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Medicine International 2022The alpha subunits (ATP1A1-3) of Na/K-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin....
The alpha subunits (ATP1A1-3) of Na/K-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin. In the present study, three pancreatic cancer cell lines, AsPC-1, Panc-1 and CFPAC-1, were used to investigate the effects of digitoxin in relation to the expression of the subunits ATP1A1 and ATP1A3. Cell viability and intracellular calcium concentrations was measured in relation to the gene and protein expression of ATP1A1 and ATP1A3. Digitoxin was used to treat the cells at concentrations of 1-100 nM, and the intracellular calcium concentrations increased in a concentration-dependent manner in the Panc-1 and in the CFPAC-1 cells with treatment at 100 nM. In the AsPC-1 cells only the supraphysiological concentration of digitoxin (100 nM) resulted in a decrease in the number of viable cells (unviable cells increased to 22%), whereas it had no effect on intracellular calcium levels. The number of viable Panc-1 and CFPAC-1 cells decreased after digitoxin treatment at 25-100 nM (unviable Panc-1 cells increased to 33-59%; unviable CFPAC-1 cells increased to 22-56%). Digitoxin treatment also affected the transcriptional expression of the and subunits. In Panc-1 cells, gene expression was negatively associated with the digitoxin concentration (25-100 nM). In the AsPC-1 and CFPAC-1 cells, the expression of the gene increased in the cells treated with the 100 nM digitoxin concentration. The protein expression of ATP1A1 and ATP1A3 was not altered with digitoxin treatment. The basal protein expression of ATP1A1 was high in the AsPC-1 and CFPAC-1 cells, compared to the Panc-1 cells, in contrast to the basal expression of ATP1A3, which was higher in the Panc-1 cells, compared to the other pancreatic cancer cells used. On the whole, the present study demonstrates that the high expression of ATP1A3 renders pancreatic cancer cells more susceptible to digitoxin-induced cell death. The findings suggest that the expression of ATP1A3 may be used as a marker for tumor sensitivity to digitoxin treatment, where a high expression of ATP1A3 is favorable for the anticancer effects of digitoxin.
PubMed: 36698913
DOI: 10.3892/mi.2022.52 -
Genes Dec 2022L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient...
L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.
Topics: Digitalis; Codon, Initiator; Regeneration; DNA; Ploidies
PubMed: 36553602
DOI: 10.3390/genes13122335 -
International Journal of Oncology Aug 2020Cervical cancer is the fourth most common gynecological malignancy affecting the health of women worldwide and the second most common cause of cancer‑related mortality...
Cervical cancer is the fourth most common gynecological malignancy affecting the health of women worldwide and the second most common cause of cancer‑related 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 double‑stranded breaks (DSBs), inhibited the cell cycle at the G2/M phase via the ataxia telangiectasia mutated serine/threonine kinase (ATM)/ATM and Rad3‑related 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/Bcl‑2, the release of cytochrome c and the sequential activation of caspases and poly(ADP‑ribose) 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.
Topics: Animals; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Proliferation; Checkpoint Kinase 1; Checkpoint Kinase 2; Digitoxin; Female; G2 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; Inhibitory Concentration 50; Mice; Mitochondria; Signal Transduction; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays; cdc25 Phosphatases
PubMed: 32468057
DOI: 10.3892/ijo.2020.5070 -
Journal of Molecular Modeling Jun 2021The interaction between SARS-CoV-2 Spike protein and angiotensin-converting enzyme 2 (ACE2) is essential to viral attachment and the subsequent fusion process....
The interaction between SARS-CoV-2 Spike protein and angiotensin-converting enzyme 2 (ACE2) is essential to viral attachment and the subsequent fusion process. Interfering with this event represents an attractive avenue for the development of therapeutics and vaccine development. Here, a hybrid approach of ligand- and structure-based virtual screening techniques were employed to disclose similar analogues of a reported antiviral phytochemical, glycyrrhizin, targeting the blockade of ACE2 interaction with the SARS-CoV-2 Spike. A ligand-based similarity search using a stringent cut-off revealed 40 FDA-approved compounds in DrugBank. These filtered hits were screened against ACE2 using a blind docking approach to determine the natural binding tendency of the compounds with ACE2. Three compounds, deslanoside, digitoxin, and digoxin, were reported to show strong binding with ACE2. These compounds bind at the H1-H2 binding pocket, in a manner similar to that of glycyrrhizin which was used as a control. To achieve consistency in the docking results, docking calculations were performed via two sets of docking software that predicted binding energy as ACE2-Deslanoside (AutoDock, -10.3 kcal/mol and DockThor, -9.53 kcal/mol), ACE2-Digitoxin (AutoDock, -10.6 kcal/mol and DockThor, -8.84 kcal/mol), and ACE2-Digoxin (AutoDock, -10.6 kcal/mol and DockThor, -8.81 kcal/mol). The docking results were validated by running molecular simulations in aqueous solution that demonstrated the stability of ACE2 with no major conformational changes in the ligand original binding mode (~ 2 Å average RMSD). Binding interactions remained quite stable with an increased potential for getting stronger as the simulation proceeded. MMGB/PBSA binding free energies were also estimated and these supported the high stability of the complexes compared to the control (~ -50 kcal/mol net MMGB/PBSA binding energy versus ~ -30 kcal/mol). Collectively, the data demonstrated that the compounds shortlisted in this study might be subjected to experimental evaluation to uncover their real blockade capacity of SARS-CoV-2 host ACE2 receptor.
Topics: Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Antiviral Agents; Binding Sites; COVID-19; Drug Discovery; Drug Repositioning; Glycyrrhizic Acid; Host-Pathogen Interactions; Humans; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Protein Conformation; Receptors, Virus; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Structure-Activity Relationship; Virus Internalization; COVID-19 Drug Treatment
PubMed: 34169390
DOI: 10.1007/s00894-021-04816-y -
In Vivo (Athens, Greece) 2020Influenza viruses, corona viruses and related pneumotropic viruses cause sickness and death partly by inducing cytokine storm, a hyper-proinflammatory host response by...
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.
Topics: Animals; Betacoronavirus; COVID-19; Coronavirus Infections; Cytokines; Digitoxin; Disease Models, Animal; Humans; Influenza, Human; Lung; Male; NF-kappa B; Pandemics; Pneumonia, Viral; Prostatic Neoplasms; Rats; SARS-CoV-2; Tumor Necrosis Factor-alpha
PubMed: 33144490
DOI: 10.21873/invivo.12221 -
PeerJ 2022Na/K-ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several...
Na/K-ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several cardiotonic steroids (CTS) has been associated with the cytotoxic effect on cancer cell lines of phytochemicals such as ouabain and digitoxin. This study evaluated the inhibitory capacity of cardenolides calotropin and corotoxigenin 3-O-glucopyranoside (C3OG) from over the Na/K-ATPase activity and . The inhibitory assays showed that calotropin and C3OG decreased the Na/K-ATPase activity with IC values of 0.27 and 0.87 μM, respectively. Furthermore, the molecules presented an uncompetitive inhibition on Na/K-ATPase activity, with K values of 0.2 μM to calotropin and 0.5 μM to C3OG. Furthermore, the molecular modeling indicated that calotropin and C3OG might interact with the Thr and Gln residues, considered essential to the interaction with the Na/K-ATPase. Besides, these cardenolides can interact with amino acid residues such as Phe, Leu, and Ala, to establish hydrophobic interactions on the binding site. Considering the results, these provide novel evidence about the mechanism of action of cardenolides from , proposing that C3OG is a novel cardenolide that deserves further consideration for cellular antiproliferative assays and studies as an anticancer molecule.
Topics: Animals; Asclepias; Cardenolides; Cardiac Glycosides; Adenosine Triphosphatases; Mammals
PubMed: 35673388
DOI: 10.7717/peerj.13524 -
Virology Jan 2024A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and...
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 ECs 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.
Topics: Humans; Cardiac Glycosides; Monensin; Ouabain; Coronavirus 229E, Human; Digitoxin; Antiviral Agents
PubMed: 37931588
DOI: 10.1016/j.virol.2023.109915 -
Cell Biology and Toxicology Dec 2023Overcoming multidrug resistance (MDR) represents a major obstacle in cancer chemotherapy. Cardiac glycosides (CGs) are efficient in the treatment of heart failure and...
Overcoming multidrug resistance (MDR) represents a major obstacle in cancer chemotherapy. Cardiac glycosides (CGs) are efficient in the treatment of heart failure and recently emerged in a new role in the treatment of cancer. ZINC253504760, a synthetic cardenolide that is structurally similar to well-known GCs, digitoxin and digoxin, has not been investigated yet. This study aims to investigate the cytotoxicity of ZINC253504760 on MDR cell lines and its molecular mode of action for cancer treatment. Four drug-resistant cell lines (P-glycoprotein-, ABCB5-, and EGFR-overexpressing cells, and TP53-knockout cells) did not show cross-resistance to ZINC253504760 except BCRP-overexpressing cells. Transcriptomic profiling indicated that cell death and survival as well as cell cycle (G2/M damage) were the top cellular functions affected by ZINC253504760 in CCRF-CEM cells, while CDK1 was linked with the downregulation of MEK and ERK. With flow cytometry, ZINC253504760 induced G2/M phase arrest. Interestingly, ZINC253504760 induced a novel state-of-the-art mode of cell death (parthanatos) through PARP and PAR overexpression as shown by western blotting, apoptosis-inducing factor (AIF) translocation by immunofluorescence, DNA damage by comet assay, and mitochondrial membrane potential collapse by flow cytometry. These results were ROS-independent. Furthermore, ZINC253504760 is an ATP-competitive MEK inhibitor evidenced by its interaction with the MEK phosphorylation site as shown by molecular docking in silico and binding to recombinant MEK by microscale thermophoresis in vitro. To the best of our knowledge, this is the first time to describe a cardenolide that induces parthanatos in leukemia cells, which may help to improve efforts to overcome drug resistance in cancer. A cardiac glycoside compound ZINC253504760 displayed cytotoxicity against different multidrug-resistant cell lines. ZINC253504760 exhibited cytotoxicity in CCRF-CEM leukemia cells by predominantly inducing a new mode of cell death (parthanatos). ZINC253504760 downregulated MEK1/2 phosphorylation and further affected ERK activation, which induced G2/M phase arrest.
Topics: Humans; Apoptosis; Phosphorylation; Cell Line, Tumor; Cardiac Glycosides; Down-Regulation; Molecular Docking Simulation; ATP Binding Cassette Transporter, Subfamily G, Member 2; Parthanatos; G2 Phase Cell Cycle Checkpoints; Neoplasm Proteins; Leukemia; Cardenolides; Mitogen-Activated Protein Kinase Kinases; Drug Resistance, Neoplasm
PubMed: 37322258
DOI: 10.1007/s10565-023-09813-w -
Rheumatology (Oxford, England) Oct 2021Direct inhibition of M1 polarization of synovial macrophages may be a useful therapeutic treatment for OA and OA-associated synovitis. Frugoside (FGS) is a cardiac...
OBJECTIVES
Direct inhibition of M1 polarization of synovial macrophages may be a useful therapeutic treatment for OA and OA-associated synovitis. Frugoside (FGS) is a cardiac glycoside compound isolated and extracted from Calotropis gigantea. Cardiac glycosides possess interesting anti-inflammatory potential. However, the corresponding activity of FGS has not been reported. Therefore, our aim was to find direct evidence of the effects of FGS on synovial macrophage M1 polarization and OA control.
METHODS
Collagenase was used to establish an experimental mouse OA model (CIOA) with considerable synovitis. Then, FGS was intra-articular administered. The mRNA and protein levels of iNOS were analysed by real-time PCR and Western blotting in vitro. Immunohistochemical and immunofluorescence staining were used to measure the expression of F4/80, iNOS, Col2α1 and MMP13 in vivo. The levels of pro-inflammatory cytokines in FGS-treated M1 macrophage culture supernatants were analysed by flow cytometry.
RESULTS
FGS attenuates synovial inflammation and delays the development of OA in CIOA mice. Further results demonstrate that FGS inhibits macrophage M1 polarization in vitro and in vivo, which subsequently decreases the secretion of IL-6 and TNF-α, in turn delaying cartilage and extracellular matrix (ECM) degradation and chondrocyte hypertrophy. FGS inhibits macrophage M1 polarization by partially downregulating miR-155 levels.
CONCLUSION
This study demonstrates that intra-articular injection of FGS is a potential strategy for OA prevention and treatment, even at an early stage of disease progression. This is a novel function of FGS and has promising future clinical applications.
Topics: Animals; Blotting, Western; Digitoxigenin; Disease Models, Animal; Disease Progression; Flow Cytometry; Fluorescent Antibody Technique; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; MicroRNAs; Osteoarthritis; Real-Time Polymerase Chain Reaction; Synovial Fluid
PubMed: 33493345
DOI: 10.1093/rheumatology/keab018 -
Electrophoresis May 2022Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication...
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.
Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Digitoxin; Digoxin; Immunoassay; Tandem Mass Spectrometry
PubMed: 35132652
DOI: 10.1002/elps.202100290