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1.
Nat Commun ; 10(1): 4731, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31636264

RESUMO

Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases.


Assuntos
Apoptose/efeitos dos fármacos , Glicosídeos Cardíacos/farmacologia , Senescência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células A549 , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Neoplasias da Mama , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Digoxina/farmacologia , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Camundongos , Osteoartrite , Ouabaína/farmacologia , Proscilaridina/farmacologia , Fibrose Pulmonar , Ensaios Antitumorais Modelo de Xenoenxerto
2.
J Recept Signal Transduct Res ; 39(3): 226-234, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31509043

RESUMO

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na+/K+-ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na+/K+-ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470 kcal/mol to 7.234 kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470 kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na+/K+-ATPase-ouabain and Na+/K+-ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100 ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.


Assuntos
Glicosídeos Cardíacos/farmacologia , Simulação de Acoplamento Molecular , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Difusão , Digoxina/química , Digoxina/farmacologia , Ligações de Hidrogênio , Ligantes , Modelos Biológicos , Ouabaína/química , Ouabaína/farmacologia , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , ATPase Trocadora de Sódio-Potássio/metabolismo
3.
Nihon Yakurigaku Zasshi ; 154(3): 103-107, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527358

RESUMO

Digitoxin and digoxin are plant-derived cardiac glycosides. They are Na+,K+-ATPase (sodium pump) inhibitors, and have been used clinically for treatment and prevention of heart failure and various tachycardia. On the other hand, some epidemiological studies showed that digoxin users have a lower cancer risk compared to the non-users, and that cancer patients who had been treated with digoxin face on improvement of their survival. In various in vitro studies, cardiac glycosides at sub-µM concentrations, which have no significant effect on enzymatic and ion-transporting activities of Na+,K+-ATPase, show anti-cancer effects. Na+,K+-ATPase is ubiquitously expressed, so it remains unclear why low concentrations of cardiac glycosides have cancer-specific effects. Recently, we found that the receptor-type Na+,K+-ATPase, which has no pumping activity, is associated with leucine-rich repeat-containing 8 family, member A(LRRC8A), one of the components of volume-regulated anion channel (VRAC), in the membrane microdomains of plasma membrane of cancer cells, and that this crosstalk contributes to the inhibition of the cancer cell growth by sub-µM cardiac glycosides. In this mechanism, cardiac glycosides bind to the receptor-type Na+,K+-ATPase, and then stimulate the production of reactive oxygen species (ROS) via NADPH oxidase. The ROS activate VRAC within the membrane microdomains, thus eliciting anti-proliferative effects. VRAC is ubiquitously expressed, and it is normally activated by cell swelling. However, VRAC is activated by cardiac glycoside without cell swelling. On the other hand, the cardiac glycosides-induced effects were not observed in non-cancer cells. Our findings can partly explain why cardiac glycosides elicit selective effects in cancer cells.


Assuntos
Glicosídeos Cardíacos/farmacologia , Neoplasias/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Digoxina/farmacologia , Humanos , Proteínas de Membrana/metabolismo , Espécies Reativas de Oxigênio/metabolismo
4.
J Vasc Res ; 56(2): 55-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31085912

RESUMO

BACKGROUND: Even though hypoxia-inducible factor-1α (HIF-1α) is among the transcriptional factors demonstrated to contribute to the formation of abdominal aortic aneurysms (AAAs), the precise mechanism has been unclear. Digoxin is known as an inhibitor of HIF-1α, and shows a protective effect against the progression of AAAs. OBJECTIVES: We tested the effect of digoxin on osteoclastogenesis (OCG) and examined the pathway through which digoxin exerts inhibition of HIF-1α. MATERIALS AND METHODS: RAW 264.7 macrophage cells were cultured and stimulated by soluble receptor activator of NF-κB ligand (sRANKL) with or without digoxin. First, we tested the effect of digoxin to attenuate macrophage activation, which led to OCG, characterized by tartrate-resistant acid phosphatase (TRAP)-positive macrophages (TPMs). RESULTS: The activation of TPMs stimulated by sRANKL was attenuated by digoxin treatment. Furthermore, the receptor activator of NF-κB (RANK)/receptor activator of NF-κB ligand (RANKL) complex signaling pathway, which is stimulated by HIF-1α, was downregulated by digoxin treatment. CONCLUSIONS: These results show that digoxin attenuates OCG. By inhibition of HIF-1α, digoxin decreases OCG through the downregulation of the RANK/RANKL signaling pathway. Therefore, digoxin is a potential candidate for medical treatment of AAAs.


Assuntos
Aneurisma da Aorta Abdominal/tratamento farmacológico , Digoxina/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Animais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/metabolismo , Camundongos , Osteoclastos/metabolismo , Células RAW 264.7 , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais
6.
PLoS One ; 14(3): e0214407, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30908540

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of morbidity and mortality in human infants. Bovine RSV infection of neonatal calves is pathologically and immunologically similar to RSV infection in infants, and is therefore a useful preclinical model for testing novel therapeutics. Treatment of severe RSV bronchiolitis relies on supportive care and may include use of bronchodilators and inhaled or systemic corticosteroids. Interleukin-17A (IL-17) is an inflammatory cytokine that plays an important role in neutrophil recruitment and activation. IL-17 is increased in children and rodents with severe RSV infection; and in calves with severe BRSV infection. It is currently unclear if IL-17 and Th17 immunity is beneficial or detrimental to the host during RSV infection. Digoxin was recently identified to selectively inhibit IL-17 production by antagonizing its transcription factor, retinoid-related orphan receptor γ t (RORγt). Digoxin inhibits RORγt binding to IL-17 and Th17 associated genes, and suppresses IL-17 production in vitro in human and murine leukocytes and in vivo in rodent models of autoimmune disease. We demonstrate here that in vitro and in vivo digoxin treatment also inhibits IL-17 production by bovine leukocytes. To determine the role of IL-17 in primary RSV infection, calves were treated prophylactically with digoxin and infected with BRSV. Digoxin treated calves demonstrated reduced signs of clinical illness after BRSV infection, and reduced lung pathology compared to untreated control calves. Digoxin treatment did not adversely affect virus shedding or lung viral burden, but had a significant impact on pulmonary inflammatory cytokine expression on day 10 post infection. Together, our results suggest that exacerbated expression of IL-17 has a negative impact on RSV disease, and that development of specific therapies targeting Th17 immunity may be a promising strategy to improve disease outcome during severe RSV infection.


Assuntos
Bronquiolite/virologia , Digoxina/administração & dosagem , Interleucina-17/metabolismo , Vírus Sincicial Respiratório Bovino/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Bovinos , Digoxina/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/virologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Profilaxia Pós-Exposição , Vírus Sincicial Respiratório Bovino/imunologia , Células Th17/imunologia , Células Th17/virologia
7.
Redox Biol ; 22: 101131, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30735911

RESUMO

Chemoresistance is a major therapeutic obstacle in the treatment of human pancreatic ductal adenocarcinoma (PDAC). As an oxidative stress responsive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of cytoprotective genes. Nrf2 not only plays a critical role in chemoprevention, but also contributes to chemoresistance. In this study, we found that digoxin markedly reversed drug resistance of gemcitabine by inhibiting Nrf2 signaling in SW1990/Gem and Panc-1/Gem cells. Further research revealed that digoxin regulated Nrf2 at transcriptional level. In in vivo study, we found that digoxin and gemcitabine in combination inhibited tumor growth more substantially when compared with gemcitabine treatment alone in SW1990/Gem-shControl cells-derived xenografts. In the meantime, SW1990/Gem-shNrf2 cells-derived xenografts responded to gemcitabine and combination treatment similarly, suggesting that digoxin sensitized gemcitabine-resistant human pancreatic cancer to gemcitabine, which was Nrf2 dependent. These results demonstrated that digoxin might be used as a promising adjuvant sensitizer to reverse chemoresistance of gemcitabine-resistant pancreatic cancer to gemcitabine via inhibiting Nrf2 signaling.


Assuntos
Desoxicitidina/análogos & derivados , Digoxina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Humanos , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cancer Biol Ther ; 20(1): 52-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30183476

RESUMO

Small cell lung cancer (SCLC) is a malignant neuroendocrine tumor with very high mortality. Effective new therapy for advanced SCLC patients is urgently needed. By screening a FDA-approved drug library, we identified a cardiac glycoside (CG), namely digoxin (an inhibitor of cellular Na+/K+ ATPase pump), which was highly effective in inhibiting SCLC cell growth. Intriguing findings showed that NaCl supplement markedly enhanced the anti-tumor activities of digoxin in both in vitro and in vivo models of SCLC. Subsequent analysis revealed that this novel combination of digoxin/NaCl caused an up-regulation of intracellular Na+ and Ca2+ levels with an induction of higher resting membrane potential of SCLC cells. We also found that this combination lead to morphological shrinking of SCLC cells, together with high levels of cytochrome C release. Lastly, our data revealed that NaCl supplement was able to induce the expression of ATP1A1 (a Na+/K+ ATPase subunit), in which contributes directly to the increased sensitivity of SCLC cells to digoxin. Thus, this is the first demonstration that NaCl is a potent supplement necessitating superior anti-cancer effects of digoxin for SCLC. Further, our study suggests that digoxin treatment could need to be combined with NaCl supplement in future clinical trial of SCLC, particularly where low Na+ is often present in SCLC patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Digoxina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Cloreto de Sódio/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Digoxina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Potenciais da Membrana/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/patologia , Sódio/sangue , Cloreto de Sódio/uso terapêutico , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
9.
J Am Heart Assoc ; 7(22): e009509, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30571484

RESUMO

Background Digoxin use was shown to be associated with an increased risk of cardiovascular events in atrial fibrillation ( AF ). We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation. Methods and Results Post hoc analysis of a prospective study of anticoagulated patients with AF . Patients were divided into 2 groups balanced for age, sex, and cardiovascular risk factors: digoxin users (n=132) and nonusers (n=388). Urinary excretion of 11-dehydro-thromboxane B2 (TxB2), a marker of platelet activation, and serum digoxin concentration ( SDC ) were measured. In vitro experiments were performed on platelets from healthy subjects and AF patients, which were incubated with scalar doses of digoxin (0.6-2.4 ng/mL) with or without prestimulation with a sub-threshold of collagen. Median 11-dehydro-TxB2 was 105.0 ( interquartile range, 60.0-190.0) ng/mg creatinine, and median SDC was 0.65 ( interquartile range, 0.40-1.00) ng/mL. Urinary 11-dehydro-TxB2 and SDC were correlated ( rs=0.350, P<0.001). Patients in the upper tertile of SDC showed higher 11-dehydro-TxB2 compared with non-digoxin users ( P=0.019). In vitro study showed an increased basal platelet activation in patients with AF compared with healthy subjects . Digoxin (2.4 ng/mL) induced calcium mobilization, PAC -1 (procaspase-activating compound 1) and platelet aggregation in AF patients but not in healthy subjects . After pretreatment with a sub-threshold of collagen, digoxin dose-dependent induced calcium mobilization, arachidonic acid release, TxB2 biosynthesis, PAC -1 and soluble platelet selectin expression, and platelet aggregation, which were inhibited by antibody against digoxin. Conclusions We found a significant in vivo correlation between SDC and platelet activation. Supratherapeutic SDC increased in vitro platelet aggregation via calcium-related phospholipase A2 phosphorylation. Our findings may have clinical implications for AF patients treated with digoxin.


Assuntos
Fibrilação Atrial/tratamento farmacológico , Cardiotônicos/efeitos adversos , Digoxina/efeitos adversos , Ativação Plaquetária/efeitos dos fármacos , Idoso , Fibrilação Atrial/sangue , Biomarcadores/urina , Plaquetas/efeitos dos fármacos , Western Blotting , Cardiotônicos/farmacologia , Estudos de Casos e Controles , Digoxina/farmacologia , Feminino , Citometria de Fluxo , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Técnicas In Vitro , Masculino , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Estudos Prospectivos , Tromboxano A2/urina
10.
Int Immunopharmacol ; 65: 174-181, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30316075

RESUMO

Recent findings have demonstrated new therapeutic functions of cardiotonic steroids, a process that is termed drug repositioning. Despite the confirmed anti-inflammatory effects of cardiotonic steroids, their clinical use has been discouraged due to toxicity related to inhibition of the Na+/K+ ATPase. A novel synthetic compound derived from digoxin, 21­benzylidene digoxin (21­BD), does not inhibit this enzyme. Herein, we evaluated the anti-inflammatory and antinociceptive effects and acute toxicity of 21­BD. Murine (Swiss mice) models of paw oedema induced by carrageenan, acetic acid-induced abdominal writhing, and formalin and acute toxicity tests were used. Oral administration of 21­BD (0.3 mg/kg) showed a significant and prolonged inhibition of paw oedema. Histological analysis demonstrated a reduction in inflammatory cells and expression of inducible nitric oxide synthase (iNOS) in footpads 6 h after administration of carrageenan. 21­BD (0.3 mg/kg) also reduced the levels of tumour necrosis factor (TNF)-α 2 and 4 h after carrageenan. 21­BD demonstrated antinociceptive activity, inhibiting abdominal writhes at all tested doses. However, in the formalin test, 21­BD did not present antinociceptive activity. In the acute toxicity test, 21­BD did not cause symptoms of toxicity or mortality. The present study demonstrated, for the first time, that 21­BD is safe and exhibits a marked anti-inflammatory activity in acute local inflammation. This effect might be a consequence of its ability to inhibit the release of the PMN leucocyte-derived mediators, including TNF-α, and iNOS expression as well as its inhibitory effect on oedema and PMN leucocyte infiltration.


Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Digoxina/análogos & derivados , Analgésicos/química , Animais , Anti-Inflamatórios/química , Carragenina/toxicidade , Digoxina/administração & dosagem , Digoxina/química , Digoxina/farmacologia , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Indometacina/farmacologia , Masculino , Camundongos , Estrutura Molecular , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Testes de Toxicidade , Fator de Necrose Tumoral alfa/metabolismo
11.
Sci Transl Med ; 10(464)2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30355798

RESUMO

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Although outcomes have improved in recent decades, new treatments are still needed to improve survival and reduce treatment-related complications. The MB subtypes groups 3 and 4 represent a particular challenge due to their intragroup heterogeneity, which limits the options for "rational" targeted therapies. Here, we report a systems biology approach to drug repositioning that integrates a nonparametric, bootstrapping-based simulated annealing algorithm and a 3D drug functional network to characterize dysregulated driver signaling networks, thereby identifying potential drug candidates. From more than 1300 drug candidates studied, we identified five members of the cardiac glycoside family as potentially inhibiting the growth of groups 3 and 4 MB and subsequently confirmed this in vitro. Systemic in vivo treatment of orthotopic patient-derived xenograft (PDX) models of groups 3 and 4 MB with digoxin, a member of the cardiac glycoside family approved for the treatment of heart failure, prolonged animal survival at plasma concentrations known to be tolerated in humans. These results demonstrate the power of a systematic drug repositioning method in identifying a potential treatment for MB. Our strategy could potentially be used to accelerate the repositioning of treatments for other human cancers that lack clearly defined rational targets.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Digoxina/uso terapêutico , Reposicionamento de Medicamentos , Meduloblastoma/tratamento farmacológico , Biologia de Sistemas , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Digoxina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Meduloblastoma/sangue , Meduloblastoma/genética , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Int J Pharm ; 551(1-2): 270-280, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30240828

RESUMO

Nonionic surfactants commonly used in pharmaceutical formulations may have P-glycoprotein (P-gp) inhibiting and/or permeation enhancing effects. The present work aims to distinguish these effects and assess the degree of cellular recovery after multiple exposures to nonionic surfactants. The investigated surfactants were polysorbates (PS): PS20, PS40, PS60, PS65, PS80 and PS85; monosaccharide-based: lauroyl methyl glucamide and n-nonyl-ß-D-glucopyranoside; or disaccharide-based: lauryl-ß-D-maltoside and trehalose 6-laurate. Bi-directional permeability studies of digoxin and mannitol, and calcein-AM efflux assay were performed in cell cultures. Cellular recovery was evaluated by continuous measurements of transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Polysorbates with one fatty acid chain decreased the efflux of digoxin through P-gp inhibition in MDCKII MDR1 cells. Mono- and di-saccharide-based surfactants, in a dose dependent manner, enhanced digoxin absorptive permeability without decreasing the secretory permeability in Caco-2 cells, suggesting that the surfactants had a transcellular permeation enhancing effect. Caco-2 cell monolayers recovered to different degrees of 60-100% of the initial TEER values. Calcein-AM assay was found to be non-predictive to surfactants influence on digoxin permeability across cell monolayers. In conclusion, these results may assist, in a mechanism-based, selection of suitable surfactants for formulating oral dosage forms to enhance the absorption of low bioavailable P-gp substrates.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Digoxina/farmacologia , Polissorbatos/farmacologia , Tensoativos/farmacologia , Animais , Células CACO-2 , Cães , Fluoresceínas/farmacologia , Humanos , Células Madin Darby de Rim Canino , Manitol/farmacologia
14.
Cell Signal ; 51: 222-232, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30102978

RESUMO

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


Assuntos
Adenocarcinoma/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , MAP Quinase Quinase 6/antagonistas & inibidores , MAP Quinase Quinase 6/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição SOX9/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Digitoxina/farmacologia , Digitoxina/uso terapêutico , Digoxina/farmacologia , Digoxina/uso terapêutico , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , MAP Quinase Quinase 6/genética , Camundongos Endogâmicos NOD , Ouabaína/farmacologia , Ouabaína/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Transcrição SOX9/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Am J Cardiovasc Drugs ; 18(6): 427-440, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30066080

RESUMO

The history of digitalis is rich and interesting, with the first use usually attributed to William Withering and his study on the foxglove published in 1785. However, some knowledge of plants with digitalis-like effects used for congestive heart failure (CHF) was in evidence as early as Roman times. The active components of the foxglove (Digitalis purpurea and Digitalis lanata) are classified as cardiac glycosides or cardiotonic steroids and include the well-known digitalis leaf, digitoxin, and digoxin; ouabain is a rapid-acting glycoside usually obtained from Strophanthus gratus. These drugs are potent inhibitors of cellular membrane sodium-potassium adenosine triphosphatase (Na+/K+-ATPase). For most of the twentieth century, digitalis and its derivatives, especially digoxin, were the available standard of care for CHF. However, as the century closed, many doubts, especially regarding safety, were raised about their use as other treatments for CHF, such as decreasing the preload of the left ventricle, were developed. Careful attention is needed to maintain the serum digoxin level at ≤ 1.0 ng/ml because of the very narrow therapeutic window of the medication. Evidence for benefit exists for CHF with reduced ejection fraction (EF), also referred to as heart failure with reduced EF (HFrEF), especially when considering the combination of mortality, morbidity, and decreased hospitalizations. However, the major support for using digoxin is in atrial fibrillation (AF) with a rapid ventricular response when a rate control approach is planned. The strongest support of all for digoxin is for its use in rate control in AF in the presence of a marginal blood pressure, since all other rate control medications contribute to additional hypotension. In summary, these days, digoxin appears to be of most use in HFrEF and in AF with rapid ventricular response for rate control, especially when associated with hypotension. The valuable history of the foxglove continues; it has been modified but not relegated to the garden or the medical history book, as some would advocate.


Assuntos
Cardiotônicos/uso terapêutico , Glicosídeos Digitálicos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Fatores Etários , Fibrilação Atrial/tratamento farmacológico , Peso Corporal , Cardiotônicos/efeitos adversos , Cardiotônicos/farmacologia , Digitalis , Glicosídeos Digitálicos/efeitos adversos , Glicosídeos Digitálicos/farmacologia , Digoxina/farmacologia , Digoxina/uso terapêutico , Interações de Medicamentos , Monitoramento de Medicamentos , Humanos , Metanálise como Assunto , Neoplasias/tratamento farmacológico , Estudos Observacionais como Assunto , Ouabaína/farmacologia , Ouabaína/uso terapêutico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Insuficiência Renal/metabolismo , Infarto do Miocárdio com Supradesnível do Segmento ST/tratamento farmacológico , Fatores Sexuais , Volume Sistólico
16.
CPT Pharmacometrics Syst Pharmacol ; 7(10): 647-659, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30091221

RESUMO

According to current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidance documents, physiologically based pharmacokinetic (PBPK) modeling is a powerful tool to explore and quantitatively predict drug-drug interactions (DDIs) and may offer an alternative to dedicated clinical trials. This study provides whole-body PBPK models of rifampicin, itraconazole, clarithromycin, midazolam, alfentanil, and digoxin within the Open Systems Pharmacology (OSP) Suite. All models were built independently, coupled using reported interaction parameters, and mutually evaluated to verify their predictive performance by simulating published clinical DDI studies. In total, 112 studies were used for model development and 57 studies for DDI prediction. 93% of the predicted area under the plasma concentration-time curve (AUC) ratios and 94% of the peak plasma concentration (Cmax ) ratios are within twofold of the observed values. This study lays a cornerstone for the qualification of the OSP platform with regard to reliable PBPK predictions of enzyme-mediated and transporter-mediated DDIs during model-informed drug development. All presented models are provided open-source and transparently documented.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Alfentanil/farmacologia , Claritromicina/farmacologia , Citocromo P-450 CYP3A/metabolismo , Digoxina/farmacologia , Itraconazol/farmacologia , Midazolam/farmacologia , Modelos Biológicos , Rifampina/farmacologia , Interações de Medicamentos , Humanos
17.
Cell Death Dis ; 9(5): 511, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29724996

RESUMO

Liver cancer is one of the most lethal malignancies with very poor prognosis once diagnosed. The most common form of liver cancer is hepatocellular carcinoma (HCC). The WW domain-containing oxidoreductase (WWOX) is a large gene that is often perturbed in a wide variety of tumors, including HCC. WWOX has been shown to act as a tumor suppressor modulating cellular metabolism via regulating hypoxia-inducible factor 1α (HIF-1α) levels and function. Given that WWOX is commonly inactivated in HCC, we set to determine whether specific targeted deletion of murine Wwox affects liver biology and HCC development. WWOX liver-specific knockout mice (Wwox ΔHep ) showed more potent liver regeneration potential and enhanced proliferation as compared with their control littermates. Moreover, WWOX deficiency in hepatocytes combined with diethylnitrosamine treatment increased the tumor burden, which was associated with increased HIF1α levels and target gene transactivation. Inhibition of HIF1α by systemic treatment with digoxin significantly delayed HCC formation. Our work suggests that WWOX inactivation has a central role in promoting HCC through rewiring of cellular metabolism and modulating proliferation.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genética , Animais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Dieta Hiperlipídica/efeitos adversos , Dietilnitrosamina/farmacologia , Digoxina/farmacologia , Modelos Animais de Doenças , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Proteínas Supressoras de Tumor/deficiência , Oxidorredutase com Domínios WW/deficiência
18.
Eur J Pharm Sci ; 121: 85-94, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-29709579

RESUMO

The presence of several binding sites for both substrates and inhibitors is yet a poorly explored thematic concerning the assessment of the drug-drug interactions risk due to interactions of multiple drugs with the human transport protein P-glycoprotein (P-gp or MDR1, gene ABCB1). In this study we measured the inhibitory behaviour of a set of known drugs towards P-gp by using three different probe substrates (digoxin, Hoechst 33,342 and rhodamine 123). A structure-based model was built to unravel the different substrates binding sites and to rationalize the cases where drugs were not inhibiting all the substrates. A separate set of experiments was used to validate the model and confirmed its suitability to either detect the substrate-dependent P-gp inhibition and to anticipate proper substrates for in vitro experiments case by case. The modelling strategy described can be applied for either design safer drugs (P-gp as antitarget) or to target specific sub-site inhibitors towards other drugs (P-gp as target).


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Modelos Moleculares , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Digoxina/farmacologia , Humanos , Rodamina 123/farmacologia
19.
Sci Rep ; 8(1): 7355, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29743548

RESUMO

Vascular disrupting agents as DMXAA inhibit tumor growth only for a short period of time followed by rapid tumor regrowth. Among others, hypoxia and presence of transcription factor HIF-1α are responsible for tumors regrowth. The aim of our study was to investigate the inhibition of murine melanoma growth by combining two agents: anti-vascular - DMXAA and the HIF-1α inhibitor - digoxin and explaining the mechanism of action of this combination. After DMXAA treatment tumor size was reduced only for a limited time. After 7 days regrowth of tumors was observed and number of vessels was increased especially in tumor's peripheral areas. DMXAA also induced an influx of immune cells: macrophages, CD8+ cytotoxic lymphocytes, NK cells, CD4+ lymphocytes. Administration of digoxin alone inhibited the growth of tumors. Administration of both agents in the proper sequence significantly inhibited the regrowth of tumors better than either agents alone. Combination therapy reduced number of newly formed vessels. In tumors of mice treated with combination therapy, the number of macrophages M1, CD8+ cytotoxic lymphocytes, NK cells and to a lesser extent CD4+ cells was increased. The combination of anti-vascular agents with HIF-1α inhibitors appears to be an effective therapeutic option.


Assuntos
Antineoplásicos/farmacologia , Digoxina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Melanoma Experimental/patologia , Melanoma/patologia , Neovascularização Patológica/tratamento farmacológico , Xantonas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Melanoma/irrigação sanguínea , Melanoma/imunologia , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/tratamento farmacológico , Camundongos , Xantonas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Cell Metab ; 27(2): 339-350.e3, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29414684

RESUMO

Sterile inflammation after tissue damage is a ubiquitous response, yet it has the highest amplitude in the liver. This has major clinical consequences, for alcoholic and non-alcoholic steatohepatitis (ASH and NASH) account for the majority of liver disease in industrialized countries and both lack therapy. Requirements for sustained sterile inflammation include increased oxidative stress and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independently of PKM2 kinase activity results in chromatin remodeling and downregulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin that can effectively protect the liver from ASH and NASH.


Assuntos
Digoxina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hepatopatia Gordurosa não Alcoólica/genética , Piruvato Quinase/metabolismo , Ativação Transcricional/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Modelos Animais de Doenças , Endotoxinas , Histonas/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Oxirredução , Ligação Proteica/efeitos dos fármacos , Piruvato Quinase/química , Células THP-1 , Transcrição Genética/efeitos dos fármacos
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