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

ABSTRACT:

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 Ca2+, Na+ and K+ changes.

Results

The specific Na+/K+-ATPase blocker ouabain (0.1 - 10 ?M) 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 ?M). 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 ?2 and ?3 subunits compared to the TMZ-sensitive cell line LN229 and normal human astrocytes. At low concentrations, ouabain selectively killed T98G cells. Knocking down the ?3 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.

SUBMITTER: Chen D

PROVIDER: S-EPMC4190379 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Publications

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

Chen Dongdong D Song Mingke M Mohamad Osama O Yu Shan Ping SP

BMC cancer 20140926

<h4>Background</h4>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.<h4>Methods</h4>In human LN229 and drug-resistant T98G glioblastoma cell cultures, cell death and signal pathways were measured using immunocytochemistry and We ...[more]

PMID: 25255962

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Project description:BackgroundMER receptor tyrosine kinase (MERTK) is expressed in a variety of malignancies, including glioblastoma multiforme (GBM). Our previous work demonstrated that inhibition of MERTK using RNA interference induced cell death and chemosensitivity in GBM cells, implicating MERTK as a potential therapeutic target. Here we investigate whether a novel MERTK-selective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines.MethodsCorrelations between expression of GAS6, a MERTK ligand, and prognosis were determined using data from the TCGA database. GBM cell lines (A172, SF188, U251) were treated in vitro with increasing doses of UNC2025 (50-400nM). Cell count and viability were determined by trypan blue exclusion. Cell cycle profiles and induction of apoptosis were assessed by flow cytometric analysis after BrdU or Po-Pro-1/propidium iodide staining, respectively. Polyploidy was detected by propidium iodide staining and metaphase spread. Cellular senescence was determined by ?-galactosidase staining and senescence-associated secretory cytokine analysis.ResultsDecreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic. All three GBM cell lines exhibited dose dependent reductions in cell number and colony formation (>90% at 200nM) after treatment with UNC2025. Cell cycle analysis demonstrated accumulation of cells in the G2/M phase and development of polyploidy. After extended exposure, 60-80% of cells underwent apoptosis. The majority of surviving cells (65-95%) were senescent and did not recover after drug removal. Thus, UNC2025 mediates anti-tumor activity in GBM by multiple mechanisms.ConclusionsThe findings described here provide further evidence of oncogenic roles for MERTK in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

Similar Datasets

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