Inhibition of Na(+)-K(+)-2Cl(-) cotransporter isoform 1 accelerates temozolomide-mediated apoptosis in glioblastoma cancer cells

Cell Physiol Biochem. 2012;30(1):33-48. doi: 10.1159/000339047. Epub 2012 Jun 8.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects*
  • Bumetanide / pharmacology*
  • Caspase 3 / metabolism
  • Caspase 8 / metabolism
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Size / drug effects
  • Chlorides / metabolism
  • Dacarbazine / analogs & derivatives*
  • Dacarbazine / pharmacology
  • Drug Synergism
  • Glioblastoma
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Minor Histocompatibility Antigens
  • Neoplastic Stem Cells / drug effects
  • Neoplastic Stem Cells / metabolism
  • Neoplastic Stem Cells / physiology
  • Potassium / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • Sodium Potassium Chloride Symporter Inhibitors / pharmacology*
  • Sodium-Potassium-Chloride Symporters / metabolism*
  • Solute Carrier Family 12, Member 2
  • Temozolomide
  • Time-Lapse Imaging
  • WNK Lysine-Deficient Protein Kinase 1


  • Chlorides
  • Intracellular Signaling Peptides and Proteins
  • Minor Histocompatibility Antigens
  • SLC12A2 protein, human
  • Sodium Potassium Chloride Symporter Inhibitors
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 2
  • Bumetanide
  • Dacarbazine
  • Protein-Serine-Threonine Kinases
  • WNK Lysine-Deficient Protein Kinase 1
  • WNK1 protein, human
  • CASP3 protein, human
  • CASP8 protein, human
  • Caspase 3
  • Caspase 8
  • Potassium
  • Temozolomide