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1.
Int J Mol Sci ; 25(14)2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39063039

ABSTRACT

Acute myeloid leukemia (AML) is characterized by the abnormal proliferation and differentiation arrest of myeloid progenitor cells. The clinical treatment of AML remains challenging. Promoting AML cell differentiation is a valid strategy, but effective differentiation drugs are lacking for most types of AML. In this study, we generated Tg(drl:hoxa9) zebrafish, in which hoxa9 overexpression was driven in hematopoietic cells and myeloid differentiation arrest was exhibited. Using Tg(drl:hoxa9) embryos, we performed chemical screening and identified four FDA-approved drugs, ethacrynic acid, khellin, oxcarbazepine, and alendronate, that efficiently restored myeloid differentiation. The four drugs also induced AML cell differentiation, with ethacrynic acid being the most effective. By an RNA-seq analysis, we found that during differentiation, ethacrynic acid activated the IL-17 and MAPK signaling pathways, which are known to promote granulopoiesis. Furthermore, we found that ethacrynic acid enhanced all-trans retinoic acid (ATRA)-induced differentiation, and both types of signaling converged on the IL-17/MAPK pathways. Inhibiting the IL-17/MAPK pathways impaired ethacrynic acid and ATRA-induced differentiation. In addition, we showed that ethacrynic acid is less toxic to embryogenesis and less disruptive to normal hematopoiesis than ATRA. Thus, the combination of ethacrynic acid and ATRA may have broader clinical applications. In conclusion, through zebrafish-aided screening, our study identified four drugs that can be repurposed to induce AML differentiation, thus providing new agents for AML therapy.


Subject(s)
Cell Differentiation , Leukemia, Myeloid, Acute , Zebrafish , Animals , Zebrafish/embryology , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/metabolism , Cell Differentiation/drug effects , Humans , Embryo, Nonmammalian/drug effects , Tretinoin/pharmacology , Ethacrynic Acid/pharmacology , Antineoplastic Agents/pharmacology
2.
Nanotechnology ; 35(34)2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38788695

ABSTRACT

Although chlorambucil (CHL) is a long-established anticancer drug, the drug failure of CHL, mediated by the intracellular defense system consisting of glutathione (GSH) and GSH S-transferase pi (GST-pi), has significantly limited the application of CHL. To overcome this issue, we first designed a GSH-responsive small-molecule prodrug (EA-SS-CHL) by combining CHL and ethacrynic acid (EA). Subsequently, drug-loaded nanoparticles (ECPP) were formed by the self-assembly between EA-SS-CHL and amphiphilic PEG-PDLLA to improve the water solubility of the prodrug and its ability to target tumor sites. Upon exposure to high intracellular GSH concentration, EA-SS-CHL gradually degrades, leading to the release of EA and CHL. The presence of EA facilitates the depletion of GSH and inhibition of GST-pi, ultimately attenuating the detoxification of the intracellular defense system to CHL. Cytotoxicity studies and apoptosis assays demonstrate that ECPP exhibits higher therapeutic efficiency than CHL. Additionally,in vivotumor suppression effects and biocompatibility provide further evidence for the superiority of ECPP. This work presents a promising strategy to enhance the efficacy of CHL in cancer therapy.


Subject(s)
Chlorambucil , Ethacrynic Acid , Glutathione , Micelles , Prodrugs , Chlorambucil/pharmacology , Chlorambucil/chemistry , Prodrugs/pharmacology , Prodrugs/chemistry , Glutathione/metabolism , Humans , Animals , Ethacrynic Acid/pharmacology , Ethacrynic Acid/chemistry , Nanoparticles/chemistry , Mice , Glutathione S-Transferase pi/metabolism , Glutathione S-Transferase pi/antagonists & inhibitors , Apoptosis/drug effects , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Polyethylene Glycols/chemistry , Glutathione Transferase/metabolism , Drug Carriers/chemistry , Drug Liberation
3.
Anticancer Agents Med Chem ; 24(6): 412-422, 2024.
Article in English | MEDLINE | ID: mdl-38204257

ABSTRACT

BACKGROUND AND OBJECTIVE: All-trans retinoic acid (ATRA), an effective differentiation inducer, has been applied clinically to treat acute promyelocytic leukemia (APL). Unfortunately, it is not as potent in other kinds of acute myeloid leukemia (AML). Ethacrynic acid (EA), a classical powerful diuretic, can increase reactive oxygen species (ROS) contents, which can assist ATRA in inducing differentiation in AML cells. Here, we investigated the effect of EA combined with ATRA (EA+RA) on some AML cells except APL. METHODS: Apoptosis and differentiation were determined by morphology, cell viability, Annexin-V assay and CD11c expression. Western blot analysis and the detection of ROS and mitochondrial transmembrane potentials (MMP) were used to investigate the mechanisms. RESULTS: AML cells exhibited differentiation and/or apoptosis after EA+RA treatment. EA+RA increased the intracellular ROS contents. EA+RA-induced apoptosis was accompanied by MMP attenuation and caspase-3/7 activation. EA+RA-induced differentiation was along with MEK/ERK and Akt activation and increased expression of PU.1, CCAAT/enhancer-binding protein ß (C/EBPß) and C/EBPε. N-acetyl-L-cysteine (NAC), an antioxidant, thoroughly reduced EA+RA-increased ROS, and also inhibited MMP attenuation, the activation of caspase- 3/7, MEK/ERK and Akt pathways, the elevation of PU.1 and C/EBPs, and apoptosis and differentiation. However, MEK or PI3K specific inhibitors only suppressed EA+RA-triggered differentiation and the elevation of PU.1 and C/EBPs, but not ROS levels. CONCLUSION: EA+RA induced cell apoptosis through ROS dependent MMP attenuation and caspase 3/7 activation while inducing differentiation by ROS-MEK/ERK-PU.1/C/EBPs and ROS-Akt-PU.1/C/EBPs pathways. In summary, it may provide innovative ATRA-based combination therapy strategies for AML patients via ROS.


Subject(s)
Antineoplastic Agents , Apoptosis , Cell Differentiation , Drug Screening Assays, Antitumor , Ethacrynic Acid , Leukemia, Myeloid, Acute , Reactive Oxygen Species , Tretinoin , Humans , Apoptosis/drug effects , Reactive Oxygen Species/metabolism , Tretinoin/pharmacology , Tretinoin/chemistry , Cell Differentiation/drug effects , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/metabolism , Ethacrynic Acid/pharmacology , Ethacrynic Acid/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Cell Survival/drug effects , Dose-Response Relationship, Drug , Molecular Structure , Cell Proliferation/drug effects , Structure-Activity Relationship , Tumor Cells, Cultured
4.
Biochem Pharmacol ; 212: 115537, 2023 06.
Article in English | MEDLINE | ID: mdl-37019184

ABSTRACT

Lung cancer is characterized by high incidence and mortality. 90% of cancer deaths are caused by metastases. The epithelial-mesenchymal transition (EMT) process in cancer cells is a prerequisite for the metastatic process. Ethacrynic acid (ECA) is a loop diuretic that inhibits the EMT process in lung cancer cells. EMT has been related to the tumour immunemicroenvironment. However, the effect of ECA on immune checkpoint molecules in the context of cancer has not been fully identified. In the present study, we found that sphingosylphosphorylcholine (SPC) and TGF-ß1, awell-known EMT inducer, induced the expression of B7-H4 in lung cancer cells. We also investigated the involvement of B7-H4 in the SPC-induced EMT process. Knockdown of B7-H4 suppressed SPC-induced EMT, while B7-H4 overexpression enhanced EMT of lung cancer cells. ECA inhibited SPC/TGF-ß1-induced B7-H4 expression via suppression of STAT3 activation. Moreover, ECA inhibits the colonization of mice lung by tail vein-injected LLC1 cells. ECA-treated mice increased the CD4-positive T cells in lung tumour tissues. In summary, these results suggested that ECA inhibits B7-H4 expression via STAT3 inhibition, leading to SPC/TGF-ß1-induced EMT. Therefore, ECA might be an immune oncological drug for B7-H4-positive cancer, especially lung cancer.


Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Animals , Mice , Transforming Growth Factor beta1/metabolism , Ethacrynic Acid/pharmacology , Ethacrynic Acid/therapeutic use , Epithelial-Mesenchymal Transition , Cell Line, Tumor , Cell Movement , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism
5.
Molecules ; 28(2)2023 Jan 16.
Article in English | MEDLINE | ID: mdl-36677966

ABSTRACT

The present study aims to report the design, synthesis, and biological activity of new ethacrynic acid (EA) analogs (6-10) obtained by the double modulation of the carboxylic acid moiety and the aromatic ring with the aim to increase the chemical reactivity of Michael acceptor of EA. All obtained compounds were characterized by 1H and 13C NMR, IR, and high-resolution mass spectrometry. The antiproliferative activity was evaluated in vitro using MMT test, in a first step, against HL60 cell line and in a second step, on a panel of human cancer cell lines such as HCT116, A549, MCF7, PC3, U87-MG, and SKOV3, and normal cell line MRC5 in comparison with positive control doxorubicin. Among all the tested compounds, the product 8 containing a propargyl and a hydroxyl groups, allowing an intramolecular hydrogen bond with the keto group of EA, exhibited a pronounced and selective activity in a nanomolar range against HL60, A549, PC3, and MCF7 with IC50 values of 15, 41.2, 68.7, and 61.5 nM, respectively. Compound 8 also showed a good selectivity index (SI) against HL60 and moderate SI against the other three human cancer cells (A549, PC3, and MCF7). The study of the structure-activity relationship showed that both modifications of the carboxylic group and the introduction of an intramolecular hydrogen bond are highly required to improve the antiproliferative activities. The molecular modeling studies of compound 8 revealed that it favorably binds to the glutathione S-transferase active site, which may explain its interesting anticancer activity. These new compounds have potential to be developed as novel therapeutic agents against various cancer types.


Subject(s)
Antineoplastic Agents , Ethacrynic Acid , Humans , Cell Line, Tumor , Ethacrynic Acid/pharmacology , Antineoplastic Agents/chemistry , Cell Proliferation , Structure-Activity Relationship , Drug Screening Assays, Antitumor , Molecular Structure
6.
BMC Pharmacol Toxicol ; 23(1): 35, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35642005

ABSTRACT

BACKGROUND: Ethacrynic acid (EA) is a loop diuretic that is approved orally and parenterally to manage edema-associated diseases. Nevertheless, it was earlier reported that it is also associated with bleeding upon its parenteral administration. In this report, we investigated the effects of EA on human factor XIIIa (FXIIIa) of the coagulation process using a variety of techniques. METHODS: A series of biochemical and computational methods have been used in this study. The potency and efficacy of human FXIIIa inhibition by EA was evaluated using a bisubstrate-based fluorescence trans-glutamination assay under near physiological conditions. To establish the physiological relevance of FXIIIa inhibition by EA, the effect on FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α2-antiplasmin complex was evaluated using SDS-PAGE experiments. The selectivity profile of EA against other coagulation proteins was assessed by evaluating EA's effect on human clotting times in the activated partial thromboplastin time (APTT) and the prothrombin time (PT) assays. We also used molecular modeling studies to put forward a putative binding mode for EA in the active site of FXIIIa. Results involving EA were the average of at least three experiments and the standard error ± 1 was provided. In determining the inhibition parameters, we used non-linear regression analysis. RESULTS: FXIIIa is a transglutaminase that works at the end of the coagulation process to form an insoluble, rigid, and cross-linked fibrin rich blood clot. In fact, inhibition of FXIIIa-mediated biological processes has been reported to result in a bleeding diathesis. Inhibition of FXIIIa by EA was investigated given the nucleophilic nature of the thiol-containing active site of the enzyme and the Michael acceptor-based electrophilicity of EA. In a bisubstrate-based fluorescence trans-glutamination assay, EA inhibited FXIIIa with a moderate potency (IC50 ~ 105 µM) and efficacy (∆Y ~ 66%). In SDS-PAGE experiments, EA appears to significantly inhibit the FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α2-antiplasmin complex which indicates that EA affects the physiological functions of FXIIIa. Interestingly, EA did not affect the clotting times of human plasma in the APTT and the PT assays at the highest concentration tested of 2.5 mM suggesting the lack of effects on the coagulation serine proteases and potentially the functional selectivity of EA with respect to the clotting process. Molecular modeling studies demonstrated that the Michael acceptor of EA forms a covalent bond with catalytic residue of Cys314 in the active site of FXIIIa. CONCLUSIONS: Overall, our studies indicate that EA inhibits the physiological function of human FXIIIa in vitro which may potentially contribute to the bleeding complications that were reported with the association of the parenteral administration of EA.


Subject(s)
Antifibrinolytic Agents , Ethacrynic Acid , Factor XIIIa , Antifibrinolytic Agents/pharmacology , Blood Coagulation , Ethacrynic Acid/pharmacology , Factor XIIIa/antagonists & inhibitors , Fibrin/chemistry , Humans
7.
Mol Biol Rep ; 49(8): 7521-7530, 2022 Aug.
Article in English | MEDLINE | ID: mdl-35585382

ABSTRACT

BACKGROUND: Despite the recent advances in chemotherapy, the outcomes and the success of these treatments still remain insufficient. Novel combination treatments and treatment strategies need to be developed in order to achieve more effective treatment. This study was designed to investigate the combined effect of ethacrynic acid and cinnamic acid on cancer cell lines. METHODS: The anti-proliferative effect of ethacrynic acid and cinnamic acid was investigated by MTT cell viability assay in three different cancer cell lines. Combination indexes were calculated using CompuSyn software. Apoptosis was assessed by flow cytometric Annexin V-FITC/PI double-staining. The effect of the inhibitors on cell cycle distribution was measured by propidium iodide staining. RESULTS: The combination treatment of ethacrynic acid and cinnamic acid decreased cell proliferation significantly, by 63%, 75% and 70% for K562, HepG2 and TFK-1 cells, respectively. A 5.5-fold increase in the apoptotic cell population was observed after combination treatment of K562 cells. The population of apoptotic cells increased by 9.3 and 0.4% in HepG2 and TFK-1 cells, respectively. Furthermore, cell cycle analysis shows significant cell cycle arrest in S and G2/M phase for K562 cells and non-significant accumulation in G0/G1 phase for TFK-1 and HepG2 cells. CONCLUSIONS: Although there is a need for further investigation, our results suggest that the inhibitors used in this study cause a decrease in cellular proliferation, induce apoptosis and cause cell cycle arrest.


Subject(s)
Ethacrynic Acid , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Apoptosis , Cell Cycle Checkpoints , Cell Proliferation , Cinnamates , Ethacrynic Acid/pharmacology , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy
8.
J Enzyme Inhib Med Chem ; 37(1): 728-742, 2022 Dec.
Article in English | MEDLINE | ID: mdl-35176963

ABSTRACT

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S-transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min-1. In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 µM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 µM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 µg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 µg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.


Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Ethacrynic Acid/pharmacology , Ethylenediamines/pharmacology , Glutathione Transferase/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Putrescine/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cisplatin/chemistry , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Ethacrynic Acid/chemistry , Ethylenediamines/chemistry , Female , Glutathione Transferase/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Mice , Mice, Nude , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Putrescine/chemistry , Structure-Activity Relationship
9.
Bioorg Chem ; 115: 105293, 2021 10.
Article in English | MEDLINE | ID: mdl-34426162

ABSTRACT

For unmet clinical needs, a novel class of ethacrynic acid (EA) derivatives containing triazole moieties (3a-i and 8) were designed, synthesized and evaluated as new anticancer agents. The in vitro anti-proliferative activities were assessed first on HL60 cell line and in a second stage, the two selected compounds 3a and 3c were tested on a panel of human cancer cell lines (A549, MCF7, PC3, U87-MG, SKOV3 and HCT116) and on a normal cell line (MCR5). Compound3c exhibited very good antitumor activities with IC50 values of 20.2, 56.5 and 76.8 nM against A549, PC3 and U87-MG cell lines respectively, which is 2.8- and 1.3-fold more active than doxorubicin on A549 and U87-MG cancer cells, respectively. In addition, compound 3c displays a very good safety index (SI) of 82 fold for A549. Compound 3a showed also good IC50 values of 50 nM on both A549 and PC3 cells and lower selectivity compared to 3c for A549 and PC3 vs. MCR5 with SI of 33 and 18 fold, respectively. The measurement of mitochondrial membrane potential on HCT116 cells after treatments by either 3a or 3c showed that both compounds induced mitochondrial dysfunctions causing thus caspase-induced apoptosis.


Subject(s)
Antineoplastic Agents/pharmacology , Ethacrynic Acid/pharmacology , Triazoles/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Ethacrynic Acid/chemical synthesis , Ethacrynic Acid/chemistry , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Structure-Activity Relationship , Triazoles/chemistry
10.
Dis Markers ; 2021: 5530673, 2021.
Article in English | MEDLINE | ID: mdl-34122668

ABSTRACT

Background. Despite afatinib as a new first-line treatment for EGFR L858R and exon 19 deletion or other rare EGFR-mutation patients, the acquired resistance or toxic effects associated with it limited its use clinically. The controlling of acquired resistance or optimization of the afatinib dosage in EGFR/T790M mutation-positive non-small-cell lung cancer (NSCLC) is still an important fundamental problem. Ethacrynic acid (EA) has been proved as a dual inhibitor of GST and WNT, and the α, ß-unsaturated-keto structure of it is similar to that of irreversible tyrosine kinase inhibitors (TKIs). However, these beneficial effects of EA combined with afatinib have never been reported in NSCLC. Therefore, the antitumor effects of afatinib combined with EA in EGFR L858R/T790M-mutated NSCLC cells and related mechanisms were analyzed. Our in vitro and in vivo results showed that EA has strong synergistic antitumor effects with afatinib in EGFR L858R/T790M-mutated NSCLC cells, but has no cytotoxic effects in NSCLC cells when used it alone, i.e., the cytotoxic effects of afatinib (IC30) plus EA (IC30) were stronger than the effects of afatinib (IC50) alone. Our functional studies found that the antitumor mechanisms of afatinib when combined with EA mainly occurred by inhibiting WNT/ß-catenin pathway activation and suppression of the secretion of anti-inflammatory factors. These results revealed that combination of afatinib with EA derivatives not only provided a new therapeutic approach for EGFR/T790M-mutated NSCLC patients but also offered a new idea for developing new drugs or optimizing the dose of afatinib in clinical use in future antitumor therapy.


Subject(s)
Afatinib/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Ethacrynic Acid/pharmacology , Lung Neoplasms/drug therapy , Wnt Signaling Pathway/drug effects , A549 Cells , Afatinib/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Drug Resistance, Neoplasm/genetics , Drug Synergism , Ethacrynic Acid/therapeutic use , Genes, erbB-1 , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Mice , Mice, Inbred BALB C , Mutation , Random Allocation
11.
Viruses ; 13(1)2021 Jan 13.
Article in English | MEDLINE | ID: mdl-33451132

ABSTRACT

In 2019 an outbreak occurred which resulted in a global pandemic. The causative agent has been identified in a virus belonging to theCoronaviridae family, similar to the agent of SARS, referred to as SARS-CoV-2. This epidemic spread rapidly globally with high morbidity and mortality. Although vaccine development is at a very advanced stage, there are currently no truly effective antiviral drugs to treat SARS-CoV-2 infection. In this study we present systematic and integrative antiviral drug repurposing effort aimed at identifying, among the drugs already authorized for clinical use, some active inhibitors of the SARS-CoV-2 main protease. The most important result of this analysis is the demonstration that ethacrynic acid, a powerful diuretic, is revealed to be an effective inhibitor of SARS-CoV-2 main protease. Even with all the necessary cautions, given the particular nature of this drug, these data can be the starting point for the development of an effective therapeutic strategy against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Ethacrynic Acid/pharmacology , Protease Inhibitors/pharmacokinetics , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Databases, Factual , Drug Repositioning , Ethacrynic Acid/chemistry , Inhibitory Concentration 50 , Molecular Docking Simulation , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology
13.
Biochem Pharmacol ; 183: 114339, 2021 01.
Article in English | MEDLINE | ID: mdl-33189676

ABSTRACT

Lung cancer is one of the leading causes of death in cancer patients. Epithelial-mesenchymal transition (EMT) plays an important role in lung cancer progression. Therefore, for lung cancer treatment, it is crucial to find substances that inhibit EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux and exerts anticancer effects. However, the effects of ECA on EMT in lung cancer remain unclear. We examined the effects of ECA on sphingosylphosphorylcholine (SPC) or TGF-ß1-induced EMT process in A549 and H1299 cells via reverse transcription polymerase chain reaction and Western blotting. We found that ECA inhibited SPC-induced EMT and SPC-induced WNT signalling in EMT. We observed that SPC induces the expression of NDP [Norrie disease protein] and WNT-2, whereas ECA suppressed their expression. SPC-induced WNT activation, EMT, migration, and invasion were suppressed by NDP small-interfering RNA (siNDP), but NDP overexpression (pNDP) enhanced these events in A549 and H1299 cells. Accordingly, NDP expression may influence lung cancer prognosis. In summary, our results revealed that ECA inhibited SPC or TGF-ß1-induced EMT in A549 and H1299 lung cancer cells by downregulating NDP expression and inhibiting WNT activation. Therefore, ECA might be a new drug candidate for lung cancer treatment.


Subject(s)
Epithelial-Mesenchymal Transition/drug effects , Ethacrynic Acid/pharmacology , Eye Proteins/pharmacology , Lung Neoplasms/metabolism , Nerve Tissue Proteins/pharmacology , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Wnt Signaling Pathway/drug effects , A549 Cells , Animals , Cell Movement/drug effects , Cell Movement/physiology , Dose-Response Relationship, Drug , Epithelial-Mesenchymal Transition/physiology , Ethacrynic Acid/therapeutic use , Eye Proteins/antagonists & inhibitors , Eye Proteins/biosynthesis , Humans , Lung Neoplasms/drug therapy , Male , Mice , Mice, Inbred NOD , Mice, SCID , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/biosynthesis , RNA, Small Interfering/pharmacology , Sodium Potassium Chloride Symporter Inhibitors/therapeutic use , Wnt Signaling Pathway/physiology
14.
Chemistry ; 26(72): 17525-17535, 2020 Dec 23.
Article in English | MEDLINE | ID: mdl-33252170

ABSTRACT

A facile route to PtII complexes doubly functionalized with bioactive molecules through a bipyridine-type ligand is described. Initially, ligands LEE (containing two ethacrynic acid units), LEF (ethacrynic acid+flurbiprofen) and LEB (ethacrynic acid+biotin) were obtained in moderate to good yields from 2,2'-bipyridine-4,4'-dicarboxylic acid. Subsequent reaction of the ligands with [PtCl2 (DMSO)2 ] afforded complexes [PtCl2 (LEE )] (2), [PtCl2 (LEF )] (3) and [PtCl2 (LEB )] (4) in high yields. All compounds were fully characterized by analytical and spectroscopic methods. Complexes 2-4 are highly stable in water/DMSO solution at 37 °C after 72 h, whereas progressive release of the bioactive fragments was detected in a cell culture medium. The compounds were assessed for their in vitro antiproliferative activity towards tumorigenic A2780, A2780cisR and Y79 cells and non-tumourigenic HEK293 cells. In particular, the combination of ethacrynic acid and flurbiprofen in 3 overcomes cisplatin-based resistance and provides strong cancer cell selectivity. Enzyme inhibition assays on human GST P1 and human COX-2 and cross-experiments with complex 1, analogous to 2-4 but lacking bio-groups, revealed a clear synergy between the PtII frame and the bioactive organic components.


Subject(s)
2,2'-Dipyridyl/chemistry , Antineoplastic Agents , Cisplatin/pharmacology , Ethacrynic Acid/pharmacology , Ovarian Neoplasms , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cisplatin/therapeutic use , Ethacrynic Acid/therapeutic use , Female , Flurbiprofen/therapeutic use , HEK293 Cells , Humans , Ovarian Neoplasms/drug therapy
15.
J Vis Exp ; (164)2020 10 10.
Article in English | MEDLINE | ID: mdl-33104076

ABSTRACT

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)-two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 ± 3.6 µM and a Ki of 23.2 ± 3.2 µM. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.


Subject(s)
Cytosol/enzymology , Drug Evaluation, Preclinical , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , Glutathione Transferase/antagonists & inhibitors , Spectrophotometry/methods , Animals , Curcumin/pharmacology , Dinitrobenzenes/metabolism , Ethacrynic Acid/pharmacology , Glutathione/metabolism , Glutathione Transferase/metabolism , Horses , Inhibitory Concentration 50 , Isoenzymes/metabolism , Kinetics , Liver/enzymology , Substrate Specificity/drug effects
16.
Bioorg Med Chem Lett ; 30(19): 127426, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32755677

ABSTRACT

A series of ethacrynic acid (2-[2,3-dichloro-4-(2-methylidenebutanoyl)phenoxy]acetic acid) (EA, Edecrin) containing sulfonamides linked via three types of linkers namely 1,2-ethylenediamine, piperazine and 4-aminopiperidine was synthesized and subsequently evaluated in vitro against HL60 and HCT116 cancer cell lines. All the EA analogs, excluding 6a and 6c, showed anti-proliferative activity with IC50s in the micromolar range (less than 4 uM). Three derivatives 6b, 7b and 7e were selected for their interesting dual activity on HL60 cell line in order to be further evaluated against a panel of cancer cell lines (HCT116, A549, MCF7, PC3, U87-MG and SKOV3) as well as on MRC5 as a normal cell line. These compounds displayed IC50 values in nanomolar range against A549, MCF7, PC3 and HCT116 cell lines, deducing the discovery that piperazine or 4-aminopiperidine is the linker's best choice to develop EA analogs with highly potent anti-proliferative activities own up to 24 nM. Besides, in terms of selectivity, those linkers are more suitable offering safety ratios of up to 63.8.


Subject(s)
Antineoplastic Agents/pharmacology , Ethacrynic Acid/analogs & derivatives , Ethacrynic Acid/pharmacology , Sulfonamides/pharmacology , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Structure-Activity Relationship , Sulfonamides/chemical synthesis
17.
Biochem Pharmacol ; 175: 113920, 2020 05.
Article in English | MEDLINE | ID: mdl-32201212

ABSTRACT

To identify signal transducer and activator of transcription factor 3 (STAT3) inhibitors, we generated STAT3-dependent gene expression signature by analyzing gene expression profiles of DU145 cancer cells treated with STAT3 inhibitor, piperlongumine and 2-hydroxycinnamaldehyde. Then we explored gene expression signature-based strategies using a connectivity map database and identified several STAT3 inhibitors, including ethacrynic acid (EA). EA is currently used as a diuretic drug. EA inhibited STAT3 activation in DU145 prostate cancer cells and consequently decreased the levels of STAT3 target genes such as cyclin A and MCL-1. Furthermore, EA treatment inhibited tumor growth in mice xenografted with DU145 cells and decreased p-STAT3 expression in tumor tissues. Knockdown of Src homology region 2 domain-containing phosphatase-2 (SHP2) or Protein tyrosine phosphatase 1B (PTP1B) gene expression by siRNA suppressed the ability of EA to inhibit STAT3 activation. When EA was combined with an activator of SHP2 or PTP1B, p-STAT3 expression was synergistically decreased; when EA was combined with an inhibitor of SHP2 or PTP1B, p-STAT3 expression was rescued. By using an affinity pulldown assay with biotinyl-EA, EA was shown to associate with SHP2 and PTP1B in vitro. Additionally, the drug affinity responsive target stability (DARTS) assay confirmed the direct binding of EA to SHP2 and PTP1B. SHP2 is activated by EA through active phosphorylation at Y580 and direct binding to SHP2. Collectively, our results suggest that EA inhibits STAT3 activity through the modulation of phosphatases such as SHP2 and PTP1B and may be a potential anticancer drug to target STAT3 in cancer progression.


Subject(s)
Ethacrynic Acid/pharmacology , Prostatic Neoplasms/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , STAT3 Transcription Factor/metabolism , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Ethacrynic Acid/therapeutic use , Female , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Prostatic Neoplasms/drug therapy , STAT3 Transcription Factor/antagonists & inhibitors , Xenograft Model Antitumor Assays/methods
18.
Sheng Li Xue Bao ; 71(6): 863-873, 2019 Dec 25.
Article in Chinese | MEDLINE | ID: mdl-31879742

ABSTRACT

The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca2+ concentration ([Ca2+]i) in ASM cells. The results showed that EA significantly inhibited the high K+ (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) µmol/L and (56.22 ± 7.62) µmol/L, respectively. EA decreased the K+ and ACh-induced elevation of [Ca2+]i from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca2+ influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca2+ influx and decreases of [Ca2+]i, leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.


Subject(s)
Ethacrynic Acid , Muscle Contraction , Muscle, Smooth , Respiratory System , Animals , Calcium/metabolism , Calcium Channels, L-Type , Enzyme Inhibitors/pharmacology , Ethacrynic Acid/pharmacology , Mice , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Respiratory System/cytology , Respiratory System/drug effects
19.
Redox Biol ; 26: 101235, 2019 09.
Article in English | MEDLINE | ID: mdl-31202080

ABSTRACT

Glutathione (GSH), the most abundant vertebrate endogenous redox buffer, plays key roles in organogenesis and embryonic development, however, organ-specific GSH utilization during development remains understudied. Monochlorobimane (MCB), a dye conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct, was used to visualize organ-specific GSH utilization in live developing zebrafish (Danio rerio) embryos. Embryos were incubated in 20 µM MCB for 1 h and imaged on an epifluorescence microscope. GSH conjugation with MCB was high during early organogenesis, decreasing as embryos aged. The heart had fluorescence 21-fold above autofluorescence at 24 hpf, dropping to 8.5-fold by 48 hpf; this increased again by 72 hpf to 23.5-fold, and stayed high till 96 hpf (18-fold). The brain had lower fluorescence (10-fold) at 24 and 48 hpf, steadily increasing to 30-fold by 96 hpf. The sensitivity and specificity of MCB staining was then tested with known GSH modulators. A 10-min treatment at 48 hpf with 750 µM tert-butylhydroperoxide, caused organ-specific reductions in staining, with the heart losing 30% fluorescence, and, the brain ventricle losing 47% fluorescence. A 24 h treatment from 24-48 hpf with 100 µM of N-Acetylcysteine (NAC) resulted in significantly increased fluorescence, with the brain ventricle and heart showing 312% and 240% increases respectively, these were abolished upon co-treatment with 5 µM BSO, an inhibitor of the enzyme that utilizes NAC to synthesize GSH. A 60 min 100 µM treatment with ethacrynic acid, a specific GST inhibitor, caused 30% reduction in fluorescence across all measured structures. MCB staining was then applied to test for GSH disruptions caused by the toxicants perfluorooctanesulfonic acid and mono-(2-ethyl-hexyl)phthalate; MCB fluorescence responded in a dose, structure and age-dependent manner. MCB staining is a robust, sensitive method to detect spatiotemporal changes in GSH utilization, and, can be applied to identify sensitive target tissues of toxicants.


Subject(s)
Brain/metabolism , Fluorescent Dyes/chemistry , Glutathione/metabolism , Pyrazoles/chemistry , Staining and Labeling/methods , Zebrafish/metabolism , Acetylcysteine/pharmacology , Alkanesulfonic Acids/toxicity , Animals , Brain/drug effects , Brain/growth & development , Diethylhexyl Phthalate/analogs & derivatives , Diethylhexyl Phthalate/toxicity , Embryo, Nonmammalian , Ethacrynic Acid/pharmacology , Fluorocarbons/toxicity , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/metabolism , Heart/drug effects , Heart/growth & development , Organogenesis/drug effects , Organogenesis/physiology , Toxicity Tests, Chronic , Zebrafish/embryology , Zebrafish/growth & development , tert-Butylhydroperoxide/pharmacology
20.
PLoS One ; 14(3): e0214160, 2019.
Article in English | MEDLINE | ID: mdl-30897163

ABSTRACT

Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 µM concentration afforded at least 50% inhibition of EcaGST A3-3. The most potent inhibitors, anthralin, sennoside A, tannic acid, and ethacrynic acid, were characterized by IC50 values in the submicromolar range when assayed with the natural substrate Δ5-androstene-3,17-dione.


Subject(s)
Enzyme Inhibitors/pharmacology , Glutathione Transferase/antagonists & inhibitors , Animals , Anthralin/pharmacology , Ethacrynic Acid/pharmacology , Glutathione Transferase/metabolism , Horses , Sennosides/pharmacology , Substrate Specificity , Tannins/pharmacology
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