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PloS One 2023Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is...
Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is evidence that low concentrations of ouabain lead to a reduction of cell viability of cancer cells independent of its inhibition of the pumping function of the Na+/K+-ATPase. Regarding the impact of ouabain on biliary tract cancer, no data is currently available. Therefore, we aimed for a first-time investigation of ouabain as a potential anti-neoplastic biliary tract cancer agent using comprehensive human biliary tract cancer in vitro models. We found that ouabain has a strong cell line-dependent cytotoxic effect with IC50 levels in the (low) nanomolar-range and that this effect was not associated with the mRNA expression levels of the Na+/K+-ATPase α, β and fxyd-subunits. Regarding the mode of cytotoxicity, we observed induction of apoptosis in biliary tract cancer cells upon treatment with ouabain. Interestingly, cytotoxic effects of ouabain at sub-saturating (< μM) levels were independent of cellular membrane depolarization and changes in intracellular sodium levels. Furthermore, using a 3D cell culture model, we found that ouabain disturbs spheroid growth and reduces the viability of biliary tract cancer cells within the tumor spheroids. In summary, our data suggest that ouabain possesses anti-biliary tract cancer potential at low μM-concentration in 2D and 3D in vitro biliary tract cancer models and encourage further detailed investigation.
Topics: Humans; Ouabain; Biliary Tract Neoplasms; Antineoplastic Agents; Apoptosis; Sodium-Potassium-Exchanging ATPase
PubMed: 37390071
DOI: 10.1371/journal.pone.0287769 -
Frontiers in Cellular and Infection... 2023Globally, liver cancer poses a serious threat to human health and quality of life. Despite numerous studies on the microbial composition of the gut in hepatocellular...
Integrated microbiome and metabolome analysis reveals the interaction between intestinal flora and serum metabolites as potential biomarkers in hepatocellular carcinoma patients.
Globally, liver cancer poses a serious threat to human health and quality of life. Despite numerous studies on the microbial composition of the gut in hepatocellular carcinoma (HCC), little is known about the interactions of the gut microbiota and metabolites and their role in HCC. This study examined the composition of the gut microbiota and serum metabolic profiles in 68 patients with HCC, 33 patients with liver cirrhosis (LC), and 34 healthy individuals (NC) using a combination of metagenome sequencing and liquid chromatography-mass spectrometry (LC-MS). The composition of the serum metabolites and the structure of the intestinal microbiota were found to be significantly altered in HCC patients compared to non-HCC patients. LEfSe and metabolic pathway enrichment analysis were used to identify two key species ( and ) and five key metabolites (ouabain, taurochenodeoxycholic acid, glycochenodeoxycholate, theophylline, and xanthine) associated with HCC, which then were combined to create panels for HCC diagnosis. The study discovered that the diagnostic performance of the metabolome was superior to that of the microbiome, and a panel comprised of key species and key metabolites outperformed alpha-fetoprotein (AFP) in terms of diagnostic value. Spearman's rank correlation test was used to determine the relationship between the intestinal flora and serum metabolites and their impact on hepatocarcinogenesis and progression. A random forest model was used to assess the diagnostic performance of the different histologies alone and in combination. In summary, this study describes the characteristics of HCC patients' intestinal flora and serum metabolism, demonstrates that HCC is caused by the interaction of intestinal flora and serum metabolites, and suggests that two key species and five key metabolites may be potential markers for the diagnosis of HCC.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Gastrointestinal Microbiome; Quality of Life; Metabolome; Biomarkers; Liver Cirrhosis; Biomarkers, Tumor
PubMed: 37260707
DOI: 10.3389/fcimb.2023.1170748 -
Cells Apr 2023Two α-isoforms of the Na,K-ATPase (α and α) are expressed in the cardiovascular system, and it is unclear which isoform is the preferential regulator of...
Two α-isoforms of the Na,K-ATPase (α and α) are expressed in the cardiovascular system, and it is unclear which isoform is the preferential regulator of contractility. Mice heterozygous for the familial hemiplegic migraine type 2 (FHM2) associated mutation in the α-isoform (G301R; α mice) have decreased expression of cardiac α-isoform but elevated expression of the α-isoform. We aimed to investigate the contribution of the α-isoform function to the cardiac phenotype of α hearts. We hypothesized that α hearts exhibit greater contractility due to reduced expression of cardiac α-isoform. Variables for contractility and relaxation of isolated hearts were assessed in the Langendorff system without and in the presence of ouabain (1 µM). Atrial pacing was performed to investigate rate-dependent changes. The α hearts displayed greater contractility than WT hearts during sinus rhythm, which was rate-dependent. The inotropic effect of ouabain was more augmented in α hearts than in WT hearts during sinus rhythm and atrial pacing. In conclusion, cardiac contractility was greater in α hearts than in WT hearts under resting conditions. The inotropic effect of ouabain was rate-independent and enhanced in α hearts, which was associated with increased systolic work.
Topics: Mice; Animals; Sodium-Potassium-Exchanging ATPase; Ouabain; Atrial Fibrillation; Protein Isoforms; Mutation; Migraine Disorders; Phenotype
PubMed: 37190017
DOI: 10.3390/cells12081108 -
Ouabain Reverts CUS-Induced Disruption of the HPA Axis and Avoids Long-Term Spatial Memory Deficits.Biomedicines Apr 2023Ouabain (OUA) is a cardiotonic steroid that modulates Na+, K+ -ATPase activity. OUA has been identified as an endogenous substance that is present in human plasma, and...
Ouabain (OUA) is a cardiotonic steroid that modulates Na+, K+ -ATPase activity. OUA has been identified as an endogenous substance that is present in human plasma, and it has been shown to be associated with the response to acute stress in both animals and humans. Chronic stress is a major aggravating factor in psychiatric disorders, including depression and anxiety. The present work investigates the effects of the intermittent administration of OUA (1.8 μg/kg) during the chronic unpredictable stress (CUS) protocol in a rat's central nervous system (CNS). The results suggest that the intermittent OUA treatment reversed CUS-induced HPA axis hyperactivity through a reduction in (i) glucocorticoids levels, (ii) CRH-CRHR1 expression, and by decreasing neuroinflammation with a reduction in iNOS activity, without interfering with the expression of antioxidant enzymes. These changes in both the hypothalamus and hippocampus may reflect in the rapid extinction of aversive memory. The present data demonstrate the ability of OUA to modulate the HPA axis, as well as to revert CUS-induced long-term spatial memory deficits.
PubMed: 37189795
DOI: 10.3390/biomedicines11041177 -
Journal of Ethnopharmacology Oct 2023Herb-induced liver injury is poorly described for African herbal remedies, such as Acokanthera oppositifolia. Although a commonly used treatment for pain, snake bites...
ETHNOPHARMACOLOGICAL RELEVANCE
Herb-induced liver injury is poorly described for African herbal remedies, such as Acokanthera oppositifolia. Although a commonly used treatment for pain, snake bites and anthrax, it is also a well-known arrow poison, thus toxicity is to be expected.
AIM OF THE STUDY
The cytotoxicity and preliminary mechanisms of toxicity in HepG2 hepatocarcinoma cells were assessed.
MATERIALS AND METHODS
The effect of hot water and methanol extracts were on cell density, oxidative status, mitochondrial membrane potential, fatty acids, caspase-3/7 activity, adenosine triphosphate levels, cell cycling and viability was assessed. Phytochemicals were tentatively identified using ultra-performance liquid chromatography.
RESULTS
The hot water extract displayed an IC of 24.26 μg/mL, and reduced proliferation (S- and G2/M-phase arrest) and viability (by 30.71%) as early as 24 h after incubation. The methanol extract had a comparable IC of 26.16 μg/mL, and arrested cells in the G2/M-phase (by 18.87%) and induced necrosis (by 13.21%). The hot water and methanol extracts depolarised the mitochondrial membrane (up to 0.84- and 0.74-fold), though did not generate reactive oxygen species. The hot water and methanol extracts decreased glutathione (0.42- and 0.62-fold) and adenosine triphosphate (0.08- and 0.26-fold) levels, while fatty acids (2.00- and 4.61-fold) and caspase-3/7 activity (1.98- and 5.82-fold) were increased.
CONCLUSION
Extracts were both cytostatic and cytotoxic in HepG2 cells. Mitochondrial toxicity was evident and contributed to reducing adenosine triphosphate production and fatty acid accumulation. Altered redox status perturbed proliferation and promoted necrosis. Extracts of A. oppositifolia may thus promote necrotic cell death, which poses a risk for inflammatory hepatotoxicity with associated steatosis.
Topics: Humans; Hep G2 Cells; Methanol; Cytostatic Agents; Caspase 3; Plant Extracts; Carcinoma, Hepatocellular; Antineoplastic Agents; Necrosis; Liver Neoplasms; Water; Apocynaceae; Adenosine Triphosphate; Apoptosis
PubMed: 37182674
DOI: 10.1016/j.jep.2023.116617 -
PloS One 2023Recently, we have developed software that allows, using a minimum of required experimental data, to find the characteristics of ion homeostasis and a list of all...
Unidirectional fluxes of monovalent ions in human erythrocytes compared with lymphoid U937 cells: Transient processes after stopping the sodium pump and in response to osmotic challenge.
Recently, we have developed software that allows, using a minimum of required experimental data, to find the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane both in a balanced state and during the transient processes. Our approach has been successfully validated in human proliferating lymphoid U937 cells during transient processes after stopping the Na/K pump by ouabain and for staurosporine-induced apoptosis. In present study, we used this approach to find the characteristics of ion homeostasis and the monovalent ion fluxes through the cell membrane of human erythrocytes in a resting state and during the transient processes after stopping the Na/K pump with ouabain and in response to osmotic challenge. Due to their physiological significance, erythrocytes remain the object of numerous studies, both experimental and computational methods. Calculations showed that, under physiological conditions, the K+ fluxes through electrodiffusion channels in the entire erythrocyte ion balance is small compared to the fluxes through the Na/K pump and cation-chloride cotransporters. The proposed computer program well predicts the dynamics of the erythrocyte ion balance disorders after stopping the Na/K pump with ouabain. In full accordance with predictions, transient processes in human erythrocytes are much slower than in proliferating cells such as lymphoid U937 cells. Comparison of real changes in the distribution of monovalent ions under osmotic challenge with the calculated ones indicates a change in the parameters of the ion transport pathways through the plasma membrane of erythrocytes in this case. The proposed approach may be useful in studying the mechanisms of various erythrocyte dysfunctions.
Topics: Humans; Sodium-Potassium-Exchanging ATPase; U937 Cells; Ouabain; Cell Membrane; Ion Transport; Sodium; Erythrocytes; Chlorides; Potassium
PubMed: 37141334
DOI: 10.1371/journal.pone.0285185 -
Biomedicine & Pharmacotherapy =... Jun 2023The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved...
The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5'-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.
Topics: Humans; Antiviral Agents; SARS-CoV-2; COVID-19; Dihydroorotate Dehydrogenase; Drug Repositioning; Dronedarone; Pandemics; Atovaquone; Mebendazole; Purines; Molecular Docking Simulation; Protease Inhibitors; Molecular Dynamics Simulation
PubMed: 37068330
DOI: 10.1016/j.biopha.2023.114614 -
International Journal of Molecular... Mar 2023Glutamate mediates photic entrainment of the central clock in the suprachiasmatic nucleus (SCN) by evoking intracellular Ca signaling mechanisms. However, the detailed...
Glutamate mediates photic entrainment of the central clock in the suprachiasmatic nucleus (SCN) by evoking intracellular Ca signaling mechanisms. However, the detailed mechanisms of glutamate-evoked Ca signals are not entirely clear. Here, we used a ratiometric Ca and Na imaging technique to investigate glutamate-evoked Ca responses. The comparison of Ca responses to glutamate (100 μM) and high (20 mM) K solution indicated slower Ca clearance, along with rebound Ca suppression for glutamate-evoked Ca transients. Increasing the length of exposure time in glutamate, but not in 20 mM K, slowed Ca clearance and increased rebound Ca suppression, a result correlated with glutamate-induced Na loads. The rebound Ca suppression was abolished by ouabain, monensin, Na-free solution, or nimodipine, suggesting an origin of activated Na/K-ATPase (NKA) by glutamate-induced Na loads. Ouabain or Na-free solution also slowed Ca clearance, apparently by retarding Na/Ca-exchanger (NCX)-mediated Ca extrusion. Together, our results indicated the involvement of glutamate-induced Na loads, NKA, and NCX in shaping the Ca response to glutamate. Nevertheless, in the absence of external Na (NMDG substituted), Ca clearance was still slower for the Ca response to glutamate than for 20 mM K, suggesting participation of additional Ca handlers to the slower Ca clearance under this condition.
Topics: Rats; Animals; Glutamic Acid; Ouabain; Sodium-Potassium-Exchanging ATPase; Sodium-Calcium Exchanger; Suprachiasmatic Nucleus; Calcium
PubMed: 37047417
DOI: 10.3390/ijms24076444 -
Biomedicines Mar 2023A lipopolysaccharide (LPS)-induced neuroinflammation rat model was used to study the effects of ouabain (OUA) at low concentrations, which can interact with the...
A lipopolysaccharide (LPS)-induced neuroinflammation rat model was used to study the effects of ouabain (OUA) at low concentrations, which can interact with the Na,K-ATPase, causing the modulation of intracellular signalling pathways in the Central Nervous System. Our study aimed to analyse the effects of OUA on glutamate transport in the hippocampus of rats with LPS-induced neuroinflammation. Adult male Wistar rats were divided into four groups: OUA (1.8 µg/kg), saline (CTR), LPS (200 µg/kg), and OUA + LPS (OUA 20 min before LPS). The animals were sacrificed after 2 h, and the hippocampus was collected for analysis. After treatment, we determined the activities of Na,K-ATPase and glutamine synthetase (GS). In addition, expression of the α1, α2, and α3 isoforms of Na,K-ATPase and the glutamate transporters, EAAT1 and EAAT2, were also analysed. Treatment with OUA caused a specific increase in the α2 isoform expression (~20%), whereas LPS decreased its expression (~22%), and treatment with OUA before LPS prevented the effects of LPS. Moreover, LPS caused a decrease of approximately 50% in GS activity compared with that in the CTR group; however, OUA pre-treatment attenuated this effect of LPS. Notably, it was found that treatment with OUA caused an increase in the expression of EAAT1 (~30%) and EAAT2 (~25%), whereas LPS caused a decrease in the expression of EAAT1 (~23%) and EAAT2 (~25%) compared with that in the CTR group. When treated with OUA, the effects of LPS were abrogated. In conclusion, the OUA pre-treatment abolished the effect caused by LPS, suggesting that this finding may be related to the restoration of the interaction between FXYD2 and the studied membrane proteins.
PubMed: 36979899
DOI: 10.3390/biomedicines11030920 -
Annali Dell'Istituto Superiore Di Sanita 2023Endogenous ouabain (EO) is a steroid hormone secreted by the adrenal glands associated with adverse cardiovascular outcomes. However, EO plays other roles as brain...
INTRODUCTION
Endogenous ouabain (EO) is a steroid hormone secreted by the adrenal glands associated with adverse cardiovascular outcomes. However, EO plays other roles as brain protection against traumatic injury and seems involved in the adaptive response to hypoxia. Recently, we detected, for the first time, EO in a healthy human group of acute hypoxia and diving animals.
METHODS
This study complements the above as we considered a human model of chronic hypoxia. The aim is to detect EO in five idiopathic pulmonary arterial hypertension patients.
RESULTS AND DISCUSSION
We found that these patients had higher plasma concentrations of EO than control subjects. In addition, EO plasma concentrations were negatively correlated with the mean pulmonary arterial pressure and total pulmonary vascular resistance. The results could suggest that high concentrations of EO are predictive of better adaptation of the right ventricular afterload.
CONCLUSION
Although the results are preliminary, they can represent a helpful hint for future investigations for possible therapeutic and diagnostic approaches.
Topics: Animals; Humans; Ouabain; Familial Primary Pulmonary Hypertension; Hypertension
PubMed: 36974708
DOI: 10.4415/ANN_23_01_11