Inhibition of Na⁺-K⁺-2Cl^–Cotransporter isoform 1 Accelerates Temozolomidemediated Apoptosis in Glioblastoma Cancer Cells

Abstract

The hallmark of apoptosis is a significant reduction in cell volume (AVD) resulting from loss of K⁺_iand 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⁺_iand Cl^–_iand 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^–_ilevels 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.

Down-Regulation of Laminin (LN)- α5 is Associated with Preeclampsia and Impairs Trophoblast Cell Viability and Invasiveness Through PI3K Signaling Pathway

“…Flow cytometric analysis was used to detect apoptosis of trophoblast by the use of a fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Key-GEN Biotech, Jiangsu, China) as previously described [22]. In brief, 1x10 6 cells were seeded in six-well plates with different groups for 48h, then the cells were harvested and washed with phosphate-buffered saline (PBS) for twice.…”

Upregulation of NHE1 protein expression enables glioblastoma cells to escape TMZ-mediated toxicity via increased H+extrusion, cell migration and survival

“…After incubation, 50 μl binding buffer was added to each sample. Annexin V positive cells were analyzed using the Nikon TiE300 microscope as described before (18).…”

Anti-angiogenic Effects of Bumetanide Revealed by DCE-MRI with a Biodegradable Macromolecular Contrast Agent in a Colon Cancer Model

“…By blocking the NKCC1 cotransporter, bumetanide has shown the ability to suppress the proper volume regulation required to support tumor growth and migration in an orthotopic glioma model (16). Bumetanide is also able to increase glioma sensitivity to chemotherapeutics by preventing the tumor’s ability to resist the significant cellular volume loss that occurs at the onset of apoptosis (17). Lastly, by inhibiting the volume increase that accompanies cell growth and division, bumetanide can effectively slow down the transition between the G1 and S phases of the cell cycle to decrease the overall rate of proliferation (18, 19).…”