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

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）：恶性神经胶质瘤是最常见和最致命的原发性脑癌，影响每 10 万人中约 5 人。尽管经过数十年的研究和包括手术、放疗和化疗在内的积极治疗，这种癌症的中位生存期仅为约 1 年。为了在这种破坏性神经系统疾病的临床治疗方面取得进展，了解神经胶质瘤独特病理生理学的研究可能会揭示新的治疗靶点。最近的研究表明离子通道与神经胶质瘤细胞积极迁移和增殖的能力有关。更具体地说，某些 K+ 和 Cl- 通道的表达赋予神经胶质瘤细胞同时排出 K+ 和 Cl- 的能力增强，导致必然的水释放和动态细胞体积变化，这对于细胞侵袭和增殖至关重要。神经胶质瘤细胞表达的对该过程至关重要的离子通道之一是 ClC-3，一种电压门控氯离子通道。 ClC-3在神经胶质瘤细胞迁移和增殖中起主要作用，但ClC-3在神经胶质瘤细胞中被激活的机制尚不清楚。多项证据表明 ClC-3 可能受到 Ca2+/钙调蛋白依赖性蛋白激酶 II（一种 Ca2+ 敏感激酶）的调节。鉴于调节神经胶质瘤 Ca2+ 水平的配体和通道在神经胶质瘤迁移和增殖中也发挥着关键作用，CaMKII 对 ClC-3 的调节特别有趣。因此，本研究的目的是了解 CaMKII 的 Ca2+ 激活导致 ClC-3 磷酸化是否会导致胶质瘤细胞迁移和增殖增强。这将通过首先使用全细胞膜片钳电生理学、免疫细胞化学和其他生化测定确定特定目标 1 中的 CaMKII 磷酸化是否增强神经胶质瘤细胞中的 ClC-3 电流来实现。接下来，在具体目标 2 中，将进行成像、基因敲除和电生理学实验，以确定细胞内 Ca2+ 的增加是否通过神经胶质瘤细胞中的 CaMKII 磷酸化激活 ClC-3 电导。最后，Specific Aim 3 旨在确定 CaMKII 介导的 ClC-3 激活是否确实在神经胶质瘤细胞的迁移和增殖中发挥作用。 CaMKII 可能是一种分子翻译器，通过 ClC-3 磷酸化将细胞内 Ca2+ 信号转化为氯离子电导的变化。因此，干扰 ClC-3 活性或 ClC-3 CaMKII 激活的新疗法可能会带来更好的临床结果。事实上，氯毒素（一种氯电流抑制剂）目前正进入治疗恶性神经胶质瘤的 III 期试验。 公共卫生相关性：尽管采取了积极的治疗，多形性胶质母细胞瘤（一种 IV 级原发性脑癌）患者的预后仍然很差。因此，了解胶质母细胞瘤生物学的独特特征将有助于确定新靶点，从而开发治疗药物，从而获得更好的临床结果。