Alonso 2013 Steroids
|Alonso E, Cano-Abad MF, Moreno-Ortega AJ, Novalbos J, Milla J, García AG, Ruiz-Nuño A (2013) Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria. Steroids 78:1110-8.|
Abstract: 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 Ca2+ concentration ([Ca2+]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca2+ 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.
Copyright © 2013 Elsevier Ltd. All rights reserved.
• Keywords: 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