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Cell Communication and Signaling : CCS Oct 2023Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem...
BACKGROUND
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored.
METHODS
Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation-one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery.
RESULTS
Primitive AML cells containing CD34 LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation.
CONCLUSIONS
Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract.
Topics: Humans; Cardiac Glycosides; Ouabain; Myeloid Cell Leukemia Sequence 1 Protein; Leukemia, Myeloid, Acute; Cell Differentiation; Stem Cells; Neoplastic Stem Cells; Apoptosis
PubMed: 37828578
DOI: 10.1186/s12964-023-01317-8 -
Nature Communications Oct 2019Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions....
Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases.
Topics: A549 Cells; Animals; Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Breast Neoplasms; Cardiac Glycosides; Cell Line, Tumor; Cell Membrane; Cellular Senescence; Chondrocytes; Digoxin; Female; Fibroblasts; Humans; Hydrogen-Ion Concentration; Mice; Osteoarthritis; Ouabain; Proscillaridin; Pulmonary Fibrosis; Xenograft Model Antitumor Assays
PubMed: 31636264
DOI: 10.1038/s41467-019-12888-x -
International Journal of Molecular... Aug 2018The first reports of cardiac Na/K-ATPase signaling, published 20 years ago, have opened several major fields of investigations into the cardioprotective action of... (Review)
Review
The first reports of cardiac Na/K-ATPase signaling, published 20 years ago, have opened several major fields of investigations into the cardioprotective action of low/subinotropic concentrations of cardiotonic steroids (CTS). This review focuses on the protective cardiac Na/K-ATPase-mediated signaling triggered by low concentrations of ouabain and other CTS, in the context of the enduring debate over the use of CTS in the ischemic heart. Indeed, as basic and clinical research continues to support effectiveness and feasibility of conditioning interventions against ischemia/reperfusion injury in acute myocardial infarction (AMI), the mechanistic information available to date suggests that unique features of CTS-based conditioning could be highly suitable, alone /or as a combinatory approach.
Topics: Animals; Cardiac Glycosides; Cardiotonic Agents; Humans; Myocardial Infarction; Ouabain; Signal Transduction; Sodium-Potassium-Exchanging ATPase
PubMed: 30096873
DOI: 10.3390/ijms19082336 -
American Journal of Therapeutics 2014Based on a wealth of mechanistic evidence supported by the fact that ouabain mimics the spleen-liver effect in this article, the hypothesis is established that the... (Review)
Review
Based on a wealth of mechanistic evidence supported by the fact that ouabain mimics the spleen-liver effect in this article, the hypothesis is established that the endogenous hormone ouabain not only mimics the effects of ischemic preconditioning but also may be an ideal drug for the prevention of ischemic diseases. Moreover, it is argued that the spleen-liver effect may represent a general protective mechanism for the protection of organisms against oxygen deficiency. Investigating the spleen-liver mechanism offers a new approach to decipher the secrets of ischemic conditioning. Preconditioning represents a basic mechanism to protect a wide variety of cells against stressful stimuli such as ischemia. The ability to undergo preconditioning is almost ubiquitous in tissues and is highly conserved across species. Reinvestigation of the "spleen-liver mechanism" will allow the study of metabolic inhibitors and hormone mimics that all could help to transform ischemic preconditioning into a cure of the epidemic ischemic heart disease. Ouabain mimics the effects of the spleen factor. Cardioprotection induced by ouabain is due to the activation of pathways that are also activated in ischemic preconditioning. Just like ischemic preconditioning, ouabain activates the reperfusion injury salvage kinase pathway. Activation of nuclear factor kappa B and other transcription factors contribute to the long lasting effects of ouabain. The endogenous hormone ouabain just like preconditioning offers multiorgan protection based on innate mechanisms, which warrants clinical investigation. Clinical studies with ouabain that correspond to current standards are warranted.
Topics: Animals; Cardiotonic Agents; Humans; Ischemic Preconditioning, Myocardial; Liver; Ouabain; Sodium-Potassium-Exchanging ATPase; Spleen
PubMed: 22713528
DOI: 10.1097/MJT.0b013e31824d622c -
BioFactors (Oxford, England) May 2018Studies in the early 1990s suggested that a hormone identical to ouabain or an isomer of ouabain is secreted by the adrenal glands into the circulation and plays a role... (Review)
Review
Studies in the early 1990s suggested that a hormone identical to ouabain or an isomer of ouabain is secreted by the adrenal glands into the circulation and plays a role in the regulation of arterial pressure and cardiac and renal function. This hormone, known as endogenous ouabain (EO), was claimed to contribute to the pathophysiology of a number of disorders including heart failure, renal failure, pregnancy-induced, and essential hypertension. However, some research groups have been unable to confirm the presence of EO in the human circulation and the issue remains in dispute. In that the implications are of considerable importance to clinicians who, like the authors, lack biochemical expertise, it would be useful if the dispute could be addressed by disinterested scientists with long-standing and acknowledged expertise in analytical chemistry who could opine as to whether the evidence is, or is not, sufficient to state categorically that EO does (or does not) exist in the circulation in man. This brief review does not present new data but, rather, recommends that adjudication is needed regarding this important issue. © 2018 BioFactors, 44(3):219-221, 2018.
Topics: Blood Pressure; Cardiotonic Agents; Dissent and Disputes; Female; Heart Failure; Humans; Hypertension; Ouabain; Pregnancy; Quackery; Renal Insufficiency; Wedge Argument
PubMed: 29524270
DOI: 10.1002/biof.1421 -
Cell Calcium Mar 2020Accumulating evidence obtained over the last three decades has revealed a neuroendocrine system in the brain that mediates long term increases in blood pressure. The... (Review)
Review
Accumulating evidence obtained over the last three decades has revealed a neuroendocrine system in the brain that mediates long term increases in blood pressure. The system involves distinct ion transport pathways including the alpha-2 isoform of the Na,K pump and epithelial sodium channels, as well as critical hormone elements such as angiotensin II, aldosterone, mineralocorticoid receptors and endogenous ouabain. Activation of this system either by circulating or central sodium ions and/or angiotensin II leads to a cascading sequence of events that begins in the hypothalamus and involves the participation of several brain nuclei including the subfornical organ, supraoptic and paraventricular nuclei and the rostral ventral medulla. Key events include heightened aldosterone synthesis and mineralocorticoid receptor activation, upregulation of epithelial sodium channels, augmented synthesis and secretion of endogenous ouabain from hypothalamic magnocellular neurons, and sustained increases in sympathetic outflow. The latter step depends upon increased production of angiotensin II and the primary amplification of angiotensin II type I receptor signaling from the paraventricular nucleus to the rostral ventral lateral medulla. The transmission of sympathetic traffic is secondarily amplified in the periphery by increased short- and long-term potentiation in sympathetic ganglia and by sustained actions of endogenous ouabain in the vascular wall that augment expression of sodium calcium exchange, increase cytosolic Ca and heighten myogenic tone and contractility. Upregulation of this multi-amplifier system participates in forms of hypertension where salt, angiotensin and/or aldosterone are elevated and contributes to adverse outcomes in heart failure.
Topics: Aldosterone; Animals; Brain; Heart Failure; Humans; Hypertension; Ouabain; Sodium Chloride, Dietary; Sodium-Potassium-Exchanging ATPase
PubMed: 31954234
DOI: 10.1016/j.ceca.2019.102151 -
Phytomedicine : International Journal... Apr 2023Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively...
BACKGROUND
Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate.
PURPOSE
To facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA).
STUDY DESIGN
The antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels.
METHODS
Isolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA).
RESULTS
Single-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence.
CONCLUSION
Our results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.
Topics: Rats; Humans; Animals; Guinea Pigs; Anti-Arrhythmia Agents; Matrines; Ouabain; Aconitine; Arrhythmias, Cardiac; Ion Channels; Myocytes, Cardiac; Isoproterenol; Potassium; Action Potentials
PubMed: 36738478
DOI: 10.1016/j.phymed.2023.154688 -
Molecules (Basel, Switzerland) May 2017Ouabain and other cardenolides are steroidal compounds originally discovered in plants. Cardenolides were first used as poisons, but after finding their beneficial... (Review)
Review
Ouabain and other cardenolides are steroidal compounds originally discovered in plants. Cardenolides were first used as poisons, but after finding their beneficial cardiotonic effects, they were rapidly included in the medical pharmacopeia. The use of cardenolides to treat congestive heart failure remained empirical for centuries and only relatively recently, their mechanisms of action became better understood. A breakthrough came with the discovery that ouabain and other cardenolides exist as endogenous compounds that circulate in the bloodstream of mammals. This elevated these compounds to the category of hormones and opened new lines of investigation directed to further study their biological role. Another important discovery was the finding that the effect of ouabain was mediated not only by inhibition of the activity of the Na,K-ATPase (NKA), but by the unexpected role of NKA as a receptor and a signal transducer, which activates a complex cascade of intracellular second messengers in the cell. This broadened the interest for ouabain and showed that it exerts actions that go beyond its cardiotonic effect. It is now clear that ouabain regulates multiple cell functions, including cell proliferation and hypertrophy, apoptosis, cell adhesion, cell migration, and cell metabolism in a cell and tissue type specific manner. This review article focuses on the cardenolide ouabain and discusses its various in vitro and in vivo effects, its role as an endogenous compound, its mechanisms of action, and its potential use as a therapeutic agent; placing especial emphasis on our findings of ouabain as a pro-cystogenic agent in autosomal dominant polycystic kidney disease (ADPKD).
Topics: Animals; Cardiotonic Agents; Cell Communication; Humans; Kidney; Ouabain; Polycystic Kidney, Autosomal Dominant; Signal Transduction
PubMed: 28467389
DOI: 10.3390/molecules22050729 -
Neuroscience Letters Jan 2021To identify the role of RIP3 in ouabain-induced necroptosis and offer clinical implications to prevent spiral ganglion neurons (SGNs) from death, ouabain was applied in...
To identify the role of RIP3 in ouabain-induced necroptosis and offer clinical implications to prevent spiral ganglion neurons (SGNs) from death, ouabain was applied in SGNs derived from fetal rats and injected into Sprague-Dawley rats to construct injury model in vitro and in vivo, respectively. The necroptosis rate of SGNs was determined by flow cytometry and MTT assays. The protein levels and phosphorylation of RIP3 were evaluated using western blotting and immunofluorescence. SGNs injury was observed using H&E staining and immunofluorescence. The hearing function of rats was evaluated by the auditory brainstem response (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) methods. Ouabain caused dose-dependent necroptosis in SGNs and significant loss of SGNs of the cochlear axis in vivo. RIP3 and pRIP3 were upregulated with SGNs injury promoted, and RIP3 overexpression promoted ouabain-induced necroptosis in SGNs in vitro, which could be suppressed by necrostatin-1. RIP3 knockdown inhibited ouabain-induced necroptosis and reduced the phosphorylation of MLKL but no RIP3-dependent effect on the level of MLKL. RIP3 inhibition in vivo protected rats from ouabain-induced hearing damage with reducing ABR threshold shifts and promoting DPOAE amplitudes, while overexpression of RIP3 enhanced ouabain-induced injury that could be partially reversed by necrostatin-1. A decrease of SGNs density and an upregulation of pRIP3 were observed with RIP3 overexpression, which was in contrast when RIP3 was silenced. Therefore, RIP3 was essential for mediating necroptosis in ouabain-induced SGNs damage. Targeting RIP3 may prevent SGNs from death in clinical practice, and finally help the treatment of sensorineural hearing loss.
Topics: Animals; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Male; Necroptosis; Neurons; Ouabain; Rats; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases; Spiral Ganglion
PubMed: 33359086
DOI: 10.1016/j.neulet.2020.135565 -
International Journal of Molecular... Feb 2018Understanding the mechanisms of sodium (Na⁺) influx, effective compartmentalization, and efflux in higher plants is crucial to manipulate Na⁺ accumulation and assure... (Review)
Review
Understanding the mechanisms of sodium (Na⁺) influx, effective compartmentalization, and efflux in higher plants is crucial to manipulate Na⁺ accumulation and assure the maintenance of low Na⁺ concentration in the cytosol and, hence, plant tolerance to salt stress. Na⁺ influx across the plasma membrane in the roots occur mainly via nonselective cation channels (NSCCs). Na⁺ is compartmentalized into vacuoles by Na⁺/H⁺ exchangers (NHXs). Na⁺ efflux from the plant roots is mediated by the activity of Na⁺/H⁺ antiporters catalyzed by the salt overly sensitive 1 (SOS1) protein. In animals, ouabain (OU)-sensitive Na⁺, K⁺-ATPase (a P-type ATPase) mediates sodium efflux. The evolution of P-type ATPases in higher plants does not exclude the possibility of sodium efflux mechanisms similar to the Na⁺, K⁺-ATPase-dependent mechanisms characteristic of animal cells. Using novel fluorescence imaging and spectrofluorometric methodologies, an OU-sensitive sodium efflux system has recently been reported to be physiologically active in roots. This review summarizes and analyzes the current knowledge on Na⁺ influx, compartmentalization, and efflux in higher plants in response to salt stress.
Topics: Animals; Calcium; Cytosol; Ion Transport; Ouabain; Plant Physiological Phenomena; Plant Roots; Plants; Salt Tolerance; Sodium; Sodium-Potassium-Exchanging ATPase; Stress, Physiological; Vacuoles
PubMed: 29495332
DOI: 10.3390/ijms19030647