Regulation of ClC-3 in Human Malignant Glioma

ClC-3 在人类恶性胶质瘤中的调控

• 批准号: 8207503
• 负责人: Vishnu Anand Cuddapah
• 金额: $ 3.46万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8207503
• 关键词: Adult; Affect; Automobile Driving; Biochemical; Biological Assay; Biology; Bradykinin Receptor; Brain; Calcium; Calcium Signaling; Cell Proliferation; Cell Shape; Cell Volumes; Cell membrane; Cells; Chloride Channels; Chloride Ion; Chlorides; Chlorotoxin; Clinical; Clinical Management; Clinical Treatment; Co-Immunoprecipitations; Development; Diffuse; Electrophysiology (science); Extracellular Space; Functional disorder; Genetic; Genetic Transcription; Glioblastoma; Glioma; Goals; Homeostasis; Human; Image; Immigration; Ion Channel; Ions; Lead; Ligands; Malignant Epithelial Cell; Malignant Glioma; Malignant Neoplasms; Malignant neoplasm of brain; Measurable; Measures; Mediating; Mitosis; Molecular; Nasopharynx Carcinoma; Operative Surgical Procedures; Outcome; Patients; Phase III Clinical Trials; Phosphorylation; Phosphotransferases; Physical condensation; Play; Process; Proliferating; Radiation therapy; Receptor Activation; Regulation; Research; Role; Shapes; Signal Transduction; Source; Testing; Therapeutic Agents; Translating; Water; calmodulin-dependent protein kinase II; cancer cell; cell motility; chemotherapy; design; immunocytochemistry; inhibitor/antagonist; interest; migration; nervous system disorder; neutrophil; novel; novel therapeutics; outcome forecast; patch clamp; public health relevance; research study; tumor; voltage; Adult; Affect; Automobile Driving; Biochemical; Biological Assay; Biology; Bradykinin Receptor; Brain; Calcium; Calcium Signaling; Cell Proliferation; Cell Shape; Cell Volumes; Cell membrane; Cells; Chloride Channels; Chloride Ion; Chlorides; Chlorotoxin; Clinical; Clinical Management; Clinical Treatment; Co-Immunoprecipitations; Development; Diffuse; Electrophysiology (science); Extracellular Space; Functional disorder; Genetic; Genetic Transcription; Glioblastoma; Glioma; Goals; Homeostasis; Human; Image; Immigration; Ion Channel; Ions; Lead; Ligands; Malignant Epithelial Cell; Malignant Glioma; Malignant Neoplasms; Malignant neoplasm of brain; Measurable; Measures; Mediating; Mitosis; Molecular; Nasopharynx Carcinoma; Operative Surgical Procedures; Outcome; Patients; Phase III Clinical Trials; Phosphorylation; Phosphotransferases; Physical condensation; Play; Process; Proliferating; Radiation therapy; Receptor Activation; Regulation; Research; Role; Shapes; Signal Transduction; Source; Testing; Therapeutic Agents; Translating; Water; calmodulin-dependent protein kinase II; cancer cell; cell motility; chemotherapy; design; immunocytochemistry; inhibitor/antagonist; interest; migration; nervous system disorder; neutrophil; novel; novel therapeutics; outcome forecast; patch clamp; public health relevance; research study; tumor; voltage

DESCRIPTION (provided by applicant): Malignant glioma, the most common and lethal type of primary brain cancer, affects about 5 in 100,000 people. Despite decades of research and aggressive treatments consisting of surgery, radiotherapy, and chemotherapy, the median survival for this cancer is only about 1 year. To make headway in the clinical management of this devastating neurological disease, research to understand the unique pathophysiology of gliomas may reveal new targets for therapy. Recent research has implicated ion channels in the ability of glioma cells to aggressively migrate and proliferate. More specifically, expression of certain K+ and Cl- channels endows glioma cells with an enhanced ability to concomitantly extrude K+ and Cl-, leading to obligated water release and dynamic cell volume change that are essential for cell invasion and proliferation. One of the ion channels expressed by glioma cells critical to this process is ClC-3, a voltage-gated chloride channel. ClC-3 plays a major role in glioma cell migration and proliferation, but the mechanism by which ClC-3 is activated in glioma cells is not known. Several lines of evidence suggest that ClC-3 may be regulated by Ca2+/calmodulin-dependent protein kinase II, a Ca2+-sensitive kinase. Regulation of ClC-3 by CaMKII is particularly interesting, given that ligands and channels regulating glioma Ca2+ levels also play critical roles in glioma migration and proliferation. Therefore the goal of the current study is to understand if Ca2+ activation of CaMKII leading to ClC-3 phosphorylation will lead to enhanced glioma cell migration and proliferation. This will be accomplished by first determining in Specific Aim 1 if ClC-3 currents in glioma cells are enhanced by CaMKII phosphorylation by using whole-cell patch clamp electrophysiology, immunocytochemistry, and other biochemical assays. Next, in Specific Aim 2, imaging, genetic knockdown, and electrophysiological experiments will be performed to determine if increases in intracellular Ca2+ activate ClC-3 conductance via CaMKII phosphorylation in glioma cells. Finally, Specific Aim 3 is designed to determine if CaMKII-mediated activation of ClC-3 actually plays a role in the migration and proliferation of glioma cells. CaMKII may be a molecular translator, converting intracellular Ca2+ signals into changes in chloride conductance via ClC-3 phosphorylation. Therefore novel therapeutics interfering with ClC-3 activity or CaMKII activation of ClC-3 may lead to better clinical outcomes. Indeed, Chlorotoxin, an inhibitor of chloride currents, is currently entering Phase III trials for the treatment of malignant gliomas. PUBLIC HEALTH RELEVANCE: Despite aggressive treatment, the prognosis for patients suffering from glioblastoma multiforme, a Grade IV primary brain cancer, is very poor. Therefore understanding the unique features of glioblastoma biology will lead to the identification of novel targets for the development of therapeutic agents leading to better clinical outcomes.

描述（由申请人提供）：最常见和致命类型的原发性脑癌的恶性神经胶质瘤影响100,000人中约有5人。尽管数十年的研究和包括手术，放疗和化学疗法组成的侵略性疗法，但该癌症的中位生存期仅约1年。为了在这种毁灭性神经系统疾病的临床管理中取得进展，了解神经胶质瘤的独特病理生理学的研究可能揭示了新的治疗靶标。最近的研究暗示了胶质瘤细胞积极迁移和增殖的能力。更具体地说，某些K+和Cl-通道的表达赋予神经胶质瘤细胞具有增强的伴随K+和Cl-的能力，从而导致水释放和动态细胞体积变化，这对于细胞浸润和增殖至关重要。对此过程至关重要的神经胶质瘤细胞表达的离子通道之一是电压门控氯化物通道Clc-3。 CLC-3在神经胶质瘤细胞迁移和增殖中起主要作用，但是在神经胶质瘤细胞中激活CLC-3的机制尚不清楚。几条证据表明，CLC-3可以由Ca2+/钙调蛋白依赖性蛋白激酶II（Ca2+敏感激酶）调节。鉴于调节神经胶质瘤Ca2+水平的配体和通道在神经胶瘤迁移和增殖中也起着关键作用，CAMKII对CLC-3的调节特别有趣。因此，当前研究的目的是了解CA2+ CAMKII是否导致CLC-3磷酸化的CA2+激活是否会导致神经胶质瘤细胞的迁移和增殖增强。如果通过使用全细胞贴片夹电物质学，免疫细胞化学和其他生化测定方法，通过CAMKII磷酸化增强了神经胶质瘤细胞中的CLC-3电流，则首先通过在特定目标1中确定这一点。接下来，将在特定的目标2中进行成像，遗传敲低和电生理实验，以确定细胞内Ca2+中通过CAMKII细胞中的CAMKII磷酸化激活CLC-3电导是否增加。最后，设计特定的目标3旨在确定CAMKII介导的CLC-3的激活是否在神经胶质瘤细胞的迁移和增殖中起作用。 CAMKII可能是分子翻译器，将细胞内Ca2+信号转换为通过CLC-3磷酸化的变化。因此，新的治疗剂会干扰CLC-3活性或CLC-3的CAMKII激活，可能会导致更好的临床结果。实际上，氯毒素是一种氯化物电流的抑制剂，目前正在进入III期试验以治疗恶性神经胶质瘤。 公共卫生相关性：尽管有积极的治疗，但患有多种胶质母细胞瘤的患者（IV级原发性脑癌）的预后非常差。因此，了解胶质母细胞瘤生物学的独特特征将导致鉴定出新的靶标，以开发治疗剂，从而获得更好的临床结局。