Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria.
The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.
Instituto 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.
Elba
E
Alonso
María F
MF
Cano-Abad
Ana J
AJ
Moreno-Ortega
Jesús
J
Novalbos
Juan
J
Milla
Antonio G
AG
García
Ana
A
Ruiz-Nuño
D017209:Apoptosis / Q000187:drug effects*; D001692:Biological Transport; D015415:Biomarkers / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology*; D013997:Time Factors
D015415:Biomarkers; D009842:Oligopeptides; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D010042:Ouabain; D006632:Histamine; D053453:Caspase 9; D002118:Calcium
AFU; Apoptosis; CG; Ca(2+); DEVD; DMEM; DMSO; Dulbecco’s minimal essential medium; ER; FBS; HeLa cells; Hist; InsP(3)R; MTT; Mitochondria; NKA; Na(+)/K(+)-ATPase; OD; Ouabain; Oub; Oub-BODIPY; RLU/s; St; TMRE; Z-LEHD-FMK; ZLF; [Ca(2+)](c); [Na(+)](c); arbitrary fluorescence units; bromide (3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyl); cardiac glycoside; cytosolic Ca(2+) concentration; cytosolic concentration of Na(+); dimethylsulfoxide; endoplasmic reticulum; fetal bovine serum; histamine; inositol-1,4,5-trisphosphate receptor; mPTP; mitochondrial permeability transition pore; optical density; ouabain; ouabain-BODIPY FL®; pNCX; plasmalemmal Na(+)/Ca(2+) exchanger; relative luminescent units per second; specific amino acid sequence Asp-Glu-Val-Asp; staurosporine; tetramethylrhodamine ethyl ester