Indolecarbinol target proteins and anti-cancer signaling in human melanoma cells

吲哚甲醇靶蛋白和人黑色素瘤细胞中的抗癌信号传导

基本信息

批准号：
8624542
负责人：
GARY L FIRESTONE
金额：
$ 21.38万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-16 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8624542
关键词：
26S proteasome 3-Dimensional Adverse effects Amino Acids Apoptosis Apoptotic Aspirin Attenuated Binding Binding Proteins Biological Assay Brassica Broccoli - dietary Cabbage - dietary Cancer Cell Growth Cell Cycle Arrest Cell Cycle Regulation Cell Survival Cells Computer Simulation Cyclin D1 Dominant-Negative Mutation Down-Regulation Drug Combinations Drug resistance Elastases Electrostatics Exhibits Foundations Gene Targeting Growth Hormone Receptor Human In Vitro Indole-3-Carbinol Indoles Lead Lipids Malignant Neoplasms Maps Mediating Melanoma Cell Molecular Nuclear Translocation Nude Mice Pathway interactions Phenotype Phosphorylation Physiological Phytochemical Property Protein phosphatase Proteins RNA Resistance Serine Protease Shapes Signal Transduction Site Skin Cancer Specificity Structure Testing Therapeutic Toxic effect Tumor Suppressor Proteins Ubiquitination Vegetables base cancer cell cancer therapy cell motility cell type chemotherapeutic agent cost cruciferous vegetable design in vivo knock-down melanoma novel pre-clinical prevent protein degradation protein function reproductive response simulation transcription factor tumor tumor growth tumor xenograft ubiquitin ligase ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Indole-3-carbinol (I3C), an indolecarbinol compound obtained from Brassica vegetables, such as broccoli, cabbage and Brussels sprouts, exhibits potent anti-proliferative properties in a wide range of human cancers with negligible levels of toxicity or side effects. We have documented that I3C, and its more potent and stable derivative 1-benzyl-I3C, triggers complementary sets of transcriptional, cell signaling, and enzymatic cascades that control cancer cell growth, apoptosis, cell migration and in vivo tumor growth of human cancer cells. Our studies originally established the serine protease elastase as the first known I3C target protein in human reproductive cancer cells, which has provided a crucial experimental foundation to define the mechanisms of indolecarbinol anti-proliferative signaling in other cancer cell types. In human melanoma cells, we have observed that I3C induces a G1 cell cycle arrest and inhibits cell survival signaling by stabilizing the PTEN tumor suppressor protein levels, which through its dual lipid/protein phosphatase activity then disrupts the Akt signaling network including the inactivation of NFkB transcriptional activity and attenuated expression of NFkB target genes such as cyclin D1. I3C triggers this response by inhibiting the NEDD4-1 mediated ubiquitination of PTEN, and in silico 3-D simulations using the crystallographic structure NEDD4-1 predicts that I3C specificity interacts with the HECT domain of NEDD4-1, which is the domain responsible for the E3 ubiquitin ligase activity. This proposal will test the hypothesis that that the NEDD4-1 E3 ubiquitin ligase, which selectively ubiquitinates and signals the 26S proteasome degradation of PTEN suppressor protein, is directly inhibited by I3C and represents a new indolecarbinol target protein that mediates I3C anti-proliferative responsiveness of human melanoma cells. One aim of this proposal will utilize in vitro ubiquitination, I3C binding and protein (or domain) interaction assays to determine the mechanism by which I3C inhibits the NEDD4-1 E3 ubiquitin ligase activity. A mutagenic strategy will alter the in silico defined I3C interaction site to define the precise amino acid requirements for I3C binding and generate novel I3C-resistant forms of NEDD4-1. The second aim will functionally characterize I3C activated anti-proliferative and pro-apoptotic cascades due to PTEN protein stabilization in human melanomas cells with distinct phenotypes. Also, more potent and selective derivatives of I3C will be identified based on their interactions with NEDD4-1 and by their ability to stabilize PTEN protein and disrupt Akt/NFkB signaling. The third aim will characterize the anti-cancer effects in melanoma cells of combinations of indolecarbinol compounds and aspirin in both cellular and in vivo contexts. Our proposed studies will establish the preclinical foundation that is needed to eventually develop novel and low cost I3C-based therapeutic strategies for human skin cancers.

描述（由申请人提供）：吲哚-3-甲醇（I3C），一种从甘蓝蔬菜中获得的吲哚核酸盐化合物，例如西兰花，卷心菜和布鲁塞尔新芽，在毒性或毒性或副作用的可忽视水平中具有有效的抗增殖性能。我们已经证明，I3C及其更有效，更稳定的衍生物1-烯唑-I3C，触发了转录，细胞信号传导和酶促级联反应的互补集，可控制癌细胞生长，细胞迁移，细胞迁移和人类癌细胞的体内肿瘤生长。我们的研究最初将丝氨酸蛋白酶弹性蛋白酶确定为人类生殖癌细胞中首个已知的I3C靶蛋白，该癌细胞为定义其他癌细胞类型中吲哚核酸棒醇抗增殖信号的机制提供了重要的实验基础。在人黑色素瘤细胞中，我们观察到I3C通过稳定PTEN肿瘤抑制蛋白水平诱导G1细胞周期停滞并抑制细胞存活信号传导，通过其双脂质/蛋白磷酸酶活性，该水平会破坏Akt信号网络，包括NFKB转录活性和nfkkk的nfkken and cent anf nfkkkkkkk的表达，并破坏Akt信号网络。 I3C通过抑制NEDD4-1介导的PTEN泛素化，以及使用晶体学结构NEDD4-1预测I3C特异性与NEDD4-1的Hect域相互作用，从而触发了这一响应，这是负责E3 Ubiquitin ligase活动的领域。该提议将检验以下假设：NEDD4-1 E3泛素连接酶有选择地泛素化并指示PTEN抑制蛋白的26S蛋白酶体降解，它直接抑制了I3C，代表了一种新的吲哚核酸糖靶蛋白，可介导I3C抗蛋白酶抗性的人类酸性蛋白质的新型吲哚酚靶蛋白。该建议的目的之一将利用体外泛素化，I3C结合和蛋白质（或域）相互作用测定法来确定I3C抑制NEDD4-1 E3 E3泛素连接酶活性的机制。一种诱变策略将改变硅定义的I3C相互作用位点，以定义I3C结合的精确氨基酸需求，并生成新颖的NEDD4-1抗I3C耐药形式。第二个目标将在功能上表征I3C激活的抗增殖性和促凋亡级联反应，这是由于PTEN蛋白质稳定在人黑色素瘤细胞中具有不同表型的稳定。同样，I3C的更有效和选择性衍生物将根据其与NEDD4-1的相互作用以及它们稳定PTEN蛋白和破坏AKT/NFKB信号传导的能力来鉴定。第三个目的将表征吲哚核酸化合物和阿司匹尼在细胞和体内环境中的抗癌细胞中的抗癌作用。我们提出的研究将建立临床前的基础，以最终为人类皮肤癌的基于I3C的新颖和低成本的治疗策略。