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: 8494596
• 负责人: Donna D Zhang
• 金额: $ 29.27万
• 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/8494596
• 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. ! "!

描述（由申请人提供）：转录因子NRF2已通过上调编码抗氧化剂的抗氧化剂反应元件（IS）的基因来作为细胞保护机制的主要调节剂，该基因编码了抗氧化剂，解毒酶，酶，异生物元素转运蛋白和压力反应蛋白。 KEAP1是Cullin3（CUL3）基于E3泛素连接酶的底物衔接蛋白，严格调节NRF2-ARE信号通路。在基础条件下，NRF2不断针对KEAP1介导的泛素化和随后的蛋白酶体降解，以保持人体器官中低的本构水平。途径激活后，抑制了Keap1-Cul3-RBx1 E3连接酶的酶促活性，从而导致NRF2稳定和NRF2下游基因的激活。自1999年发现NRF2-KEAP1-ARE信号通路以来，NRF2被视为“良好”转录因子，可保护我们免受包括氧化应激相关疾病的侵害，包括癌症。以下两个事实已经充分证明了NRF2的化学预防特性：（i）通过激活NRF2-ARE信号传导途径，许多经过良好研究的化学预防化合物引起了它们的活动，并且（ii）NRF2-NULL小鼠非常易于化学碳而受到化学癌的易受保护的化合物，并且不再受到化学碳的保护。矛盾的是，NRF2的“阴暗面”最近被揭示。例如，在几种类型的肿瘤和癌症系，尤其是非小细胞细胞肺癌（NSCLC）中鉴定出了破坏NRF2的KEAP1介导的NRF2负调控的体细胞突变。此外，出现了越来越多的证据，表明NRF2有助于化学抗性，这是癌症治疗的主要障碍。 NRF2的“阴暗面”的发现清楚地说明了迫切需要鉴定NRF2抑制剂并将其发展为可药物的化合物，以增强癌症治疗的功效。我们使用实验室中建立的稳定细胞系MDA-MB-231-ARE-LUC筛选了大量天然产物，以抑制其对L-Luciferase活性的抑制作用，其中包含Ane（来自GST-YA）依赖性的荧光素酶基因。使用这种方法，我们已经确定了能够抑制葡萄酸酶活性的植物提取物。此外，已从提取物中分离出一种纯化合物Brusatol，并被发现可以抑制NRF2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Brusatol在体外对化学治疗药物的几个癌细胞敏感度敏感，并且更重要的是，Brusatol在NRF2依赖于NRF2依赖性的方式中使肺癌的异胞植物对肺癌的异位移植物在体内敏感。基于brusatol特异性抑制NRF2并将培养的癌细胞和异种移植物敏化的能力，我们假设Brusatol可以通过抑制NRF2依赖性的保护机制来抑制化学治疗药物，从而通过使癌细胞对化学治疗药物的敏感性提高当前癌细胞的疗效。拟议的研究的目的是使用临床前肺癌模型进一步表征Brusatol的抗癌特性，并描述Brusatol的分子靶标和机械作用。拟议的研究不仅将为将该NRF2抑制剂开发到治疗药物中以对抗化学抗性，还为该领域的基础研究提供了第一个NRF2抑制剂，这两种抑制剂都将对全球人类健康产生深远的影响。因此，将追求以下三个目标：目标＃1。表征布鲁萨尔介导的NRF2抑制NRF2的机械作用主要由蛋白质水平的KEAP1-CUL3-RBX1 E3连接酶通过泛素化和降解来调节。因此，我们将研究布鲁萨尔对可能增强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的动态组装/拆卸的调节蛋白。目标＃2。确定布鲁萨尔的分子靶标将确定和验证布鲁萨尔的靶蛋白。这些蛋白质的生物学功能，尤其是它们与NRF2信号通路的串扰。这些蛋白最有可能直接或间接调节KEAP1-CUL3-RBX1 E3连接酶。目标＃3。使用LSL-KRASG12D/+ Mouselung Cancer模型评估Brusatol的体内功效，将使用Brusatol作为增强当前癌症治疗的辅助药物的可行性将在这种临床上的临床上进行测试，以重新调节人类Lung lung lung cancer的发育和进展。呢“！