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Author Notes:

Correspondence: spyu@emory.edu

DC performed cell culture procedures, cell death assays, flow cytomatry assay, and Western blot analysis.

MS participated in cell culture and cell death experiments.

OM participated in molecular biological experiments and helped to draft the manuscript.

SY conceived of the study, developed the hypothesis and wrote the manuscript.

All authors read and approved the final manuscript.

The authors declare that they have no competing interests.


Research Funding:

This investigation was supported by grants from National Institute of Health, USA (NS057255 to SY); American Heart Association (Grant-in-Aid Award GRNT12060222 to SY, Predoctoral Fellowship PRE4430032 to OM, and Postdoctoral Fellowship POST12080252 to MS).

It was also supported by the O. Wayne Rollins Endowed Chair appointment to SY.


  • Science & Technology
  • Life Sciences & Biomedicine
  • Oncology
  • Na+ pump
  • Glioblastomas
  • Apoptosis
  • Hybrid cell death
  • K+ homeostasis
  • Intracellular Ca2+
  • Temozolomide

Inhibition of Na+/K+-ATPase induces hybrid cell death and enhanced sensitivity to chemotherapy in human glioblastoma cells

Journal Title:

BMC Cancer


Volume 14


Type of Work:

Article | Final Publisher PDF


Background: Glioblastoma multiforme (GBM) is very difficult to treat with conventional anti-cancer/anti-apoptotic drugs. We tested the hypothesis that inhibition of Na<sup>+</sup>/K<sup>+</sup>-ATPase causes a mixed or hybrid form of concurrent apoptosis and necrosis and therefore should enhance anti-cancer effects of chemotherapy on glioblastoma cells. Methods: In human LN229 and drug-resistant T98G glioblastoma cell cultures, cell death and signal pathways were measured using immunocytochemistry and Western blotting. Fluorescent dyes were applied to measure intracellular Ca<sup>2+</sup>, Na<sup>+</sup> and K<sup>+</sup> changes. Results: The specific Na<sup>+</sup>/K<sup>+</sup>-ATPase blocker ouabain (0.1 - 10 μM) induced cell death and disruption of K<sup>+</sup> homeostasis in a time- and concentration-dependent manner. Annexin-V translocation and caspase-3 activation indicated an apoptotic component in ouabain cytoxicity, which was accompanied with reduced Bcl-2 expression and mitochondrial membrane potential. Ouabain-induced cell death was partially attenuated by the caspase inhibitor Z-VAD (100 μM). Consistently, the K<sup>+</sup> ionophore valinomycin initiated apoptosis in LN229 cells in a K<sup>+</sup> efflux-dependent manner. Ouabain caused an initial cell swell, which was followed by a sustained cell volume decrease. Electron microscopy revealed ultrastructural features of both apoptotic and necrotic alterations in the same cells. Finally, human T98G glioblastoma cells that are resistant to the chemotherapy drug temozolomide (TMZ) showed a unique high expression of the Na<sup>+</sup>/K<sup>+</sup>-ATPase α2 and α3 subunits compared to the TMZ-sensitive cell line LN229 and normal human astrocytes. At low concentrations, ouabain selectively killed T98G cells. Knocking down the α3 subunit sensitized T98G cells to TMZ and caused more cell death. Conclusion: This study suggests that inhibition of Na<sup>+</sup>/K<sup>+</sup>-ATPase triggers hybrid cell death and serves as an underlying mechanism for an enhanced chemotherapy effect on glioblastoma cells.

Copyright information:

© 2014 Chen et al.; licensee BioMed Central Ltd.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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