-
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 -
Journal of Natural Products Dec 2019Ten cardiac glycosides (-) including six 20,22-dihydrodigitoxigenin and four gitoxigenin glycosides were isolated from the stems of together with six known triterpenoid...
Ten cardiac glycosides (-) including six 20,22-dihydrodigitoxigenin and four gitoxigenin glycosides were isolated from the stems of together with six known triterpenoid cinnamates. Spectroscopic data of these previously undescribed compounds are reported. All isolates were evaluated for their growth inhibitory activities against three cancer cell lines, and compound was the most active against KB cells with an IC value of 0.03 ± 0.001 μM. Also, compounds , , , and and the triterpenoid cinnamates - showed inhibitory activity (IC < 10 μM) for one or more of the cell lines used.
Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Digitoxigenin; Drug Screening Assays, Antitumor; Glycosides; Humans; Molecular Structure; Plant Stems; Spectrum Analysis
PubMed: 31820973
DOI: 10.1021/acs.jnatprod.9b00803 -
Oncology Letters Oct 2020Hepatocellular carcinoma (HCC) remains a challenge in the medical field due to its high malignancy and mortality rates particularly for HCC, which has developed...
Hepatocellular carcinoma (HCC) remains a challenge in the medical field due to its high malignancy and mortality rates particularly for HCC, which has developed multidrug resistance. Therefore, the identification of efficient chemotherapeutic drugs for multidrug resistant HCC has become an urgent issue. Natural products have always been of significance in drug discovery. In the present study, a cell-based method was used to screen a natural compound library, which consisted of 78 compounds, and the doxorubicin-resistant cancer cell line, HepG2/ADM, as screening tools. The findings of the present study led to the shortlisting of one of the compounds, digitoxin, which displayed an inhibitory effect on HepG2/ADM cells, with 50% inhibitory concentration values of 132.65±3.83, 52.29±6.26, and 9.13±3.67 nM for 24, 48, and 72 h, respectively. Immunofluorescence, western blotting and cell cycle analyses revealed that digitoxin induced G/M cell cycle arrest via the serine/threonine-protein kinase ATR (ATR)-serine/threonine-protein kinase Chk2 (CHK2)-M-phase inducer phosphatase 3 (CDC25C) signaling pathway in HepG2/ADM cells, which may have resulted from a DNA double-stranded break. Digitoxin also induced mitochondrial apoptosis, which was characterized by changes in the interaction between Bcl-2 and Bax, the release of cytochrome , as well as the activation of the caspase-3 and -9. To the best of our knowledge, the present study is the first report that digitoxin displays an anti-HCC effect on HepG2/ADM cells through G/M cell cycle arrest, which was mediated by the ATR-CHK2-CDC25C signaling pathway and mitochondrial apoptosis. Therefore, digitoxin could be a promising chemotherapeutic agent for the treatment of patients with HCC.
PubMed: 32863904
DOI: 10.3892/ol.2020.11932 -
Biomedicine & Pharmacotherapy =... Apr 2021Cancer is one of the main causes of death in the world and thus a global public health problem. Among the treatments available for cancer are surgery, radiotherapy, and...
Cancer is one of the main causes of death in the world and thus a global public health problem. Among the treatments available for cancer are surgery, radiotherapy, and chemotherapy. Currently, there is increased interest in the combination of two or more antitumor agents to achieve a synergistic effect in cancer therapy. Doxorubicin (DOX), a chemotherapeutic which has a potent antineoplastic action, has been used in the treatment of various tumors. However, the use of DOX is limited, mainly due to the cardiotoxicity. Therefore, nanostructured systems, such as liposomes, have been developed to carry this drug and target the tumor region, since tumor tissues present enhanced permeability and retention for nanosystems. Cardiac glycosides, such as digitoxin, have recently shown great antitumor potential despite the low therapeutic index which may limit their use. Furthermore, some compounds of this class have low water solubility, which makes their in vivo administration difficult. In this context, liposomes represent a valid strategy to carry simultaneously antitumor drugs allowing their intravenous administration. In this study, liposomes loaded with glucoevatromonoside containing peracetylated glucose hydroxyl groups (GEVPG) and DOX at molar ratio of 1:1 (SpHL-GEVPG:DOX 1:1) were developed, and their chemical and physicochemical properties were evaluated. This formulation presented a combination index (CI) lower than 1 at inhibitory concentration of 90 % growth (IC) for three human breast tumor lines evaluated (0.52 ± 0.39 for MDA-MB-231, 0.19 ± 0.13 for MCF-7, and 0.99 ± 0.09 for SKBR-3). These results indicate a synergistic cytotoxic effect of the GEVPG and DOX combination encapsulated in liposomes. In addition, SpHL-GEVPG:DOX 1:1 presented selectivity towards these cancer cells. Long-term in vitro cytotoxicity studies demonstrated that MDA-MB-231 surviving cells after treatment with SpHL-GEVPG:DOX 1:1 did not recover proliferation capacity after 21 d. From the studies of cell cycle and death pathway evaluation, it was observed that SpHL-GEVPG:DOX 1:1 arrested the cell cycle in the G2/M phase and similarly induced apoptosis and necrosis. However, SpHL-GEVPG:DOX at molar ratio of 1:1 showed lower induction of both apoptotic and necrotic pathways compared to free DOX and SpHL-DOX, suggesting that the mechanism of death involved may not be related to necrosis or apoptosis. Lastly, SpHL-GEVPG:DOX 1:1 showed a good storage stability for 90 d at 4 °C. Therefore, the results of the present work indicate the potential use of SpHL-GEVPG:DOX 1:1 as a new anticancer formulation.
Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cardenolides; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Drug Compounding; Drug Synergism; Female; Humans; Lipids; Liposomes; MCF-7 Cells; Necrosis; Time Factors
PubMed: 33486211
DOI: 10.1016/j.biopha.2020.111123 -
Journal of Fluorescence Sep 2023The interaction of orphenadrine hydrochloride (ORD) with the model protein, bovine serum albumin (BSA), was investigated using a variety of spectroscopic techniques such...
The interaction of orphenadrine hydrochloride (ORD) with the model protein, bovine serum albumin (BSA), was investigated using a variety of spectroscopic techniques such as steady-state fluorescence, ultraviolet-visible, Fourier transform infrared, 3-D spectroscopy, and electrochemical methods under physiological conditions. Stern-Volmer plots were used to calculate fluorescence quenching at various temperatures. The findings point to a static quenching mechanism between ORD and BSA. At various reaction times, the binding sites (n) and binding constants (K) of ORD to BSA were recorded. Thermodynamic parameters ∆H, ∆S and ∆G between ORD and BSA were calculated and reported. The average binding distance (r) between the donor (BSA) and acceptor (ORD) molecules was predicted using Förster's theory. Three-dimensional fluorescence spectra, Fourier transform infrared spectra, and synchronous fluorescence studies all supported the alternations in protein structure following the interaction with ORD. A displacement study using site probes such as warfarin, ibuprofen, and digitoxin confirmed ORD binding at Sudlow's site I of BSA. The effect of common metal ions such as Cu, Ni, Ca, Co, and Zn on binding constant values was investigated and reported.
Topics: Serum Albumin, Bovine; Orphenadrine; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Binding Sites; Thermodynamics; Protein Binding; Circular Dichroism
PubMed: 36976401
DOI: 10.1007/s10895-023-03199-y -
Pharmacological Research Jan 2023Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various...
Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3'-epi-12β-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.
Topics: Animals; Humans; Mice; Apoptosis; Autophagy; Cell Line, Tumor; Immunogenic Cell Death; Receptor-Interacting Protein Serine-Threonine Kinases; Triple Negative Breast Neoplasms; Tumor Microenvironment
PubMed: 36535569
DOI: 10.1016/j.phrs.2022.106613 -
Science & Justice : Journal of the... Mar 2022Detection of seed-based toxins is a need for forensic chemists when suspected poisonings occur. The evidence that is found is often physically unidentifiable, as the...
Detection of seed-based toxins is a need for forensic chemists when suspected poisonings occur. The evidence that is found is often physically unidentifiable, as the seeds are mashed to extract the toxin. This work investigates potential strategies for rapid detection of seed-based toxins and seed mashes containing these toxins using chemical signatures obtained by direct analysis in real time mass spectrometry (DART-MS). Seven toxins (digoxin, digitoxin, hypaconitine, hyoscyamine, lanatoside, oleandrin, and scopolamine) and six seeds containing these toxins were studied. While detection of four of the toxins was readily attainable, detection of digoxin, digitoxin, and lanatoside was hindered by the inability to thermally desorb these larger compounds under normal operating conditions. The use of DART-MS variants capable of higher desorption temperatures (thermal desorption (TD)-DART-MS and infrared thermal desorption (IRTD)-DART-MS) enabled detection of these compounds. Detection of toxins from direct analysis of seed mashes and methanolic seed mash extracts was found to be compound and technique dependent. Principal component analysis (PCA) of generated mass spectra enabled differentiation of seed species, even in cases where the toxins were undetectable.
Topics: Digitoxin; Digoxin; Humans; Mass Spectrometry; Principal Component Analysis; Seeds
PubMed: 35277227
DOI: 10.1016/j.scijus.2021.12.004 -
Chemico-biological Interactions Jun 2022Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na/K ATPase and have similar effects as cardiac...
Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na/K ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term "Myc active pathway" according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed "MYC_TARGETS_V2" with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as "TNFA_SIGNALING_VIA_NFKB", "APOPTOSIS", and "TGF_BETA_SIGNALING" were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.
Topics: Apoptosis; Bufanolides; Cell Line, Tumor; Cell Proliferation; Humans; Leukemia, Myeloid, Acute
PubMed: 35447139
DOI: 10.1016/j.cbi.2022.109936 -
Anti-cancer Drugs Jun 2020Cardiac glycosides (CGs) are natural compounds traditionally used for the treatment of heart disorders, and recently new therapeutic possibilities were proposed. Their...
Cardiac glycosides (CGs) are natural compounds traditionally used for the treatment of heart disorders, and recently new therapeutic possibilities were proposed. Their antitumor reports and clinical trials have notably enhanced, including those targeted for lung cancer, the most lethal type that lacks of new treatment agents, instigating the research of these molecules. The CGs studied here, named C10 {3β-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin} and C18 (3β-(aminoacetyl)amino-3-deoxydigitoxigenin), are semisynthetic derivatives prepared from digitoxigenin scaffold. Both compounds demonstrated high cytotoxicity for different cancer cell lines, especially H460 lung cancer cells, and their cytotoxic effects were deeply investigated using different methodological approaches. C10 induced cell death at lower concentrations and during shorter periods of treatment than C18, and increased the number of small and irregular nuclei, which are characteristics of apoptosis. This type of cell death was confirmed by caspase-3/7 assay. Both compounds reduced H460 cells proliferative potential by long-term action, and C10 showed the strongest potential. Moreover, these compounds induced a significant decrease of the area and viability of H460 spheroids providing preclinical favorable profiles to develop new chemotherapeutic agents.
Topics: Antineoplastic Agents; Apoptosis; Cell Proliferation; Digitoxigenin; Humans; Lung Neoplasms; Tumor Cells, Cultured
PubMed: 32079825
DOI: 10.1097/CAD.0000000000000872 -
Biochemical and Biophysical Research... Feb 2020Ebola virus (EBOV), pathogen of Ebola hemorrhagic fever (EHF), is an enveloped filamental RNA virus. Recently, the EHF crisis occurred in the Democratic Republic of the...
Ebola virus (EBOV), pathogen of Ebola hemorrhagic fever (EHF), is an enveloped filamental RNA virus. Recently, the EHF crisis occurred in the Democratic Republic of the Congo again highlights the urgency for its clinical treatments. However, no Food and Drug Administration (FDA)-approved therapeutics are currently available. Drug repurposing screening is a time- and cost-effective approach for identifying anti-EBOV therapeutics. Here, by combinatorial screening using pseudovirion and minigenome replicon systems we have identified several FDA-approved drugs with significant anti-EBOV activities. These potential candidates include azithromycin, clomiphene, chloroquine, digitoxin, epigallocatechin-gallate, fluvastatin, tetrandrine and tamoxifen. Mechanistic studies revealed that fluvastatin inhibited EBOV pseudovirion entry by blocking the pathway of mevalonate biosynthesis, while the inhibitory effect of azithromycin on EBOV maybe due to its intrinsic cationic amphiphilic structure altering the homeostasis of later endosomal vesicle similar as tamoxifen. Moreover, based on structure and pathway analyses, the anti-EBOV activity has been extended to other family members of statins, such as simvastatin, and multiple other cardiac glycoside drugs, some of which exhibited even stronger activities. More importantly, in searching for drug interaction, we found various synergy between several anti-EBOV drug combinations, showing substantial and powerful synergistic against EBOV infection. In conclusion, our work illustrates a successful and productive approach to identify new mechanisms and targets for treating EBOV infection by combinatorial screening of FDA-approved drugs.
Topics: Antiviral Agents; Azithromycin; Cardiac Glycosides; Cell Line; Cholesterol; Combinatorial Chemistry Techniques; Drug Approval; Drug Evaluation, Preclinical; Drug Synergism; Ebolavirus; Fluvastatin; Humans; Mevalonic Acid; Models, Biological; Surface-Active Agents; Virion; Virus Internalization; Virus Replication
PubMed: 31806372
DOI: 10.1016/j.bbrc.2019.11.065