that disrupt the Keap1-mediated negative regulation of Nrf2, resulting in a high constitutive level of Nrf2, have been identified in several types of tumors and cancer cel lines, especially non-small cell lung carcinoma (NSCLC). Furthermore, mounting evidence has emerged, indicating that Nrf2 contributes to chemoresistance, the major obstacle in cancer treatment. The discovery of the "dark side" of Nrf2 has clearly illustrated the urgent need to identify Nrf2 inhibitors and develop them into druggable compounds to enhance the efficacy of cancer treatments. We have screened a large number of natural products for their inhibition of ARE-luciferase activity using a stable cell line established in our lab, MDA-MB-231-ARE-Luc, containing an ARE (from GST-Ya)-dependent luciferase gene. Using this method, we have identified a plant extract that is able to inhibit ARE-luciferase activity. Furthermore, a pure compound, brusatol, has been isolated from the extract and has been found to inhibit the protein level of Nrf2 and exhibit potent anti-cancer activities. So far, we have obtained a substantial amount of preliminary data demonstrating that brusatol sensitizes several cancer cell lines to chemotherapeutic drugs in vitro and, more significantly, brusatol sensitizes lung cancer xenografts to cisplatin in vivo in an Nrf2-dependent manner. Based on the ability of brusatol to specifically inhibit Nrf2 and sensitize cultured cancer cells and xenografts to cisplatin treatments, we hypothesize that brusatol can enhance the efficacy of current cancer treatments by sensitizing cancer cells to chemotherapeutic drugs through inhibition of the Nrf2-dependant protective mechanism. The goal of the proposed research is to further characterize the anti-cancer properties of brusatol using a preclinical lung cancer model and delineate the molecular targets and mechanistic actions of brusatol. The proposed study will not only provide a framework for the development of this Nrf2 inhibitor into a therapeutic drug to combat chemoresistance, but also provide the first Nrf2 inhibitor for basic research in the field, both of which will have profound impacts on human health worldwide. Therefore, the following three aims will be pursued: Aim #1. Characterize the mechanistic actions of brusatol-mediated Nrf2 inhibition Nrf2 is primarily regulated by the Keap1-Cul3-Rbx1 E3 ligase at the protein level through ubiquitination and degradation. Therefore, we will investigate the effects of brusatol on the proteins that may enhance the activity of the E3 ligase, such as the protein subunits of the E3 ligase, as well as regulatory proteins that control the dynamic assembly/disassembly of the ligase complex. Aim #2. Determine the molecular targets of brusatol The target proteins of brusatol will be identified and verified. The biological functions of these proteins, in particular their crosstalk with the Nrf2 signaling pathway, wil be investigated. Most likely, these proteins will directly or indirectly regulate the Keap1-Cul3-Rbx1 E3 ligase. Aim #3. Evaluate the in vivo efficacy of brusatol using an LSL-KrasG12D/+ mouselung cancer model The feasibility of using brusatol as an adjuvant to enhance current cancer treatments and to combat both intrinsic and acquired resistance will be tested in this preclinical model that recapitulates the development and progression of human lung cancer. ! "! PUBLIC HEALTH RELEVANCE: Lung Cancer is the leading cause of cancer-related death worldwide. Little progress has been made in the treatment of lung cancer due to high resistance of lung cancer cells to chemotherapeutic treatments. Nrf2 is a protein that regulates one of the most important cellular defense mechanisms to cope with environmental toxins and chemotherapeutic drugs. Therefore, inhibition of Nrf2 represents a novel mechanism to sensitize cancer cells to chemotherapeutic drugs. This proposal focuses on the discovery and development of a phytochemical, brusatol, into a druggable compound that inhibits the Nrf2- mediated protective pathway, enhancing the effectiveness of a broad range of cancer treatments. In addition, mechanistic actions of brusatol will also be investigated.

描述(由申请人提供)：转录因子Nrf2通过上调携带抗氧化剂反应元件(ARE)的基因，编码抗氧化酶、解毒酶、外源转运体和应激反应蛋白，成为细胞保护机制的主要调节因子。Keap1是一种基于CUL3的E3泛素连接酶的底物适配蛋白，严格调控Nrf2-ARE信号通路。在基础条件下，Nrf2总是被Keap1介导的泛素化和随后的蛋白酶体降解作为靶点，以维持人体器官中的低组成水平。一旦激活该途径，Keap1-CUL3-Rbx1 E3连接酶的酶活性被抑制，导致Nrf2的稳定和Nrf2下游基因的激活。自从1999年发现Nrf2-Keap1-ARE信号通路以来，Nrf2一直被认为是一种“良好的”转录因子，可以保护我们免受氧化应激相关疾病的侵袭，包括癌症。Nrf2的化学预防作用已经被以下两个事实很好地证明：(I)许多研究很好的化学预防化合物通过激活Nrf2-ARE信号通路来激发它们的活性，以及(Ii)Nrf2基因缺失的小鼠对化学致癌物高度敏感，不再受到化学预防化合物的保护。矛盾的是，NRF2的“阴暗面”最近被揭露了。例如，在几种类型的肿瘤和癌细胞系中，已经发现了破坏Keap1介导的Nrf2负调控的体细胞突变，导致Nrf2的高组成水平，特别是非小细胞肺癌(NSCLC)。此外，越来越多的证据表明，Nrf2导致了化疗耐药，这是癌症治疗的主要障碍。Nrf2黑暗面的发现清楚地表明，迫切需要识别Nrf2抑制剂，并将其开发成可药物化合物，以增强癌症治疗的疗效。我们利用我们实验室建立的稳定的细胞系MDA-MB-231-ARE-LUC，筛选了大量天然产物对ARE-荧光素酶活性的抑制作用，该细胞系含有依赖于ARE(来自GST-Ya)的荧光素酶基因。利用这种方法，我们已经鉴定出一种能够抑制ARE-荧光素酶活性的植物提取物。此外，从提取物中分离出一种纯化合物Brusatol，并发现它能抑制Nrf2的蛋白水平，并显示出强大的抗癌活性。到目前为止，我们已经获得了大量的初步数据表明，灯盏花醇在体外使几种癌细胞对化疗药物增敏，更重要的是，灯盏花醇在体内以Nrf2依赖的方式增敏肺癌异种移植瘤对顺铂的敏感性。基于Brusatol特异性抑制Nrf2并使培养的癌细胞和异种移植瘤对顺铂治疗增敏的能力，我们假设Brusatol可以通过抑制Nrf2依赖的保护机制使癌细胞对化疗药物增敏来增强当前癌症治疗的疗效。这项研究的目的是利用临床前肺癌模型进一步表征灯盏花醇的抗癌特性，并描绘灯盏花醇的分子靶点和作用机制。这项拟议的研究不仅将为将这种Nrf2抑制剂开发成对抗化疗耐药的治疗药物提供框架，还将为该领域的基础研究提供第一个Nrf2抑制剂，这两项研究都将对全球人类健康产生深远影响。因此，我们将致力于以下三个方面的研究：目的1.研究Brusatol介导的Nrf2抑制作用的机制，Nrf2主要由Keap1-CUL3-Rbx1 E3连接酶在蛋白质水平上通过泛素化和降解来调节。因此，我们将研究Brusatol对可能增强E3连接酶活性的蛋白质的影响，如E3连接酶的蛋白亚基，以及控制连接酶复合体动态组装/拆卸的调节蛋白。目的#2.确定鸦胆子醇的分子靶标，以鉴定和验证鸦胆子醇的靶蛋白。这些蛋白质的生物学功能，特别是它们与Nrf2信号通路的串扰，将被研究。最有可能的是，这些蛋白质将直接或间接调节Keap1-CUL3-Rbx1 E3连接酶。目的#3.用LSL-KrasG12D/+小鼠肺癌模型评价Brusatol的体内疗效使用Brusatol作为佐剂来加强目前的癌症治疗并对抗固有和获得性耐药的可行性将在这个概括人类肺癌发展和进展的临床前模型中进行测试。！“！ 公共卫生相关性：肺癌是全球癌症相关死亡的主要原因。由于肺癌细胞对化疗药物的高耐药性，肺癌的治疗进展甚微。Nrf2是一种蛋白质，调节着应对环境毒素和化疗药物的最重要的细胞防御机制之一。因此，抑制Nrf2代表了一种使癌细胞对化疗药物增敏的新机制。这项提议的重点是发现和开发一种植物化学物质布鲁索醇，使其成为一种可药物化合物，抑制Nrf2介导的保护途径，增强广泛癌症治疗的有效性。此外，还将对鸦胆子醇的机械作用进行研究。