-
Biochemical Pharmacology Apr 2024We previously showed that digitoxin inhibits angiogenesis and cancer cell proliferation and migration and these effects were associated to protein tyrosine kinase 2...
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.
Topics: Humans; Reactive Oxygen Species; Digitoxin; Human Umbilical Vein Endothelial Cells; Focal Adhesion Kinase 1; Phosphorylation; Cell Movement; NADPH Oxidases; rhoA GTP-Binding Protein; rho-Associated Kinases
PubMed: 38342347
DOI: 10.1016/j.bcp.2024.116049 -
Frontiers in Molecular Biosciences 2023Digitoxin is a cardiac glycoside used to treat heart failure and heart arrhythmia. However, its therapeutic concentration range is very narrow. High doses of digitoxin...
Digitoxin is a cardiac glycoside used to treat heart failure and heart arrhythmia. However, its therapeutic concentration range is very narrow. High doses of digitoxin are associated with severe side effects; therefore, it is necessary to develop the delivery system which can control the plasma levels of it. In this context, the binding of lysozyme, an important protein having many applications, with digitoxin has been studied to see the ability of the former as a carrier. The studies were carried out using both experimental and computational methods. The intrinsic fluorescence of lysozyme increased on the addition of digitoxin. Fluorescence results suggested that there was a strong interaction between lysozyme and digitoxin which was favored, mainly, by hydrophobic forces. Further, digitoxin affected the secondary structure of lysozyme slightly by causing the partial unfolding of lysozyme. The preferred binding site of digitoxin within lysozyme was the large cavity of the protein. Molecular docking studies also established the principal role of hydrophobic forces in the binding with a significant support of hydrogen bonding. Frontier molecular orbitals of free digitoxin and in complexation with lysozyme were also computed and discussed. The findings from molecular dynamics simulation studies elucidate that, when contrasted with the first and third conformations of the digitoxin-bound lysozyme complex, the second conformation promotes structural stability, reduces flexibility, and enhances the compactness and folding properties of lysozyme. The overall study shows that lysozyme could act as a potential carrier for digitoxin in pharmaceutical formulations.
PubMed: 38187092
DOI: 10.3389/fmolb.2023.1327740 -
International Immunopharmacology Feb 2024Rheumatoid arthritis (RA) is an autoimmune disease that affects joints, causing inflammation, synovitis, and erosion of cartilage and bone. Periplogenin is an active...
Rheumatoid arthritis (RA) is an autoimmune disease that affects joints, causing inflammation, synovitis, and erosion of cartilage and bone. Periplogenin is an active ingredient in the anti-rheumatic and anti-inflammatory herb, cortex periplocae. We conducted a study using a CIA model and an in vitro model of fibroblast-like synoviocytes (FLS) induced by Tumor Necrosis Factor-alpha (TNF-α) stimulation. We evaluated cell activity, proliferation, and migration using the CCK8 test, EDU kit, and transwell assays, as well as network pharmacokinetic analysis of periplogenin targets and RA-related effects. Furthermore, we measured inflammatory factors and matrix metalloproteinases (MMPs) expression using ELISA and qRT-PCR assays. We also evaluated joint destruction using HE and Safranin O-Fast Green Staining and examined the changes in the JAK2/3-STAT3 pathway using western blot. The results indicated that periplogenin can effectively inhibit the secretion of inflammatory factors, suppress the JAK2/3-STAT3 pathway, and impede the proliferation and migration of RA FLS. Thus, periplogenin alleviated the Synovial inflammatory infiltration of RA.
Topics: Humans; Animals; Arthritis, Rheumatoid; Synoviocytes; Inflammation; Cell Proliferation; Fibroblasts; Synovial Membrane; Cells, Cultured; Arthritis, Experimental; Janus Kinase 2; STAT3 Transcription Factor; Digitoxigenin
PubMed: 38183911
DOI: 10.1016/j.intimp.2024.111487 -
Clinical Research in Cardiology :... Aug 2023The present study aimed to develop a simple dosing score when starting the cardiac glycoside digitoxin in heart failure with reduced ejection fraction (HFrEF) employing... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
The present study aimed to develop a simple dosing score when starting the cardiac glycoside digitoxin in heart failure with reduced ejection fraction (HFrEF) employing first data from the randomized, double-blinded DIGIT-HF trial.
METHODS AND RESULTS
In DIGIT-HF, digitoxin was started with a dose of 0.07 mg once daily (o.d.) in all patients. For score derivation, 317 patients were analyzed who had been randomized to digitoxin. In these patients, after scheduled determination of serum levels at study week 6, the digitoxin dose had remained unchanged or had been reduced to 0.05 mg o.d. (97% of patients) to achieve serum concentrations within a predefined range (10.5-23.6 nmol/l). In logistic regression analyses, sex, age, body mass index (BMI), and estimated glomerular filtration rate (eGFR) were associated with need for dose reduction and, therefore, selected for further developing the dosing score. Optimal cut-points were derived from ROC curve analyses. Finally, female sex, age ≥ 75 years, eGFR < 50 ml/min/1.73 m, and BMI < 27 kg/m each were assigned one point for the digitoxin dosing score. A score of ≥ 1 indicated the need for dose reduction with sensitivity/specificity of 81.6%/49.7%, respectively. Accuracy was confirmed in a validation data set including 64 patients randomized to digitoxin yielding sensitivity/specificity of 87.5%/37.5%, respectively.
CONCLUSION
In patients with HFrEF, treatment with digitoxin should be started at 0.05 mg o.d. in subjects with either female sex, eGFR < 50 ml/min/1.73m, BMI < 27 kg/m, or age ≥ 75 years. In any other patient, digitoxin may be safely started at 0.07 mg o.d.
Topics: Humans; Female; Aged; Heart Failure; Digitoxin; Stroke Volume; ROC Curve; Sensitivity and Specificity
PubMed: 37087503
DOI: 10.1007/s00392-023-02199-z -
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 -
The Journal of Organic Chemistry Aug 2023The Mislow-Evans rearrangement was used as a key reaction to construct digitoxose-derived glycals. The same rearrangement was iteratively performed on di- and...
The Mislow-Evans rearrangement was used as a key reaction to construct digitoxose-derived glycals. The same rearrangement was iteratively performed on di- and trisaccharides to form the digoxose glycal donor component present in the cardenolides digitoxin, digoxin, and gitoxin. The scalability of the trisaccharide synthesis was shown by performing the reactions on a multigram scale. Glycosylation reactions were also performed between the synthesized digoxin glycal donor and aglycons digoxigenin and gitoxigenin to synthesize novel cardenolide derivatives.
PubMed: 37555372
DOI: 10.1021/acs.joc.3c01067 -
European Journal of Pharmaceutical... Apr 2024In this communication, the solubility of digitoxin drug in supercritical CO was studied at different operating conditions (311 < T (K) < 343, 120 < P (bar) < 300). The...
In this communication, the solubility of digitoxin drug in supercritical CO was studied at different operating conditions (311 < T (K) < 343, 120 < P (bar) < 300). The results revealed digitoxin drug solubility (in mole fraction) was between 0.095 × 10 to 1.12 × 10. In the case of thermodynamic solubility modeling, cubic and non-cubic equation of states i.e. SAFT (statistical associating fluid theory), SRK (Soave-Redlich-Kwong) and sPC-SAFT (simplified perturbed chain SAFT) EoSs and six density-based correlations (Chrastil, Kumar-Johnston (KJ), Mendez-Santiago-Teja (MST), Garlapati and Madras (GM), Bartle et al. and Sung-Shim models) were considered. All used equations indicated reasonable behavior with appropriate accuracy for the solubility of the digitoxin drug. Meanwhile, sPC-SAFT EoS and Kumar-Johnston correlation with AARD% set to 8.96 % and 6.25 %, respectively exhibited greater accuracy in fitting the solubility data. Moreover, total, solvation and vaporization enthalpies of the digitoxin/supercritical carbon dioxide binary mixture were calculated based on KJ, Chrastil and Bartle et al. models.
Topics: Carbon Dioxide; Solubility; India; Thermodynamics
PubMed: 38387711
DOI: 10.1016/j.ejps.2024.106731 -
Cellular and Molecular Biology... Jun 2024Colorectal cancer (CRC) poses a significant global health challenge with high morbidity and mortality rates. This study investigates the role of LY6G6D, a member of the...
Colorectal cancer (CRC) poses a significant global health challenge with high morbidity and mortality rates. This study investigates the role of LY6G6D, a member of the LY6/uPAR superfamily, in CRC. Employing a bioinformatic approach, we analyzed LY6G6D expression across different cancer types, compared it with known oncogenes in CRC, explored the involved genomic alterations, and assessed associated clinicopathological characteristics. LY6G6D exhibited aberrant expression, particularly elevated in CRC adenocarcinoma and highly specific to tumor tissues when compared with other oncogenes, despite its comparatively low frequency of genomic alteration. Subsequently, tumor immune infiltration analysis revealed distinct associations, primarily indicating a negative correlation, suggesting immune down-regulation. Survival analysis in context of LY6G6D was conducted with Kaplan-Meier (KM) curves, indicating a 10% risk of disease recurrence in the case of elevated expression. Additionally, we constructed a 3D protein model of LY6G6D through ab-inito approach. The protein model was validated, followed by conservation analysis and active site identification. Active site identification of LY6G6D's final predicted model revealed some similar sites that were estimated to be conserved. Target-guided drug molecules were collected and molecular docking was executed, proposing Cardigin (Digitoxin) and Manzamine A as potential therapeutic candidates. In conclusion, LY6G6D emerges as a significant biomarker for diagnostic and therapeutic applications in CRC, highlighting its multifaceted role in tumorigenesis. The proposed drugs present avenues for further investigations.
Topics: Colorectal Neoplasms; Humans; Biomarkers, Tumor; Gene Expression Regulation, Neoplastic; Molecular Docking Simulation; Antigens, Ly; GPI-Linked Proteins
PubMed: 38836687
DOI: 10.14715/cmb/2024.70.6.3