THE MOLECULAR TARGET OF GAP JUNCTION ENHANCERS

间隙连接增强剂的分子靶点

• 批准号: 8364918
• 负责人: DUY H HUA
• 金额: $ 12.36万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364918
• 关键词: Ablation; Animals; Attenuated; Binding; Breast Cancer Cell; Cancer Center; Cancer Patient; Cell Communication; Cell Proliferation; Cell Survival; Cells; Clinical; Code; Collaborations; Connexin 43; Connexins; Data; Defect; Disease; Disease Progression; Drug Delivery Systems; EGF gene; Enhancers; Epithelial; Estrogen Therapy; Estrogens; Etiology; Exhibits; Fibroblast Growth Factor; Funding; Gap Junctions; Genes; Goals; Grant; Growth; Growth Factor; Homeostasis; Hormones; Inhibitory Concentration 50; Intercellular Junctions; Ions; Laboratories; Lead; Link; Maintenance; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Mediating; Molecular Target; Molecular Weight; Mus; Names; National Center for Research Resources; Neoplasm Metastasis; Normal Cell; Pathology; Phosphorylation; Principal Investigator; Proteins; RNA; Reporting; Research; Research Infrastructure; Resources; Signal Transduction; Source; Surface; Surface Plasmon Resonance; T47D; Therapeutic; Time; Tissues; Tumorigenicity; United States National Institutes of Health; Woman; Xenograft procedure; animal tissue; antitumor agent; cancer cell; carcinogenesis; cell growth regulation; cost; dalton; drug sensitivity; extracellular; gap junction channel; high throughput screening; intercellular communication; malignant breast neoplasm; mortality; neoplastic; neoplastic cell; novel; quinoline; small molecule; therapy development; trafficking; tumor; tumor growth; ubiquitin-protein ligase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Breast cancer is the most common cancer in women worldwide and mortality from breast cancer is consistent due to tumor metastasis. Breast cancer patients initially respond to estrogen ablation therapy, but estrogen-independent cells almost always aggressively emerge. The disease eventually progresses to estrogen-independent breast cancer. Tumor is no longer responsive to estrogen ablation therapy and unrestrained progression of the disease is inevitable. Progress in understanding the etiology of this disease and developing therapies has been slow due to multiple deregulations of various genes. The additive effects against mammary tumor cells might be achieved by combining antitumor agents directed against one or more altered mechanisms in cancer. Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). Cell communication is important in the cooperation between neighboring cells. It is mediated through extracellular signals such as hormones (i.e. estrogen) and growth factors (i.e. IGF, EGF, and FGF), or through cell to cell interaction between adjacent cells. One type of cell communication is gap junctional intercellular communication (GJIC), considered to be of fundamental importance. Gap junction (GJ) channels allow the transfer of small molecules, which involve in the regulation of cell growth, differentiation, and function. Gap junctions are the only communicating junctions found in animal tissues, in all species, which are responsible for the direct traffic of ions and molecules with molecular weights less than 1,200 Daltons. These traffic ways are formed by the interaction between two hemichannels on the surface of opposing cells. These hemichannels are formed by the association of six proteins, the connexins. Because of the importance of intercellular junctions in the maintenance of the cellular homeostasis, the modulation of intercellular junctions and expression of connexin is involved in carcinogenesis. Most normal cells have functional GJIC, while most, if not all, tumors cells have dysfunctional GJIC. It is believed that restoring GJIC is linked to drug sensitivity and reduction of tumorigenicity. Thus, increasing gap junction activity or enhancing GJIC in tumor cells provides the targets to enhance anti-neoplastic therapies. Several GJIC enhancers have been reported; however, an effective clinical drug targeting gap junction is not available at this time. Recently, we synthesized a new class of substituted quinolines (code name: PQ) and found that they possess potent inhibitory activities against T47D breast cancer cells (IC50 value of PQ7 is 16 nM and PQ1 is 119 nM) through the enhancement of GJIC. Our data showed that PQ1 significantly increases gap junction activity and inhibits cell viability and colony growth of T47D breast cancer cells. Moreover, PQ1 and PQ7 decrease 71% and 99%, respectively, of xenograft breast tumors bearing mice. Interestingly, PQ-treated animals have a normal histological pathology. Furthermore, PQ1 and PQ7 have no effect on normal primary epithelial mammary cells. We have found that PQ1 has a very strong binding with Nedd4 (from surface plasmon resonance studies), an E3 ubiquitin ligase, and decreases the interaction of Nedd4 and connexin 43 (Cx43) and the phosphorylation of Cx43, which lead to the enhancement of GJIC. Thus, the principle hypothesis of this proposal is that PQs can 1) increase gap junction activities, 2) inhibit the interaction of Nedd4 and Cx43 and phosphorylation of Cx43, and 3) attenuate tumor growth. The goal of this application is to identify the molecular target(s) of this novel class of molecules through Si RNA high-throughput screening and study the morphological changes of cells treated with PQs through a collaboration with Dr. Rathnam Chaguturu at the KU High-Throughput Screening Laboratory.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 乳腺癌是全世界女性最常见的癌症，由于肿瘤转移，乳腺癌的死亡率是一致的。乳腺癌患者最初对雌激素消融治疗有反应，但雌激素非依赖性细胞几乎总是积极出现。这种疾病最终会发展为雌激素非依赖性乳腺癌。肿瘤对雌激素消融治疗不再有反应，疾病的无节制发展是不可避免的。由于各种基因的多重失控，在了解这种疾病的病因和开发治疗方法方面进展缓慢。抗乳腺肿瘤细胞的相加效应可能是通过联合针对癌症中的一个或多个改变机制的抗肿瘤药物来实现的。 癌细胞在细胞通讯方面表现出许多缺陷，导致组织内稳态的丧失(细胞过度增殖、侵袭和转移)。在相邻小区之间的协作中，小区通信是非常重要的。它通过激素(如雌激素)和生长因子(如IGF、EGF和FGF)等细胞外信号或通过相邻细胞之间的细胞间相互作用来调节。细胞通讯的一种类型是缝隙连接细胞间通讯(GJIC)，被认为是至关重要的。缝隙连接(GJ)通道允许小分子的转运，参与细胞生长、分化和功能的调节。缝隙连接是在所有物种的动物组织中发现的唯一通讯连接，负责分子量小于1,200道尔顿的离子和分子的直接运输。这些交通方式是由相对细胞表面的两个半通道相互作用形成的。这些半突是由六种蛋白质--连接蛋白结合而成的。由于细胞间连接在维持细胞内环境平衡中的重要作用，细胞间连接的调节和连接蛋白的表达参与了肿瘤的发生。 大多数正常细胞具有GJIC功能，而大多数(如果不是全部)肿瘤细胞GJIC功能障碍。据认为，恢复GJIC与药物敏感性和降低致瘤性有关。因此，增加肿瘤细胞的缝隙连接活性或增强GJIC为加强抗肿瘤治疗提供了靶点。已经报道了几种GJIC增强剂；然而，目前还没有一种有效的针对缝隙连接的临床药物。最近，我们合成了一类新的取代喹啉类化合物(代号：PQ)，通过增强GJIC发现它们对T47D乳腺癌细胞具有很强的抑制活性(PQ7的IC50值为16 nM，PQ1的IC50值为119 nM)。我们的数据显示，PQ1显著增加了T47D乳腺癌细胞的缝隙连接活性，抑制了细胞活力和集落生长。此外，PQ1和PQ7分别使荷瘤小鼠的PQ1和PQ7下降了71%和99%。有趣的是，PQ处理的动物有正常的组织病理学。此外，PQ1和PQ7对正常乳腺原代上皮细胞没有影响。我们发现PQ1与Nedd4(一种E3泛素连接酶)有很强的结合，并减少了Nedd4与连接蛋白43(Cx43)的相互作用和Cx43的磷酸化，从而导致GJIC的增强。因此，这一建议的基本假设是PQS可以1)增加缝隙连接活性，2)抑制Nedd4和Cx43的相互作用和Cx43的磷酸化，3)抑制肿瘤生长。这项应用的目标是通过Si RNA高通量筛选确定这类新型分子的分子靶标(S)，并与KU高通量筛选实验室的Rathnam Chguturu博士合作，研究PQS处理后细胞的形态变化。