Na-K-Cl cotransporter in Glioblastoma Multiforme

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是世界卫生组织IV级癌症，是恶性程度最高的胶质瘤，中位生存时间不到1年。联合替莫唑胺（TMZ）介导的放化疗仅适度改善GBM患者的生存率[2年生存率为27%]，80%的完全切除GBM复发。治疗中的关键挑战是GBM癌细胞亚群的增加，这些细胞对细胞凋亡具有抗性。因此，需要新的策略来提高目前GBM治疗的效率。TMZ引起DNA O 6-甲基鸟嘌呤损伤，其触发DNA修复，耗尽酶O 6-甲基鸟嘌呤甲基转移酶，并导致凋亡性细胞死亡。细胞凋亡的标志是由K+1和Cl-1的损失导致的细胞体积的急剧减小，称为细胞凋亡体积减小“AVD”。AVD是细胞凋亡的普遍特征，不依赖于死亡刺激。细胞体积的损失和细胞内总离子强度的降低（通过K+和Cl-的损失）发生在任何其他可检测的凋亡特征之前。细胞内离子强度的降低被认为在半胱天冬酶的激活和触发整个半胱天冬酶级联反应和凋亡机制中起允许的作用。通常，细胞通过激活体积调节机制来响应体积扰动。萎缩细胞恢复正常体积的过程称为调节性体积增加（RVI）。RVI只能通过增加Na+、K+和Cl-等具有药理活性的溶质来调节。Na+-K+-2Cl-共转运蛋白亚型1（NKCC 1）是RVI中响应高渗或等渗细胞收缩的主要细胞体积调节蛋白，其将1个Na+、1个K+和2个Cl-离子转运到细胞内。因此，NKCC 1介导的RVI将促进细胞存活。然而，NKCC 1介导的K+、Cl-蓄积是否可以抵消AVD、恢复细胞内离子强度、减少半胱天冬酶介导的凋亡和促进细胞存活以响应TMZ介导的DNA损伤，仍有待探索。我们的初步数据表明，NKCC 1是在GBM癌细胞中调节Cl-1和RVI的最重要的离子转运机制。有趣的是，用其强效抑制剂布美他尼药理学阻断NKCC 1活性增强了TMZ介导的细胞凋亡。这使我们假设NKCC 1活性在TMZ处理的细胞中被刺激，并且其抑制可以使胶质瘤对TMZ介导的凋亡敏感。这项研究的完成将揭示基于TMZ的联合治疗与NKCC 1抑制是否提供了一种新的治疗策略，这可能会增加目前化疗的效率。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is a World Health Organization Grade IV cancer, the most malignant category of glial tumors with median survival time less than one year. The combined temozolomide (TMZ)-mediated chemoradiotherapy only modestly improves survival of GBM patients [2-yr survival rate of 27%] and 80% of totally resected GBM recur. The key challenge in the treatment is an increase of a subpopulation of GBM cancer cells which are resistant to apoptosis. Therefore, new strategies are needed to improve the efficiency of the current therapies for GBM. TMZ causes a DNA O6-methylguanine lesion which triggers DNA repair, depletes the enzyme O6-methylguanine methyltransferase, and leads to apoptotic cell death. The hallmark of apoptosis is a drastic reduction in cell volume resulting from loss of K+i and Cl-i, termed apoptotic volume decrease "AVD". AVD is an ubiquitous characteristic of apoptosis which is independent of the death stimuli. Loss of cell volume and reduction of total intracellular ionic strength (via loss of K+ and Cl-) occur before any other detectable characteristics of apoptosis. The reduction of intracellular ionic strength has been suggested to play a permissive role in activation of caspases and triggering the entire caspase cascade and apoptotic machinery. Normally, cells respond to volume perturbations by activating volume regulatory mechanisms. The process by which shrunken cells return to normal volume is termed regulatory volume increase (RVI). RVI can only be regulated by the gain of osmotically active solutes such as Na+, K+ and Cl-. Na+-K+-2Cl- co-transporter isoform 1 (NKCC1), which transports 1 Na+, 1 K+ and 2 Cl- ions into the cell, is the primary cell volume regulatory protein in RVI in response to either hypertonic or isotonic cell shrinkage. Therefore, NKCC1-mediated RVI will promote cell survival. However, it remains unexplored whether NKCC1-mediated K+, Cl- accumulation can counteract AVD, restore intracellular ionic strength, reduce caspase-mediated apoptosis, and promote cell survival in response to TMZ-mediated DNA damage. Our preliminary data illustrate that NKCC1 is the most important ion transport mechanism in regulating Cl-i and RVI in GBM cancer cells. Interestingly, pharmacological blockade of NKCC1 activity with its potent inhibitor bumetanide enhanced TMZ- mediated apoptosis. This led us to hypothesize that NKCC1 activity is stimulated in the TMZ-treated cells and its inhibition can sensitize glioma to TMZ-mediated apoptosis. Completion of this study will shed light on whether a combined TMZ-based therapy with NKCC1 inhibition presents a novel therapeutic strategy, which may increase the efficiency of the current chemotherapy.