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

Background: Glioblastoma multiforme (GBM) is very difficult to treat with conventional anti-cancer/anti-apoptotic drugs. We tested the hypothesis that inhibition of Na/K-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, Na and K changes. Results: The specific Na/K-ATPase blocker ouabain (0.1 - 10 μM) induced cell death and disruption of K 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 ionophore valinomycin initiated apoptosis in LN229 cells in a K 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/K-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/K-ATPase triggers hybrid cell death and serves as an underlying mechanism for an enhanced chemotherapy effect on glioblastoma cells.