Foliar application of esculin and digitoxin improve the yield quality of salt-stressed flax by improving the antioxidant defense system.

BACKGROUND: Secondary metabolites of several plants, including esculin and digitoxin, which are cardiac glycosides, were previously employed for their therapeutic effects. The current study aims to investigate the functions of the main Na+ /K+ transport inhibitor digitoxin and the antioxidant esculin for enhancing flax plant growth and production under salinity. METHODOLOGY: Flax plants were irrigated with distilled water supplemented with 0.0 and 5000 mg/L salt solution starting from 15 DAS from sowing. Then exogenous treatment with digitoxin and esculin with 50 mg L- 1 and 100 mg L- 1 were used for this work. RESULTS: According to the results of this work, foliar spraying of esculin or digitoxin increased the salinity tolerance of flax plants.The foliar application of either esculin or digitoxin induced an elevation in the contents of photosynthetic pigments, osmolytes including soluble sugar and proline as well as the total phenols in salt-stressed flax plants. Moreover, esculin and digitoxin in particular counteract oxidative stress by increasing the activity of antioxidant enzymes including superoxide dismutase, catalase, peroxidase, phenylalanine ammonia-lyase, and tyrosine ammonia lyase, leading to a decrease in reactive oxygen species and lipid peroxidation levels and electrolyte leakage. The efficiency of esculin and digitoxin to sustain ion homeostasis by inhibiting Na+ absorption and increasing potassium, calcium, and phosphorus in flax plants may be the reason for their protective actions towards salinity.As a consequence, esculin and digitoxin increased yield quantity and quality as shown by increases in all investigated yield criteriaas shoot height, root length, their fresh and dry weights as well asseed yield/plant (g), and 1000 seeds weight, especially those that improved the desired oil properties. CONCLUSION: In conclusion, this study concluded that digitoxin was more effective in inhibiting Na+ build-up and increasing flax salinity tolerance, particularly at the high investigated dose as compared to esculin. In this study, we reported the recent findings of exogenousapplication of either digitoxin or esculin glycosides which are new investigated salt alleviators never used before for improving the salt tolerance in flax plants.

[Cloning and functional analysis of 3ß-hydroxysteroid dehydrogenase gene involved in biosynthesis of digitoxin].

Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3ß-Hydroxysteroid dehydrogenase(3ßHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3ßHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3ßHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3ßHSD2 was 774 bp and encoded 257 residues. Dp3ßHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3ßHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3ßHSD1 had stronger catalytic capacity than Dp3ßHSD2. The expression level of Dp3ßHSD1 was much higher than that of Dp3ßHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3ßHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.

Interaction of Odoroside A, A Known Natural Cardiac Glycoside, with Na+/K+-ATPase.

The nature of odoroside A, a cardiac glycoside (CG) extracted from Nerium oleander, as well as its chemical structure is quite similar to a well-known CG, ouabain possessing a steroid skeleton, a five-membered unsaturated lactone ring, and a sugar moiety as a common structure. Like ouabain, odoroside A inhibits the activity of Na+/K+-ATPase (NKA) and shows significant anticancer activity, however its inhibitory mechanism remains unknown. CGs show various physiological activities, including cardiotonic and anticancer activities, through the inhibition of NKA by direct interaction. Additionally, X-ray crystallographic analysis revealed the inhibitory mechanism of ouabain and digoxin in relation to NKA. By using different molecular modeling techniques, docking simulation of odoroside A and NKA was conducted based on the results of these X-ray crystallographic analyses. Furthermore, a comparison of the results with the binding characteristics of three known CGs (ouabain, digoxin, and digitoxin) was also conducted. Odoroside A fitted into the CG binding pocket on the α-subunit of NKA revealed by X-ray crystallography. It had key interactions with Thr797 and Phe783. Also, three known CGs showed similar interactions with Thr797 and Phe783. Interaction modes of odoroside A were quite similar to those of ouabain, digoxin, and digitoxin. Docking simulations indicated that the sugar moiety enhanced the interaction between NKA and CGs, but did not show enhanced NKA inhibitory activity because the sugar moiety was placed outside the entrance of active site. Thus, these results suggest that the inhibitory mechanism of odoroside A to NKA is the same as the known CGs.

Development of a new ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay.

Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC-MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid-liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC-MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances.

Digitalis therapy in patients with ventricular tachyarrhythmias.

Objective. The study sought to assess the prognostic value of treatment with digitalis on long-term prognosis in patients with ventricular tachyarrhythmias and atrial fibrillation (AF) and/or heart failure (HF). Background. Data regarding the outcome of digitalis therapy following ventricular tachyarrhythmias is limited. Methods. A large retrospective registry was used including consecutive patients with episodes of ventricular tachycardia (VT) or fibrillation (VF) from 2002 to 2015. Patients treated with digitalis were compared to patients without. The primary prognostic endpoint was all-cause mortality at 3 years, secondary endpoints comprised a composite arrhythmic endpoint (i.e. recurrences of ventricular tachyarrhythmias, appropriate implantable cardioverter defibrillator (ICD) therapies, sudden cardiac death) and cardiac rehospitalization. Kaplan Mayer survival curves, multivariable cox regression, and time trend analyses were applied for statistics. Results. Eight hundred and thirty-one patients were included (20% treated with digitalis and 80% without). At 3 years, digitalis treatment was not associated with all-cause mortality following ventricular tachyarrhythmias (24 vs. 21%, log-rank p = .736; HR = 1.063; 95% CI 0.746-1.515; p = .736). However, digitalis therapy was associated with an increased risk of the composite arrhythmic endpoint (38 vs. 23%; log-rank p = .001; HR = 1.719; 95% CI 1.279-2.311; p = .001) and cardiac rehospitalization (31 vs. 18%; log-rank p = .001; HR = 1.829; 95% CI 1.318-2.538; p = .001), which was still evident within multivariable Cox regression analyses. Finally, digitoxin may be associated with a worse prognosis than digoxin. Conclusion. Digitalis therapy was not associated with mortality in patients with ventricular tachyarrhythmias, but with increased risk of the composite arrhythmic endpoint and cardiac rehospitalization at 3 years.

Digitoxin Affects Metabolism, ROS Production and Proliferation in Pancreatic Cancer Cells Differently Depending on the Cell Phenotype.

Digitoxin has repeatedly shown to have negative effects on cancer cell viability; however, the actual mechanism is still unknown. In this study, we investigated the effects of digitoxin (1-100 nM) in four pancreatic cancer cell lines, BxPC-3, CFPAC-1, Panc-1, and AsPC-1. The cell lines differ in their KRAS/BRAF mutational status and primary tumor or metastasis origin. We could detect differences in the basal rates of cell proliferation, glycolysis, and ROS production, giving the cell lines different phenotypes. Digitoxin treatment induced apoptosis in all four cell lines, but to different degrees. Cells derived from primary tumors (Panc-1 and BxPC-3) were highly proliferating with a high proportion of cells in the S/G2 phase, and were more sensitive to digitoxin treatment than the cell lines derived from metastases (CFPAC-1 and AsPC-1), with a high proportion of cells in G0/G1. In addition, the effects of digitoxin on the rate of glycolysis, ROS production, and proliferation were dependent on the basal metabolism and origin of the cells. The KRAS downstream signaling pathways were not altered by digitoxin treatment, thus the effects exerted by digitoxin were probably disconnected from these signaling pathways. We conclude that digitoxin is a promising treatment in highly proliferating pancreatic tumors.

In Silico Study towards Repositioning of FDA-Approved Drug Candidates for Anticoronaviral Therapy: Molecular Docking, Molecular Dynamics and Binding Free Energy Calculations.

The SARS-CoV-2 targets were evaluated for a set of FDA-approved drugs using a combination of drug repositioning and rigorous computational modeling methodologies such as molecular docking and molecular dynamics (MD) simulations followed by binding free energy calculations. Six FDA-approved drugs including, Ouabain, Digitoxin, Digoxin, Proscillaridin, Salinomycin and Niclosamide with promising anti-SARS-CoV-2 activity were screened in silico against four SARS-CoV-2 proteins-papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), SARS-CoV-2 main protease (Mpro), and adaptor-associated kinase 1 (AAK1)-in an attempt to define their promising targets. The applied computational techniques suggest that all the tested drugs exhibited excellent binding patterns with higher scores and stable complexes compared to the native protein cocrystallized inhibitors. Ouabain was suggested to act as a dual inhibitor for both PLpro and Mpro enzymes, while Digitoxin bonded perfectly to RdRp. In addition, Salinomycin targeted PLpro. Particularly, Niclosamide was found to target AAK1 with greater affinity compared to the reference drug. Our study provides comprehensive molecular-level insights for identifying or designing novel anti-COVID-19 drugs.

Successful Treatment of Severe Digitoxin Intoxication with CytoSorb® Hemoadsorption.

While several intoxications can be successfully treated with specific antidotes, intoxications with the steroid glycoside digitoxin still represent a major challenge. Besides conventional approaches, CytoSorb® hemoadsorption might be another treatment option. We report on an 81-year-old female patient treated in our intensive care unit (ICU) with severe digitoxin intoxication, acute renal failure, and urinary tract infection (UTI). As physiological digitoxin elimination kinetics are known to appear slow, and also in regard to the renal failure, the decision was made to initiate continuous renal replacement therapy combined with CytoSorb hemoadsorption. The patient was hemodynamically stabilized within the first 4 h of treatment and initially required catecholamines to be stopped within 24 h of treatment. Pre- and post-adsorber drug level measurements showed a rapid elimination of digitoxin. Antibiotic treatment with piperacillin/tazobactam was initiated, and despite CytoSorb hemoadsorption therapy and its known potential to reduce plasma concentrations of several drugs, the UTI was successfully treated. After 3 days of CytoSorb treatment, digitoxin plasma levels were stable and almost normalized, and no clinical signs of intoxication were present. Five days after presentation, the patient was transferred from the ICU in a stable condition. CytoSorb hemoadsorption may be an easily available, efficient, and less cost-intensive therapy option than treatment with the Fab fragment, which is the currently recommended therapy for digitalis intoxications. Therefore, the use of CytoSorb might represent an alternative treatment for life-threatening complications of digitoxin intoxications.

Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation.

Maintenance of Na+ and K+ gradients across the cell plasma membrane is an essential process for mammalian cell survival. An enzyme responsible for this process, sodium-potassium ATPase (NKA), has been currently extensively studied as a potential anticancer target, especially in lung cancer and glioblastoma. To date, many NKA inhibitors, mainly of natural origin from the family of cardiac steroids (CSs), have been reported and extensively studied. Interestingly, upon CS binding to NKA at nontoxic doses, the role of NKA as a receptor is activated and intracellular signaling is triggered, upon which cancer cell death occurs, which lies in the expression of different NKA isoforms than in healthy cells. Two major CSs, digoxin and digitoxin, originally used for the treatment of cardiac arrhythmias, are also being tested for another indication-cancer. Such drug repositioning has a big advantage in smoother approval processes. Besides this, novel CS derivatives with improved performance are being developed and evaluated in combination therapy. This article deals with the NKA structure, mechanism of action, activity modulation, and its most important inhibitors, some of which could serve not only as a powerful tool to combat cancer, but also help to decipher the so-far poorly understood NKA regulation.

Repurposing Cardiac Glycosides: Drugs for Heart Failure Surmounting Viruses.

Drug repositioning is a successful approach in medicinal research. It significantly simplifies the long-term process of clinical drug evaluation, since the drug being tested has already been approved for another condition. One example of drug repositioning involves cardiac glycosides (CGs), which have, for a long time, been used in heart medicine. Moreover, it has been known for decades that CGs also have great potential in cancer treatment and, thus, many clinical trials now evaluate their anticancer potential. Interestingly, heart failure and cancer are not the only conditions for which CGs could be effectively used. In recent years, the antiviral potential of CGs has been extensively studied, and with the ongoing SARS-CoV-2 pandemic, this interest in CGs has increased even more. Therefore, here, we present CGs as potent and promising antiviral compounds, which can interfere with almost any steps of the viral life cycle, except for the viral attachment to a host cell. In this review article, we summarize the reported data on this hot topic and discuss the mechanisms of antiviral action of CGs, with reference to the particular viral life cycle phase they interfere with.

The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner.

Desmosomal proteins are components of the intercalated disc and mediate cardiac myocyte adhesion. Enhancement of cardiac myocyte cohesion, referred to as "positive adhesiotropy", was demonstrated to be a function of sympathetic signaling and to be relevant for a sufficient inotropic response. We used the inotropic agent digitoxin to investigate the link between inotropy and adhesiotropy. In contrast to wild-type hearts, digitoxin failed to enhance pulse pressure in perfused mice hearts lacking the desmosomal protein plakoglobin which was paralleled with abrogation of plaque thickening indicating that positive inotropic response requires intact desmosomal adhesion. Atomic force microscopy revealed that digitoxin increased the binding force of the adhesion molecule desmoglein-2 at cell-cell contact areas. This was paralleled by enhanced cardiac myocyte cohesion in both HL-1 cardiac myocytes and murine cardiac slices as determined by dissociation assays as well as by accumulation of desmosomal proteins at cell-cell contact areas. However, total protein levels or cytoskeletal anchorage were not affected. siRNA-mediated depletion of desmosomal proteins abrogated increase of cell cohesion demonstrating that intact desmosomal adhesion is required for positive adhesiotropy. Mechanistically, digitoxin caused activation of ERK1/2. In line with this, inhibition of ERK1/2 signaling abrogated the effects of digitoxin on cell-cell adhesion and desmosomal reorganization. These results show that the positive inotropic agent digitoxin enhances cardiac myocyte cohesion with reorganization of desmosomal proteins in an ERK1/2-dependent manner. Desmosomal adhesion seems to be important for a sufficient positive inotropic response of digitoxin treatment, which can be of medical relevance for the treatment of heart failure.

Bibliometric profile of global scientific research on digoxin toxicity (1849-2015).

Digoxin is a cardiac glycoside derived from the common foxglove digitalis purpurea and has been available for several centuries as a medicinal agent. Despite extensive patient experience over many years, there remains some controversy regarding the possibility that digoxin might have a deleterious effect on survival. This study was constructed to assess trends in digoxin toxicity research using well-established qualitative and quantitative bibliometric indicators. The current study is based on publications that have been indexed in Scopus. Articles referring to the subject of digoxin toxicity between 1849 and 2015 were assessed according to the document type, publication language, countries/territories, institutions, journal, impact factors, total number of citations, h-index, average number of citations per publication, and international collaborations. There were 2900 publications that included 2542 (87.7%) original research articles, while 5.3% were reviews and 4.6% letters. The country of origin was the USA in 849 publications, Germany in 241, the UK in 150, and France in 143. The USA and the UK had the highest number of international collaborations. The average number of citations per publications related to digoxin toxicity was 8.1, and the h-index was 59. The USA and Canada had the highest h-indices by country at 46 and 22, respectively. This study presents the first bibliometric analysis on digoxin toxicity publications. The USA was the most important contributors to digoxin toxicity literature with the greatest international collaboration, largest number of articles and highest h-index, followed by Germany and the UK. There has been a trend towards reduced publication numbers related to digoxin toxicity at global level, although it is still an important issue and we present the current research themes related to digoxin toxicity that were identified.

Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation.

Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na+,K+/ATPase α1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the α1 subunit was required for autophagy induction, since in α1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (α1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition.IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the α1 subunit of the Na+,K+/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in α1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.

Gene therapy-emulating small molecule treatments in cystic fibrosis airway epithelial cells and patients.

BACKGROUND: Several small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy. METHODS: IB3-1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[wildtype]CFTR-treated IB3-1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients. RESULTS: CF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures. CONCLUSIONS: Adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.

Digitalis Therapy and Risk of Recurrent Ventricular Tachyarrhythmias and ICD Therapies in Atrial Fibrillation and Heart Failure.

OBJECTIVE: This study sought to assess the impact of treatment with digitalis on recurrences of ventricular tachyarrhythmias in implantable cardioverter defibrillator (ICD) recipients with atrial fibrillation (AF) and heart failure (HF). BACKGROUND: The data regarding outcomes of digitalis therapy in ICD recipients are limited. METHODS: A large retrospective registry was used, including consecutive ICD recipients with episodes of ventricular tachyarrhythmia between 2002 and 2016. Patients treated with digitalis were compared to patients without digitalis treatment. The primary prognostic outcome was first recurrence of ventricular tachyarrhythmia at 5 years. Kaplan-Meier and multivariable Cox regression analyses were applied. RESULTS: A total of 394 ICD recipients with AF and/or HF was included (26% with digitalis treatment and 74% without). Digitalis treatment was associated with decreased freedom from recurrent ventricular tachy-arrhythmias (HR = 1.423; 95% CI 1.047-1.934; p = 0.023). Accordingly, digitalis treatment was associated with decreased freedom from appropriate ICD therapies (HR = 1.622; 95% CI 1.166-2.256; p = 0.004) and, moreover, higher rates of rehospitalization (38 vs. 21%; p = 0.001) and all-cause mortality (33 vs. 20%; p = 0.011). CONCLUSION: Among ICD recipients suffering from AF and HF, treatment with digitalis was associated with increased rates of recurrent ventricular tachyarrhythmias and ICD therapies. However, the endpoints may also have been driven by interactions between digitalis, AF, and HF.

Suppression of Adenovirus Replication by Cardiotonic Steroids.

The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies. IMPORTANCE: Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

Stepwise frontal affinity chromatography model for drug and protein interaction.

Frontal affinity chromatography is an efficient technique that combines affinity interaction and high-performance liquid chromatography, and frontal analysis has been used in studying the interaction between drugs and proteins. Based on frontal analysis, stepwise frontal analysis has been established. The present study aimed to use the Lineweaver-Burk plot in stepwise frontal analysis by taking the weighted average of time data. Commercial human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP) columns were used as an affinity column. Warfarin and digitoxin were chosen as model drugs for the HSA column, whereas verapamil and tamsulosin were selected as model drugs for the AGP column. The time data obtained by frontal analysis and stepwise frontal analysis were compared, and the results revealed good correlation (r2 = 0.9946-0.9998). Frontal analysis and stepwise frontal analysis were also used to analyze the equilibrium dissociation constants (Kd) of model drugs on the HSA and AGP columns. The Kd values were compared with literature values, which revealed the same order of magnitude. These results illustrate that conversion of the time data is reasonable and feasible. The Lineweaver-Burk plot can be used in the stepwise frontal analysis model to study the characteristics of the interaction between drugs and proteins. Graphical abstract ᅟ.

Overgangen fra digitoksin til digoksin i årene 2011­13.

BACKGROUND: The withdrawal of digitoxin and subsequent substitution with digoxin around 2012 may have led to an increased health risk for patients. The aim of this study was to follow individual patients during the switch. MATERIAL AND METHOD: Serum concentrations of digitoxin and digoxin, measured at the Department of Clinical Pharmacology at St Olavs University Hospital in the period 1 January 2011-31 December 2013 were reviewed. Patients who had switched from digitoxin to digoxin and whose serum concentrations of both drugs had been measured during this period were included. RESULTS: A total of 304 patients, 1686 samples and 1858 serum concentration analyses were included in the study. Therapeutic serum concentrations were measured in 171 patients (56.3 %) before the switch and 176 (57.9 %) after this had taken place. Altogether 108 patients (35.5 %) had therapeutic concentrations both before and after the change. For 58.9 % of the patients, the change resulted in a reduction in serum concentration of digitalis, calculated as digoxin equivalents. The proportion of patients with assumed supratherapeutic concentrations fell from 43.1 % to 33.9 %; however, the proportion of patients with toxic serum concentrations rose from 0.3 % to 3.0 %. INTERPRETATION: Although the switch led to a reduction in dose and serum concentration for many, a significant number of patients may have been put in harm's way.

Chemoresistance of Lung and Breast Cancer Cells Growing Under Prolonged Periods of Serum Starvation.

The efficacy of chemotherapy is hindered by both tumor heterogeneity and acquired or intrinsic multi-drug resistance caused by the contribution of multidrug resistance proteins and stemness-associated prosurvival markers. Therefore, targeting multi-drug resistant cells would be much more effective against cancer. In this study, we characterized the chemoresistance properties of adherent (anchorage-dependent) lung H460 and breast MCF-7 cancer cells growing under prolonged periods of serum starvation (PPSS). We found that under PPSS, both cell lines were highly resistant to Paclitaxel, Colchicine, Hydroxyurea, Obatoclax, Wortmannin, and LY294002. Levels of several proteins associated with increased stemness such as Sox2, MDR1, ABCG2, and Bcl-2 were found to be elevated in H460 cells but not in MCF-7 cells. While pharmacological inhibition of either MDR1, ABCG2, Bcl-2 with Verapamil, Sorafenib, or Obatoclax, respectively decreased the levels of their target proteins under routine culture conditions as expected, such inhibition did not reverse PX resistance in PPSS conditions. Paradoxically, treatment with inhibitors in serum-starved conditions produced an elevation of their respective target proteins. In addition, we found that Digitoxin, an FDA approved drug that decrease the viability of cancer cells growing under PPSS, downregulates the expression of Sox2, MDR1, phospho- AKT, Wnt5a/b, and ß-catenin. Our data suggest that PPSS-induced chemoresistance is the result of extensive rewiring of intracellular signaling networks and that multi-resistance can be effectively overcome by simultaneously targeting multiple targets of the rewired network. Furthermore, our PPSS model provides a simple and useful tool to screen drugs for their ability to target multiple pathways of cancer resistance. J. Cell. Physiol. 232: 2033-2043, 2017. © 2016 Wiley Periodicals, Inc.

Anti-tumorigenic effects of a novel digitoxin derivative on both estrogen receptor-positive and triple-negative breast cancer cells.

While there are targeted treatments for triple positive breast cancers, lack of specific biomarkers for triple-negative breast cancers (TNBC) has hindered the development of therapies for this subset of cancers. In this study, we evaluated the anticancer properties of cardiac glycoside Digitoxin (Dtx) and its synthetic analog MonoD on breast cancer cell lines MCF-7 (estrogen receptor-positive breast cancer) and MDA-MB-468 (triple-negative breast cancer). Both cardiac glycosides, at concentrations within the therapeutic range, increased the fraction of cells in the G0/G1 phase of the cell cycle, decreased viability, and inhibited the migration of MCF-7 and MDA-MB-468 cells. Both cardiac glycosides increased production of superoxide and induced apoptosis in both cell types. Reduced protein levels of nuclear factor kappa B and IkappaB kinase-beta were found in cardiac glycoside-treated cells, indicating that the cellular effects of these compounds are mediated via nuclear factor kappa B pathway. This study demonstrates the cytotoxic potential of digitoxin, and more importantly its synthetic analog MonoD, in the treatment of triple-positive breast cancer and more importantly the aggressive triple-negative breast cancer. Collectively, this study provides a basis for the reevaluation of cardiac glycosides in the treatment of breast cancer and more importantly reveals their potential in the treatment of triple-negative breast cancers.