Digitoxin inhibits ICC cell properties via the NF­κB/ST6GAL1 signaling pathway.

Intrahepatic cholangiocarcinoma (ICC) is a type of liver cancer associated with poor prognosis and increased mortality; the limited treatment strategy highlights the urgent need for investigation. Traditional Chinese Medicine (TCM), used alone or in combination with other treatments, can enhance therapeutic efficacy, improve life quality of patients and extend overall survival. In total, two rounds of screening of a TCM library of 2,538 active compounds were conducted using a Cell Counting Kit­8 assay and ICC cell lines. Cell proliferation and migration abilities were assessed through colony formation, 5­ethynyl­2'­deoxyuridine, would healing and Transwell assays. The impact of digitoxin (DT) on signaling pathways was initially investigated using RNA sequencing and further validated using reverse transcription­quantitative PCR, western blotting, lectin blotting and flow cytometry. ICC cells stably overexpressing ST6 ß­galactoside α­2,6­sialyltransferase 1 (ST6GAL1) were generated through lentiviral transfection. It was shown that DT emerged as a highly effective anti­ICC candidate from two rounds high­throughput library screening. DT could inhibit the proliferation and migration of ICC cells by suppressing NF­κB activation and reducing nuclear phosphorylated­NF­κB levels, along with diminishing ST6GAL1 mRNA and protein expression. The aforementioned biological effects and signal pathways of DT could be counteracted by overexpressing ST6GAL1 in ICC cells. In conclusion, DT suppressed ICC cell proliferation and migration by targeting the NF­κB/ST6GAL1 signaling axis. The findings of the present study indicated the promising therapeutic effects of DT in managing ICC, offering new avenues for treatment strategies.

The antiangiogenic effect of digitoxin is dependent on a ROS-elicited RhoA/ROCK pathway activation.

We previously showed that digitoxin inhibits angiogenesis and cancer cell proliferation and migration and these effects were associated to protein tyrosine kinase 2 (FAK) inhibition. Considering the interactions between FAK and Rho GTPases regulating cell cytoskeleton and movement, we investigated the involvement of RhoA and Rac1 in the antiangiogenic effect of digitoxin. Phalloidin staining of human umbilical vein endothelial cells (HUVECs) showed the formation of stress fibers in cells treated with 10 nM digitoxin. By Rhotekin- and Pak1- pull down assays, detecting the GTP-bound form of GTPases, we observed that digitoxin (10-25 nM) induced sustained (0.5-6 h) RhoA activation with no effect on Rac1. Furthermore, inhibition of HUVEC migration and capillary-like tube formation by digitoxin was counteracted by hindering RhoA-ROCK axis with RhoA silencing or Y-27632 treatment. Digitoxin did not decrease p190RhoGAP phosphorylation at Tyr1105 (a site targeted by FAK), suggesting that RhoA activation was independent from FAK inhibition. Because increasing evidence points to a redox regulation of RhoA, we measured intracellular ROS and found that digitoxin treatment enhanced ROS levels in a concentration-dependent manner (1-25 nM). Notably, the flavoprotein inhibitor DPI or the pan-NADPH oxidase (NOX) inhibitor VAS-2870 antagonized both ROS increase and RhoA activation by digitoxin. Our results provide evidence that inhibition of HUVEC migration and tube formation by digitoxin is dependent on ROS production by endothelial NOX, which leads to the activation of RhoA/ROCK pathway. Digitoxin effects on proteins regulating cytoskeletal organization and cell motility could have a wider impact on cancer progression, beyond the antiangiogenic activity.

Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

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.

Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration and infectivity in human lung cells.

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/ß/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the ß [E484K], Mink [Y453F] and α/ß/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

Quo vadis Cardiac Glycoside Research?

Cardiac glycosides (CGs), toxins well-known for numerous human and cattle poisoning, are natural compounds, the biosynthesis of which occurs in various plants and animals as a self-protective mechanism to prevent grazing and predation. Interestingly, some insect species can take advantage of the CG's toxicity and by absorbing them, they are also protected from predation. The mechanism of action of CG's toxicity is inhibition of Na+/K+-ATPase (the sodium-potassium pump, NKA), which disrupts the ionic homeostasis leading to elevated Ca2+ concentration resulting in cell death. Thus, NKA serves as a molecular target for CGs (although it is not the only one) and even though CGs are toxic for humans and some animals, they can also be used as remedies for various diseases, such as cardiovascular ones, and possibly cancer. Although the anticancer mechanism of CGs has not been fully elucidated, yet, it is thought to be connected with the second role of NKA being a receptor that can induce several cell signaling cascades and even serve as a growth factor and, thus, inhibit cancer cell proliferation at low nontoxic concentrations. These growth inhibitory effects are often observed only in cancer cells, thereby, offering a possibility for CGs to be repositioned for cancer treatment serving not only as chemotherapeutic agents but also as immunogenic cell death triggers. Therefore, here, we report on CG's chemical structures, production optimization, and biological activity with possible use in cancer therapy, as well as, discuss their antiviral potential which was discovered quite recently. Special attention has been devoted to digitoxin, digoxin, and ouabain.

Inhibitory Effects of Digoxin and Digitoxin on Cell Growth in Human Ovarian Cancer Cell Line SKOV-3.

BACKGROUND: Cardiac glycosides (CGs) possess a chemical structure similar to steroids, and are inhibitors of the sodium potassium pump. An anti-tumor effect of CGs in breast and prostate cancers has been reported, but the effect of CGs on ovarian cancer is still unclear. AIMS: In this study, the effects of CGs on proliferation, cytotoxicity and cell cycle of ovarian cancer cell line (SKOV-3) have been investigated. PROCEDURE: The cell proliferation and cytotoxicity were detected by MTT assay and LDH activity assay, respectively. CGs, at concentrations higher than IC50, decreased cell proliferation and showed increased cytotoxicity toward SKOV-3 cells. The colony-formation ability was reduced after treatment with digoxin and digitoxin for 10 days. Furthermore, we explored the effect of digoxin and digitoxin on the distribution of cell cycle by flow cytometry. RESULTS: Results revealed that both digoxin and digitoxin led to cell cycle arrest in G0/G1 phase with 24 or 48 hours, but the arrest of G0/G1 phase was not observed at 72 hours. We evaluated the percentage of hypodiploid cell population as an index of the cellular fragments through flow cytometry. The data indicated that cellular fragments were significantly increased by treating with digitoxin at the concentrations of IC50 and 10-6 M for 72 hours. CONCLUSION: Taken together, these data suggest that CGs decreased cell proliferation and increased cytotoxicity through cell cycle arrest at the G0/G1 phase. CGs have anti-tumor effect in SKOV-3 cells and might be a potential therapeutic drug for ovarian cancer. Since this study is a preliminary investigation of CGs on SKOV-3 cells, more experiments might be performed in the future. Furthermore, more ovarian cancer cell lines might also be employed in the future studies to confirm the effect of CGs in ovarian cancer.

Classical Drug Digitoxin Inhibits Influenza Cytokine Storm, With Implications for Covid-19 Therapy.

BACKGROUND/AIM: Influenza viruses, corona viruses and related pneumotropic viruses cause sickness and death partly by inducing cytokine storm, a hyper-proinflammatory host response by immune cells and cytokines in the host airway. Based on our in vivo experience with digitoxin as an inhibitor of TNFα-driven NFĸB signaling for cytokine expression in prostate cancer in rats and in cystic fibrosis in humans, we hypothesize that this drug will also block a virally-activated cytokine storm. Materials Methods: Digitoxin was administered intraperitoneally to cotton rats, followed by intranasal infection with 107TCID50/100 g of cotton rat with influenza strain A/Wuhan/H3N2/359/95. Daily digitoxin treatment continued until harvest on day 4 of the experiment. RESULTS: The cardiac glycoside digitoxin significantly and differentially suppressed levels of the cytokines TNFα, GRO/KC, MIP2, MCP1, and IFNγ, in the cotton rat lung in the presence of influenza virus. CONCLUSION: Since cytokine storm is a host response, we suggest that digitoxin may have a therapeutic potential not only for influenza and but also for coronavirus infections.

Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells.

BACKGROUND: Cardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes. METHODS: We treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration. RESULTS: Digitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling. CONCLUSIONS: Our study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity. SIGNIFICANCE: Discovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.

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.

Digitoxin inhibits HeLa cell growth through the induction of G2/M cell cycle arrest and apoptosis in vitro and in vivo.

Cervical cancer is the fourth most common gynecological malignancy affecting the health of women worldwide and the second most common cause of cancer­related mortality among women in developing regions. Thus, the development of effective chemotherapeutic drugs for the treatment of cervical cancer has become an important issue in the medical field. The application of natural products for the prevention and treatment of various diseases, particularly cancer, has always attracted widespread attention. In the present study, a library of natural products composed of 78 single compounds was screened and it was found that digitoxin exhibited the highest cytotoxicity against HeLa cervical cancer cells with an IC50 value of 28 nM at 48 h. Furthermore, digitoxin exhibited extensive antitumor activities in a variety of malignant cell lines, including the lung cancer cell line, A549, the hepatoma cell line, MHCC97H, and the colon cancer cell line, HCT116. Mechanistically, digitoxin caused DNA double­stranded breaks (DSBs), inhibited the cell cycle at the G2/M phase via the ataxia telangiectasia mutated serine/threonine kinase (ATM)/ATM and Rad3­related serine/threonine kinase (ATR)­checkpoint kinase (CHK1)/checkpoint kinase 2 (CHK2)­Cdc25C pathway and ultimately triggered mitochondrial apoptosis, which was characterized by the disruption of Bax/Bcl­2, the release of cytochrome c and the sequential activation of caspases and poly(ADP­ribose) polymerase (PARP). In addition, the in vivo anticancer effect of digitoxin was confirmed in HeLa cell xenotransplantation models. On the whole, the findings of the present study demonstrate the efficacy of digitoxin against cervical cancer in vivo and elucidate its molecular mechanisms, including DSBs, cell cycle arrest and mitochondrial apoptosis. These results will contribute to the development of digitoxin as a chemotherapeutic agent in the treatment of cervical cancer.

Digitoxin Attenuates Heart Failure, Reduces Myocardial Hypertrophy, and Preserves the Calcium-Binding Proteins in Infarcted Rats.

We previously showed that digitoxin prolongs the survival of rats with heart failure due to myocardial infarction (MI). In this study, we evaluated the effect of digitoxin on myocardial structure, ventricular function, and proteins involved in calcium kinetics. Seventy-two rats with MI >35% of the left ventricle were randomly assigned to 4 treatment groups: sham (n = 15), digitoxin (n = 11), infarction (n = 20), and infarction + digitoxin (n = 26). The rats were assessed 120 days after surgery by echocardiogram, hemodynamics, papillary muscle mechanics, collagen content, cardiomyocyte nuclear volume, and Western blot analysis of proteins involved in calcium kinetics. Digitoxin was administered via the rat chow. Two-way analysis of variance was used for comparisons. Myocardial infarction caused inotropic impairment, pulmonary congestion, increase of nuclear volume, myocardial collagen, and Na+/Ca2+ exchanger levels, and decreased SERCA2 and phosphorylated phospholamban levels. Treatment with digitoxin showed improvements in cardiac remodeling, inotropism, ventricular performance, pulmonary congestion, collagen accumulation, nuclear volume, and proteins involved in calcium kinetics. In rats with heart failure due to MI, long-term treatment with digitoxin attenuates congestive heart failure, mitigates myocardial remodeling and contractile impairment, and preserves myocardial levels of proteins involved in calcium kinetics.

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.

Introduction of Adonis aestivalis as a new source of effective cytotoxic cardiac glycoside.

Cardiac glycosides are used for treatment of irregular heartbeats, cardiac arrhythmia and congestive heart failures. In this research, digitoxin as a cardiac glycoside was identified and isolated for the first time in the world from Adonis aestivalis and investigated for its cytotoxic activity against cervical cancer cell (HeLa) lines and human lymphocytes by MTT test. Digitoxin extracted from the aerial parts of the plant collected from west of Iran and purified by column and thin layer chromatographic techniques. The structure of isolated cardiac glycoside was identified by IR, 1H NMR and 13C NMR methods and so the presence of digitoxin was established. The half maximal inhibitory concentration values for cervical cancer and lymphocyte cells were obtained to be 5.62 and 412.94 µg/mL. The results of this study introduced the new resource of digitoxin which has considerable cytotoxic effects against HeLa cancer cells but did not damage normal human lymphocyte cells.

Digitoxin Suppresses Store Operated Calcium Entry by Modulating Phosphorylation and the Pore Region of Orai1.

BACKGROUND: Store-operated calcium entry (SOCE), primarily mediated by Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that has been linked to human diseases including myopathy, epilepsy, immunodeficiency, and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms underlying SOCE activation, the development of therapies against dysfunctional SOCE significantly lags behind, partly due to the lack of more specific pharmacological tools and poor understanding of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin. OBJECTIVE AND METHODS: Capitalizing on Ca2+ imaging and pharmacological tools, we aimed to systemically delineate the mechanism of action of digitoxin by defining how it impinges on Orai1 to exert its suppressive effect on SOCE. RESULTS: The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27-S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1. CONCLUSIONS: Our study identified two regions that are critical for the inhibition on Orai1 channels, providing valuable hotspots for future design of SOCE inhibitors.

Pharmacological targeting of p38 MAP-Kinase 6 (MAP2K6) inhibits the growth of esophageal adenocarcinoma.

Drug repurposing with a better understanding of the underlying mechanism has provided new avenues to find treatment for malignancies. Esophageal adenocarcinoma (EAC) is a rapidly increasing cancer with a dismal 5-year survival rate of <15%. Lack of efficient treatment options contributes to the high mortality rate of EAC. To find new therapy against EAC we performed unbiased drug screening of an FDA-approved drug library and identified that the cardiac glycosides including Ouabain, Digoxin and Digitoxin efficiently inhibit the proliferation of EAC cell lines (OE33 and OE19) both in vitro and in vivo. RNA-Sequencing analysis combined with RNAi screening revealed that Ouabain suppresses the proliferation of EAC cells through downregulation of p38 MAP-Kinase 6 (MAP2K6, also known as MKK6). Consistently, shRNA-mediated knockdown of MKK6 reduced the proliferation of EAC cells and tumor growth. Further analysis demonstrated that MKK6 inhibition leads to the reduced levels of the transcription factor SOX9. In line with this finding, deletion of SOX9 with CRISPR/Cas9 resulted in decreased proliferation of EACs in 3D organoid culture and reduced tumor growth. Together these findings establish a druggable axis that can be harnessed for therapeutic gain against EAC.

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling.

Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20. Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.

Effects of digitoxin on cell migration in ovarian cancer inflammatory microenvironment.

Clinical and experimental evidence supports a role for cardiac glycosides (CGs) as potential novel anticancer drugs. However, there are no studies reporting the effect of CGs on the inflammatory tumor microenvironment (TME), which plays a central role in tumor progression and invasiveness. We investigated whether digitoxin affects a) specific pathways involved in motility and/or activation of different cell types shaping TME, and b) cancer cell growth and invasiveness in response to TME-associated factors. To test our hypothesis, conditioned media (CM) from polarized macrophages, and apoptotic or non-apoptotic ovarian cancer cells (SKOV3) were tested as chemoattractants for endothelial cells, monocytes and cancer cells. We demonstrated that CM from M1 (LPS/IFNγ) and M2 (IL-4/IL-13) polarized macrophages, which mimic inflammatory TME, increased both HUVEC migration and tubularization. Treatment of HUVECs with digitoxin at concentrations within its plasma therapeutic range counteracted these effects. Digitoxin affected the expression of neither M1 (CD80/CD68) nor M2 (CD206/CD163) activation markers, nor the amount of cell-bound IL-1ß and CCL22. Accordingly, HUVEC migration in response to CM from digitoxin-treated activated macrophages was unchanged. These data point to a direct effect of digitoxin on HUVEC signaling rather than on the modulation of the cytokine profile released from activated macrophages. At variance with what observed for HUVECs, digitoxin did not prevent monocyte migration induced by SKOV3 CM. In addition, digitoxin significantly impaired SKOV3 growth and migration in response to M1 or M2 macrophage CM. Finally, we showed that digitoxin inhibited FAK phosphorylation in SKOV3 but not PYK2 phosphorylation in monocytes, thus providing a molecular mechanism accounting for the observed differential anti-migratory effect. Overall, digitoxin counteracted salient features of the inflammatory ovarian cancer microenvironment, laying the ground for potential digitoxin repositioning as an anticancer drug.

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.