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级肿瘤，是恶性程度最高的胶质肿瘤，中位生存期不足一年。联合替莫唑胺（TMZ）介导的放化疗仅能适度提高GBM患者的生存率[2年生存率为27%]，80%的完全切除的GBM复发。治疗中的关键挑战是GBM癌细胞亚群的增加，这些细胞对凋亡具有抗性。因此，需要新的策略来提高目前治疗GBM的效率。TMZ引起DNA o6 -甲基鸟嘌呤损伤，触发DNA修复，耗尽o6 -甲基鸟嘌呤甲基转移酶，导致细胞凋亡。细胞凋亡的标志是由于K+i和Cl-i的损失导致细胞体积急剧减少，称为凋亡体积减少（AVD）。AVD是细胞凋亡的普遍特征，与死亡刺激无关。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）发生在凋亡的任何其他可检测特征之前。细胞内离子强度的降低已被认为在半胱天冬酶的激活和触发整个半胱天冬酶级联和凋亡机制中起许可作用。正常情况下，细胞通过激活体积调节机制来响应体积扰动。收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Na+、K+和Cl-等渗透活性溶质的增加来调节。Na+-K+- 2cl -共转运蛋白异构体1 （NKCC1）可将1na +、1k +和2cl -离子转运到细胞中，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。因此，nkcc1介导的RVI会促进细胞存活。然而，nkcc1介导的K+， Cl-积累是否可以对抗AVD，恢复细胞内离子强度，减少caspase介导的细胞凋亡，促进细胞存活，以应对tmz介导的DNA损伤，尚不清楚。我们的初步数据表明，NKCC1是调控GBM癌细胞cl - 1和RVI的最重要的离子转运机制。有趣的是，用有效抑制剂布美他尼阻断NKCC1活性可增强TMZ介导的细胞凋亡。这使我们假设NKCC1活性在tmz处理的细胞中被刺激，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以tmz为基础的联合NKCC1抑制疗法是否提供了一种新的治疗策略，可能会提高当前化疗的效率。

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.