-
Journal of Virology Mar 2018Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac...
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. 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.
Topics: AMP-Activated Protein Kinases; Aminoimidazole Carboxamide; Autophagy; Autophagy-Related Protein-1 Homolog; Cells, Cultured; Cytomegalovirus; Cytomegalovirus Infections; Digitoxin; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Phosphatidylinositol 3-Kinases; Ribonucleotides; Sodium-Potassium-Exchanging ATPase; TOR Serine-Threonine Kinases; Virus Replication
PubMed: 29321306
DOI: 10.1128/JVI.01861-17 -
Organic Letters Jan 2018A concise and scalable enantioselective total synthesis of the natural cardenolides cannogenol and cannogenol-3-O-α-l-rhamnoside has been achieved in 18 linear steps....
A concise and scalable enantioselective total synthesis of the natural cardenolides cannogenol and cannogenol-3-O-α-l-rhamnoside has been achieved in 18 linear steps. The synthesis features a Cu(II)-catalyzed enantioselective and diastereoselective Michael reaction/tandem aldol cyclization and a one-pot reduction/transposition, which resulted in a rapid (6 linear steps) assembly of a functionalized intermediate containing C19 oxygenation that could be elaborated to cardenolide cannogenol. In addition, a strategy for achieving regio- and stereoselective glycosylation at the C3 position of synthetic cannogenol was developed and applied to the preparation of cannogenol-3-O-α-l-rhamnoside.
Topics: Aldehydes; Cyclization; Digitoxigenin; Molecular Structure; Stereoisomerism
PubMed: 29244520
DOI: 10.1021/acs.orglett.7b03513 -
Therapeutics and Clinical Risk... 2017Digitalis therapy (digoxin or digitoxin) in patients with heart failure is subject to an ongoing debate. Recent data suggest an increased mortality in patients receiving...
OBJECTIVES
Digitalis therapy (digoxin or digitoxin) in patients with heart failure is subject to an ongoing debate. Recent data suggest an increased mortality in patients receiving digitalis. This study investigated the effects of chronic digitalis therapy prior to heart transplantation (HTX) on posttransplant outcomes.
PATIENTS AND METHODS
This was a retrospective, observational, single-center study. It comprised 530 adult patients who were heart-transplanted at Heidelberg University Hospital between 1989 and 2012. Patients with digitalis prior to HTX (≥3 months) were compared to those without (no or <3 months of digitalis). Patients with digitalis were further subdivided into patients receiving digoxin or digitoxin. Primary outcomes were early posttransplant atrial fibrillation and mortality.
RESULTS
A total of 347 patients (65.5%) had digitalis before HTX. Of these, 180 received digoxin (51.9%) and 167 received digitoxin (48.1%). Patients with digitalis before HTX had a significantly lower 30-day (=0.0148) and 2-year (=0.0473) survival. There was no significant difference between digoxin and digitoxin in 30-day (=0.9466) or 2-year (=0.0723) survival. Multivariate analysis for posttransplant 30-day mortality showed pretransplant digitalis therapy as an independent risk factor (hazard ratio =2.097, CI: 1.036-4.248, =0.0397). Regarding atrial fibrillation in the early posttransplant period, there was neither a statistically significant difference between patients with and without digitalis (=0.1327) nor between patients with digoxin or digitoxin (=0.5867).
CONCLUSION
Digitalis in patients before HTX is an independent risk factor for increased posttransplant mortality.
PubMed: 29075124
DOI: 10.2147/TCRM.S147062 -
British Journal of Pharmacology Sep 2017Cardiac glycosides are Na /K -ATPases inhibitors used to treat congestive heart failure and cardiac arrhythmias. Epidemiological studies indicate that patients on...
BACKGROUND AND PURPOSE
Cardiac glycosides are Na /K -ATPases inhibitors used to treat congestive heart failure and cardiac arrhythmias. Epidemiological studies indicate that patients on digitalis therapy are more protected from cancer. Evidence of a selective cytotoxicity against cancer cells has suggested their potential use as anticancer drugs. The effect on angiogenesis of clinically used cardiac glycosides has not been extensively explored.
EXPERIMENTAL APPROACH
We studied the effect of digoxin, digitoxin and ouabain on early events of the angiogenic process in HUVECs. We determined HUVEC viability, proliferation, migration and differentiation into capillary tube-like structures. We also tested drug activity using an in vivo angiogenesis model. Activation of protein tyrosine kinase 2 (FAK) and signalling proteins associated with the Na /K -ATPase signalosome was determined by Western blotting.
KEY RESULTS
Digitoxin and ouabain (1-100 nM) inhibited HUVEC migration, concentration-dependently, without affecting cell viability, while digoxin induced apoptosis at the same concentrations. Digitoxin antagonized growth factor-induced migration and tubularization at concentrations (1-25 nM) within its plasma therapeutic range. The anti-angiogenic effect of digitoxin was confirmed also by in vivo studies. Digitoxin induced Src, Akt and ERK1/2 phosphorylation but did not affect FAK autophosphorylation at Tyr . However, it significantly inhibited growth factor-induced FAK phosphorylation at Tyr .
CONCLUSIONS AND IMPLICATIONS
Therapeutic concentrations of digitoxin inhibited angiogenesis and FAK activation by several pro-angiogenic stimuli. These novel findings suggest a potential repositioning of digitoxin as a broad-spectrum anti-angiogenic drug for diseases where pathological angiogenesis is involved.
Topics: Angiogenesis Inhibitors; Apoptosis; Cell Differentiation; Cell Movement; Cell Proliferation; Cell Survival; Digitoxin; Dose-Response Relationship, Drug; Focal Adhesion Protein-Tyrosine Kinases; Human Umbilical Vein Endothelial Cells; Humans; Intercellular Signaling Peptides and Proteins; Neovascularization, Pathologic; Protein Kinase Inhibitors; Structure-Activity Relationship
PubMed: 28688145
DOI: 10.1111/bph.13944 -
Tumour Biology : the Journal of the... Jun 2017While there are targeted treatments for triple positive breast cancers, lack of specific biomarkers for triple-negative breast cancers (TNBC) has hindered the...
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 G/G 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.
Topics: Animals; Carcinogenesis; Cardiac Glycosides; Cell Cycle; Cell Movement; Cell Proliferation; Digitoxin; Female; Humans; MCF-7 Cells; Mice; NF-kappa B; Receptors, Estrogen; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays
PubMed: 28618929
DOI: 10.1177/1010428317705331 -
PloS One 2017Cardiac glycosides (CGs) including digitalis, digoxin and digitoxin are used in the treatment of congestive heart failure and atrial fibrillation. Pre-clinical studies... (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
Cardiac glycosides (CGs) including digitalis, digoxin and digitoxin are used in the treatment of congestive heart failure and atrial fibrillation. Pre-clinical studies have investigated the anti-neoplastic properties of CGs since 1960s. Epidemiological studies concerning the association between CGs use and cancer risk yielded inconsistent results. We have performed a systematic review and meta-analysis to summarize the effects of CGs on cancer risk and mortality.
METHODS
PubMed, Scopus, Cochrane library, Medline and Web of Knowledge were searched for identifying relevant studies. Summary relative risks (RR) and 95% confidence intervals (CI) were calculated using random-effects model.
RESULTS
We included 14 case-control studies and 15 cohort studies published between 1976 and 2016 including 13 cancer types. Twenty-four studies reported the association between CGs and cancer risk and six reported the association between CGs and mortality of cancer patients. Using CGs was associated with a higher risk of breast cancer (RR = 1.330, 95% CI: 1.247-1.419). Subgroup analysis showed that using CGs increased the risk of ER+ve breast cancer but not ER-ve. Using CGs wasn't associated with prostate cancer risk (RR = 1.015, 95% CI: 0.868-1.87). However, CGs decreased the risk in long term users and showed a protective role in decreasing the risk of advanced stages. CGs use was associated with increased all-cause mortality (HR = 1.35, 95% CI: 1.248-1.46) but not cancer-specific mortality (HR = 1.075, 95% CI: 0.968-1.194).
CONCLUSION
The anti-tumor activity of CGs observed in pre-clinical studies requires high concentrations which can't be normally tolerated in humans. However, the estrogen-like activity of CGs could be responsible for increasing the risk of certain types of tumors.
Topics: Cardiac Glycosides; Female; Humans; Male; Neoplasms; Observational Studies as Topic; Risk Factors
PubMed: 28591151
DOI: 10.1371/journal.pone.0178611 -
Oncotarget Jun 2017Tissue hypoxia contributes to solid tumor pathogenesis by activating a series of adaptive programs. We previously showed that hypoxia promotes the preferential expansion...
Tissue hypoxia contributes to solid tumor pathogenesis by activating a series of adaptive programs. We previously showed that hypoxia promotes the preferential expansion and maintenance of CD133 positive human glioma stem cells (GSC) in a hypoxia inducible factor 1 alpha (HIF-1α)-dependent mechanism. Here, we examined the activity of digitoxin (DT), a cardiac glycoside and a putative inhibitor of HIF-1α, on human GSC in vitro and in vivo. During hypoxic conditions (1% O2), we observed the effect of DT on the intracellular level of HIF-1α and the extracellular level of vascular endothelial growth factor (VEGF) in human GSC. We found that DT at clinically achievable concentrations, suppressed HIF-1α accumulation during hypoxic conditions in human GSC and established glioma cell lines. DT treatment also significantly attenuated hypoxia-induced expression of VEGF, a downstream target of HIF-1α. Exposure to DT also reduced hypoxia-induced activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Furthermore, DT potently inhibited neurosphere formation, and decreased CD133 expression even at concentrations that were not overtly cytotoxic. Lastly, treatment with DT reduced GSC engraftment in an in vivo xenograft model of glioblastoma. Intraperitoneal injections of DT significantly inhibited the growth of established glioblastoma xenografts, and suppressed expression of HIF-1α and carbonic anhydrase (CA9), a surrogate marker of hypoxia. Taken together, these results suggest that DT at clinically achievable concentration functions as an inhibitor of HIF-1α, worthy of further investigations in the therapy of glioblastoma.
Topics: AC133 Antigen; Animals; Carbonic Anhydrases; Digitoxin; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, SCID; Neoplastic Stem Cells; RNA Interference; RNA, Small Interfering; Spheroids, Cellular; Tumor Cells, Cultured; Tumor Hypoxia; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays
PubMed: 28410215
DOI: 10.18632/oncotarget.16714 -
Tumour Biology : the Journal of the... Mar 2017Multiple factors including tumor heterogeneity and intrinsic or acquired resistance have been associated with drug resistance in lung cancer. Increased stemness and the...
Multiple factors including tumor heterogeneity and intrinsic or acquired resistance have been associated with drug resistance in lung cancer. Increased stemness and the plasticity of cancer cells have been identified as important mechanisms of resistance; therefore, treatments targeting cancer cells independent of stemness phenotype would be much more effective in treating lung cancer. In this article, we have characterized the anticancer effects of the antibiotic Nigericin in cells displaying varying degrees of stemness and resistance to anticancer drugs, arising from (1) routine culture conditions, (2) prolonged periods of serum starvation. These cells are highly resistant to conventional anticancer drugs such as Paclitaxel, Hydroxyurea, Colchicine, Obatoclax, Wortmannin, and LY294002, and the multidrug-resistant phenotype of cells growing under prolonged periods of serum starvation is likely the result of extensive rewiring of signaling pathways, and (3) lung tumorspheres that are enriched for cancer stem-like cells. We found that Nigericin potently inhibited the viability of cells growing under routine culture conditions, prolonged periods of serum starvation, and lung tumorspheres. In addition, we found that Nigericin downregulated the expression of key proteins in the Wnt canonical signaling pathway such as LRP6, Wnt5a/b, and β-catenin, but promotes β-catenin translocation into the nucleus. The antitumor effects of Nigericin were potentiated by the Wnt activator HLY78 and by therapeutic levels of the US Food and Drug Administration-approved drug Digitoxin and its novel synthetic analog MonoD. We believe that Nigericin may be used in a co-therapy model in combination with other novel chemotherapeutic agents in order to achieve potent inhibition of cancers that display varying degrees of stemness, potentially leading to sustained anticancer effects.
Topics: Antineoplastic Agents; Benzodioxoles; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Neoplastic Stem Cells; Nigericin; Phenanthridines; Wnt Signaling Pathway
PubMed: 28351327
DOI: 10.1177/1010428317694310