Inhibition of Na+-K+-2Cl– Cotransporter isoform 1 Accelerates Temozolomidemediated Apoptosis in Glioblastoma Cancer CellsAlgharabil J.2 · Kintner D.B.2 · Wang Q.3 · Begum G.1 · Clark P.A.2 · Yang S.-S.4 · Lin S.-H.4 · Kahle K.T.5 · Kuo J.S.2 · Sun D.1,2
1Dept. of Neurology, Univ. of Pittsburgh, Pittsburgh, PA; 2Dept. of Neurosurgery, 3Cellular and Molecular Pathology Training Program, Univ. of Wisconsin School of Medicine and Public Health, Madison; 4Division of Nephrology, Dept. of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; 5Dept. of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA Corresponding Author
Dandan Sun, M.D., Ph.D., Department of Neurology University of Pittsburgh Medical School, S-598 South Biomedical Science Tower (BST) 3500 Terrace St. Pittsburgh PA 15213 (USA), Tel. (412) 624-0418, Fax: (412) 648-3321, E-Mail firstname.lastname@example.org
The hallmark of apoptosis is a significant reduction in cell volume (AVD) resulting from loss of K+i and Cl-i. Loss of cell volume and lowering of ionic strength of intracellular K+ and Cl-occur before any other detectable characteristics of apoptosis. In the present study, temozolomide (TMZ) triggered loss of K+i and Cl–i and AVD in primary glioblastoma multiforme (GBM) cancer cells (GC) and GC cancer stem cells (GSC). We hypothesize that Na+-K+-2Cl–cotransporter isoform 1 (NKCC1) counteracts AVD during apoptosis in GBM cancer cells by regulating cell volume and Cl-homeostasis. NKCC1 protein was expressed in both GC and GSC and played an essential role in regulatory volume increase (RVI) in response to hypertonic cell shrinkage and isotonic cell shrinkage. Blocking NKCC1 activity with its potent inhibitor bumetanide abolished RVI. These cells maintained a basal [Cl–]i ( ∼ 68 mM) above the electrochemical equilibrium for Cl–i. NKCC1 also functioned to replenish Cl–i levels following the loss of Cl–i. TMZ-treated cells exhibited increased phosphorylation of NKCC1 and its up-stream novel Cl–/volume-sensitive regulatory kinase WNK1. Inhibition of NKCC1 activity with bumetanide accelerated AVD, early apoptosis, as well as activation of caspase-3 and caspase-8. Taken together, this study strongly suggests that NKCC1 is an essential mechanism in GBM cells to maintain K+, Cl–, and volume homeostasis to counteract TMZ-induced loss of K+, Cl– and AVD. Therefore, blocking NKCC1 function augments TMZ-induced apoptosis in glioma cells.
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