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: 8676718
• 负责人: Donna D Zhang
• 金额: $ 30.2万
• 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/8676718
• 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; MDA MB 231; 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. ! "!

描述（由申请人提供）：转录因子Nrf 2通过上调编码抗氧化酶、解毒酶、外源性物质转运蛋白和应激反应蛋白的抗氧化反应元件（ARE）携带基因，成为细胞保护机制的主要调节因子。Keap 1是一种基于Cullin 3（Cul 3）的E3泛素连接酶的底物衔接蛋白，严格调节Nrf 2-ARE信号通路。在基础条件下，Nrf 2不断靶向Keap 1介导的泛素化和随后的蛋白酶体降解，以维持人体器官中的低组成水平。在激活该途径后，Keap 1-Cul 3-Rbx 1 E3连接酶的酶活性被抑制，导致Nrf 2的稳定和Nrf 2下游基因的激活。自1999年发现Nrf 2-Keap 1-ARE信号通路以来，Nrf 2一直被认为是一种“好”的转录因子，可以保护我们免受氧化应激相关疾病的影响，包括癌症。Nrf 2的化学预防性质已通过以下两个事实得到充分证明：（i）许多充分研究的化学预防化合物通过活化Nrf 2-ARE信号传导途径引发其活性，和（ii）Nrf 2缺失小鼠对化学致癌物高度敏感并且不再受化学预防化合物保护。讽刺的是，Nrf 2的“黑暗面”最近被揭露出来。例如，已经在几种类型的肿瘤和癌细胞系，特别是非小细胞肺癌（NSCLC）中鉴定了破坏Keap 1介导的Nrf 2负调控的体细胞突变，导致高组成性水平的Nrf 2。此外，越来越多的证据表明，Nrf 2有助于化疗耐药性，这是癌症治疗的主要障碍。Nrf 2“黑暗面”的发现清楚地说明了迫切需要鉴定Nrf 2抑制剂并将其开发成可药用化合物以提高癌症治疗的疗效。我们已经筛选了大量的天然产物的ARE-荧光素酶活性的抑制使用在我们的实验室，MDA-MB-231-ARE-Luc，含有一个ARE（从GST-Ya）依赖性荧光素酶基因建立的稳定的细胞系。使用这种方法，我们已经鉴定了能够抑制ARE-荧光素酶活性的植物提取物。此外，已从提取物中分离出纯化合物brusatol，并发现其抑制Nrf 2的蛋白质水平并表现出有效的抗癌活性。到目前为止，我们已经获得了大量的初步数据表明，布鲁沙托在体外使几种癌细胞系对化疗药物敏感，更重要的是，布鲁沙托在体内以Nrf 2依赖性方式使肺癌异种移植物对顺铂敏感。基于布鲁沙醇特异性抑制Nrf 2并使培养的癌细胞和异种移植物对顺铂治疗敏感的能力，我们假设布鲁沙醇可以通过抑制Nrf 2依赖性保护机制使癌细胞对化疗药物敏感来增强当前癌症治疗的疗效。拟议研究的目标是使用临床前肺癌模型进一步表征布鲁沙醇的抗癌特性，并描述布鲁沙醇的分子靶点和机制作用。这项拟议的研究不仅将为将这种Nrf 2抑制剂开发成治疗药物以对抗化疗耐药性提供框架，还将为该领域的基础研究提供第一种Nrf 2抑制剂，这两者都将对全球人类健康产生深远影响。因此，将追求以下三个目标：目标1。表征布鲁沙托介导的Nrf 2抑制作用的机制Nrf 2主要由Keap 1-Cul 3-Rbx 1 E3连接酶通过泛素化和降解在蛋白水平上调节。因此，我们将研究布鲁沙醇对可能增强E3连接酶活性的蛋白质的影响，例如E3连接酶的蛋白质亚基，以及控制连接酶复合物动态组装/拆卸的调节蛋白。目标2。确定布鲁沙醇的分子靶点将对布鲁沙醇的靶蛋白进行鉴定和验证。这些蛋白质的生物学功能，特别是它们与Nrf 2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Keap 1-Cul 3-Rbx 1 E3连接酶。目标3。使用LSL-KrasG 12 D/+小鼠肺癌模型评估布鲁沙醇的体内疗效将在该临床前模型中测试使用布鲁沙醇作为佐剂以增强当前癌症治疗并对抗内在和获得性耐药的可行性，该模型重现了人类肺癌的发展和进展。! "!