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Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death
Published on: December 27, 2016
Ouabain activates signaling pathways associated with cell death in human neuroblastoma
Andrey Kulikov1, Alexander Eva, Ulrike Kirch
1Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Frankfurter Str. 100, D-35392 Giessen, Germany.
Abstract:
Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na(+),K(+)-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 microM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca(2+)] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 microM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).
Insights
Cardiotonic steroids (CTS) like ouabain impact SH-SY5Y cells by initiating both pro-survival and pro-death signaling pathways. Ouabain triggers reactive oxygen species and calcium influx, but also promotes apoptosis and inhibits neurite outgrowth, highlighting complex cellular responses.
Area of Science:
- Cellular and Molecular Biology
- Neuroscience
- Pharmacology
Background:
- Cardiotonic steroids (CTS), such as ouabain, are known inhibitors of the sodium pump (Na(+),K(+)-ATPase).
- These compounds can also modulate intracellular signaling pathways in a manner similar to hormones.
- Neuroblastoma cells (SH-SY5Y) provide a model to study these complex cellular effects.
Purpose of the Study:
- To investigate the multifaceted signaling events triggered by ouabain in SH-SY5Y neuroblastoma cells.
- To elucidate the dual role of ouabain in promoting both anti-apoptotic and pro-apoptotic pathways.
- To understand the impact of ouabain on cell viability and neuritogenesis.
Main Methods:
- Incubation of SH-SY5Y cells with varying concentrations of ouabain (10 nM to 10 microM).
- Measurement of reactive oxygen species (ROS) and cytosolic calcium ([Ca(2+)]).
- Analysis of protein phosphorylation (Erk1/2, Akt, Bad, p53), protein abundance (Bcl-XL, Bcl-2), cytochrome c release, caspase 3 activation, and cell number determination.
Main Results:
- Ouabain induced rapid increases in ROS and cytosolic calcium within 30 minutes.
- At later time points (6 hours), ouabain promoted anti-apoptotic signaling (Erk1/2, Akt, Bad phosphorylation) but also inhibited neuritogenesis and induced cell death.
- Ouabain decreased anti-apoptotic proteins (Bcl-XL, Bcl-2), triggered cytochrome c release, activated caspase 3, and phosphorylated p53, leading to significant cell number reduction at higher concentrations.
Conclusions:
- Ouabain elicits complex and opposing signaling cascades in SH-SY5Y cells, affecting cell survival and death pathways.
- The study highlights the dual nature of CTS, with potential implications for their therapeutic use and side effects.
- Further research is needed to identify the specific receptors and fully delineate the signaling networks influenced by ouabain and other CTS.
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