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Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria

Elba Alonso1, María F Cano-Abad, Ana J Moreno-Ortega

  • 1Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain; Servicio de Farmacología Clínica, Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain.

Steroids
|August 13, 2013
PubMed

Insights

Ouabain, a steroid Na(+)/K(+) ATPase (NKA) blocker, induces apoptosis in HeLa cells by directly targeting mitochondria. This mechanism involves caspase activation, ATP depletion, and cytochrome c release, leading to cell death.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Pharmacology

Background:

  • The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain exhibits pro-apoptotic effects, but the underlying mechanism remains unclear.
  • Ouabain and digoxin are cardiac glycosides with known cellular effects.

Purpose of the Study:

  • To elucidate the mechanism by which ouabain induces apoptosis in HeLa cells.
  • To investigate the potential mitochondrial involvement in ouabain-induced cell death.

Main Methods:

  • HeLa cells were incubated with nanomolar concentrations of ouabain or digoxin.
  • Caspase activation (caspase-3/7, -9, -8) was assessed.
  • Mitochondrial function, including ATP levels and cytochrome c release, was analyzed.
  • Fluorescent ouabain was used to track its cellular localization.
  • Calcium signaling and endoplasmic reticulum (ER) function were evaluated.

Main Results:

  • Ouabain and digoxin induced apoptotic death in 30-50% of HeLa cells after 24h.
  • Ouabain activated caspases-3/7 and -9, but not caspase-8.
  • Mitochondrial pathway involvement was suggested by Z-LEHD-FMK's inhibition of apoptosis and caspase-9 activation.
  • Ouabain treatment led to ATP depletion, mitochondrial cytochrome c release, and mitochondrial disruption.
  • Fluorescent ouabain localized tightly with mitochondria, indicating a direct mitochondrial interaction.
  • Ouabain treatment also affected ER calcium stores.

Conclusions:

  • Ouabain is taken up by HeLa cells and directly interacts with mitochondria.
  • The pro-apoptotic effects of ouabain are mediated through a mitochondrially-dependent pathway.
  • Ouabain may also impact endoplasmic reticulum calcium homeostasis, contributing to its cytotoxic effects.
Keywords:
AFUApoptosisCGCa(2+)DEVDDMEMDMSODulbecco’s minimal essential mediumERFBSHeLa cellsHistInsP(3)RMTTMitochondriaNKANa(+)/K(+)-ATPaseODOuabainOubOub-BODIPYRLU/sStTMREZ-LEHD-FMKZLF[Ca(2+)](c)[Na(+)](c)arbitrary fluorescence unitsbromide (3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyl)cardiac glycosidecytosolic Ca(2+) concentrationcytosolic concentration of Na(+)dimethylsulfoxideendoplasmic reticulumfetal bovine serumhistamineinositol-1,4,5-trisphosphate receptormPTPmitochondrial permeability transition poreoptical densityouabainouabain-BODIPY FL®pNCXplasmalemmal Na(+)/Ca(2+) exchangerrelative luminescent units per secondspecific amino acid sequence Asp-Glu-Val-Aspstaurosporinetetramethylrhodamine ethyl ester

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