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1.
Molecules ; 24(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505816

RESUMO

Due to the poor prognosis of metastatic osteosarcoma, chemotherapy is usually employed in the adjuvant situation to improve the prognosis and the chances of long-term survival. 4-[3,5-Bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl]-4-oxo-2-butenoic acid (CLEFMA) is a synthetic analog of curcumin and possesses anti-inflammatory and anticancer properties. To further obtain information regarding the apoptotic pathway induced by CLEFMA in osteosarcoma cells, microculture tetrazolium assay, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. CLEFMA dose-dependently decreased the cell viabilities of human osteosarcoma U2OS and HOS cells and significantly induced apoptosis in human osteosarcoma cells. In addition to the effector caspase 3, CLEFMA significantly activated both extrinsic caspase 8 and intrinsic caspase 9 initiators. Moreover, CLEFMA increased the phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), CLEFMA's increases of cleaved caspases 3, 8, and 9 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Conclusively, CLEFMA activates both extrinsic and intrinsic apoptotic pathways in human osteosarcoma cells through JNK and p38 signaling. These findings contribute to a better understanding of the mechanisms responsible for CLEFMA's apoptotic effects on human osteosarcoma cells.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Benzilideno/farmacologia , Proliferação de Células/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Piperidonas/farmacologia , Caspases/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética
2.
Redox Biol ; 19: 375-387, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30237126

RESUMO

It is generally recognized that hepatic fibrogenesis is an end result of increased extracellular matrix (ECM) production from the activation and proliferation of hepatic stellate cells (HSCs). An in-depth understanding of the mechanisms of HSC necroptosis might provide a new therapeutic strategy for prevention and treatment of hepatic fibrosis. In this study, we attempted to investigate the effect of curcumol on necroptosis in HSCs, and further to explore the molecular mechanisms. We found that curcumol ameliorated the carbon tetrachloride (CCl4)-induced mice liver fibrosis and suppressed HSC proliferation and activation, which was associated with regulating HSC necroptosis through increasing the phosphorylation of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3). Moreover, curcumol promoted the migration of RIPK1 and RIPK3 into necrosome in HSCs. RIPK3 depletion impaired the anti-fibrotic effect of curcumol. Importantly, we showed that curcumol-induced RIPK3 up-regulation significantly increased mitochondrial reactive oxygen species (ROS) production and mitochondrial depolarization. ROS scavenger, N-acetyl-L-cysteine (NAC) impaired RIPK3-mediated necroptosis. In addition, our study also identified that the activation of c-Jun N-terminal kinase1/2 (JNK1/2) was regulated by RIPK3, which mediated curcumol-induced ROS production. Down-regulation of RIPK3 expression, using siRIPK3, markedly abrogated JNK1/2 expression. The use of specific JNK1/2 inhibitor (SP600125) resulted in the suppression of curcumol-induced ROS production and mitochondrial depolarization, which in turn, contributed to the inhibition of curcumol-triggered necroptosis. In summary, our study results reveal the molecular mechanism of curcumol-induced HSC necroptosis, and suggest a potential clinical use of curcumol-targeted RIPK1/RIPK3 complex-dependent necroptosis via JNK1/2-ROS signaling for the treatment of hepatic fibrosis.


Assuntos
Células Estreladas do Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Necrose/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Sesquiterpenos/farmacologia , Acetilcisteína/farmacologia , Animais , Tetracloreto de Carbono/toxicidade , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
3.
Biomed Pharmacother ; 106: 1063-1071, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30119172

RESUMO

JNK1/2 and NF-κB signal are essential signaling pathways that mediate a variety of cellular processes, including cell survival, apoptosis, inflammation and angiogenesis. JNK1/2 activation and NF-κBp65 nuclear translocation have been found in ischemia/reperfusion (I/R)-induced injury. However, the regulation of JNK1/2-NF-κBp65 signaling pathway remains unclear. To examine the function and possible mechanism of HMGB2 in I/R-induced cell injury, human AC16 cardiomyocytes transfected with pLVX-Puro-HMGB2 were treated with SP600125 (JNK1/2 inhibitor) or PDTC (NF-κB inhibitor) and that following I/R injury were transfected with HMGB2-shRNA. The cell proliferation and apoptosis were measured by CCK-8, flow cytometry and TUNEL, respectively. The expression of HMGB2, Cleaved PARP and Caspase-3, Bax and Bcl-2 and activity of MAPKs and NF-κBp65 were measured by Western blot. Here, we found that I/R time-dependently induced the increase in the expression of HMGB2 in AC16 cardiomyocytes. HMGB2 silencing significantly inhibited I/R-induced the cell proliferation reduction, cell apoptosis, activation of ERK1/2, JNK1/2 and NF-κBp65, increased Bax, Cleaved PARP and Caspase-3 and decreased Bcl-2 expression. HMBG2 overexpression mimicked the effect of I/R-induced injury in AC16 cardiomyocytes, which was reversed by treatment with SP600125 or PDTC. Moreover, PDTC treatment in rats following I/R injury also showed decreases in the cell apoptosis, HMGB2, Cleaved PARP and Caspase-3 and Bax expression, and JNK1/2 activation. Taken together, our findings suggest that HMBG2 overexpression promotes I/R-induced cell apoptosis through activating the JNK1/2-NF-κBp65 signaling in AC16 cardiomyocytes.


Assuntos
Apoptose , Proteína HMGB2/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/enzimologia , Fator de Transcrição RelA/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antracenos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Feminino , Proteína HMGB2/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirrolidinas , Ratos Sprague-Dawley , Transdução de Sinais , Tiocarbamatos , Fatores de Tempo , Fator de Transcrição RelA/antagonistas & inibidores , Adulto Jovem
4.
Int J Cardiol ; 254: 265-271, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29407107

RESUMO

BACKGROUND: Flavonols, including 3',4'-dihydroxyflavonol (DiOHF), reduce myocardial ischemia and reperfusion (I/R) injury but their mechanism remains uncertain. To better understand the mechanism of the cardioprotective actions of flavonols we investigated the effect of DiOHF on cardiac function and the activation of protective and injurious signalling kinases after I/R in rat isolated hearts. METHODS: We assessed the effect of global ischemia (20min) and reperfusion (5-30min) on cardiac function and injury in rat isolated, perfused hearts in the absence or presence of DiOHF (10µM) during reperfusion. Western blotting was used to assess changes in the phosphorylation state of kinases known to be involved in injury or protection. RESULTS: DiOHF improved cardiac contractility and reduced perfusion pressure and cell death in the isolated hearts. Phosphorylation of p38MAPK and CaMKII increased during ischemia with no further increase during reperfusion. Phosphorylation of other kinases increased during reperfusion. Phosphorylation of phospholamban (PLN) peaked at 5min of reperfusion whereas phosphorylation of Akt, Erk, STAT3 and JNK2 was highest after 30min. The presence of DiOHF during reperfusion significantly inhibited the activation of PLN and JNK without affecting phosphorylation of the protective kinases Erk1/2 and STAT3. Experiments in vitro demonstrated that DiOHF inhibited CaMKII by competing with ATP but not Ca2+/calmodulin. CONCLUSIONS: It is proposed that DiOHF confers protection against myocardial reperfusion injury by inhibiting CaMKII and subsequent PLN-induced leak of Ca2+ from the sarcoplasmic reticulum as well as by inhibiting JNK2 activation to reduce apoptosis.


Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Cardiotônicos/uso terapêutico , Flavonóis/uso terapêutico , Preparação de Coração Isolado , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Flavonóis/farmacologia , Preparação de Coração Isolado/métodos , Masculino , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , Ratos Sprague-Dawley
5.
Cell Physiol Biochem ; 44(5): 1842-1855, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29224011

RESUMO

BACKGROUND/AIMS: GLP-1 and ghrelin are common appetite-regulating hormones. Both have multiple functions beyond metabolic regulation. However, the effects of GLP-1 and ghrelin on endothelial biology are not fully understood. Here, we investigate the roles of GLP-1 and ghrelin in microvascular endothelial apoptosis and senescence. METHODS: Human microvascular endothelial cells (HMECs) were exposed to high glucose/high lipid (HG/HL) conditions and treated with GLP-1 or ghrelin. Cellular apoptosis, senescence, and mitochondrial function were measured. In addition, the MAPK and Akt signaling pathways were examined. RESULTS: Both GLP-1 and ghrelin treatment decreased the number of TUNEL-positive cells and inhibited caspase-3 and PARP cleavage and mitochondrial dysfunction in HG/HL-exposed HMECs. GLP-1, but not ghrelin decreased the number of ß-galactosidase (ß-gal)-positive cells. Furthermore, GLP-1 and ghrelin inhibited ERK1/2, JNK1/2, and p38 signaling. GLP-1 suppressed Akt signaling, but ghrelin had no effect. Moreover, JNK1/2 and p38 inhibitors, but not ERK1/2 and Akt inhibitors, decreased the number of TUNEL-positive cells. Additionally, only the Akt inhibitor decreased the number of ß-gal-positive cells. CONCLUSION: These results demonstrate that GLP-1 and ghrelin inhibit mitochondrial dysfunction under HG/HL conditions, and suppress endothelial apoptosis via inhibiting JNK1/2 and p38 signaling; moreover, GLP-1 alleviates endothelial senescence via inactivating Akt signaling.


Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Grelina/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/farmacologia , Lipídeos/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Microvasos/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
J Am Heart Assoc ; 6(11)2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29138180

RESUMO

BACKGROUND: Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. METHODS AND RESULTS: After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. CONCLUSIONS: Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity.


Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Endotélio Vascular/fisiopatologia , Derivação Gástrica/efeitos adversos , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Obesidade/cirurgia , Peptídeos/administração & dosagem , Vasodilatação/fisiologia , Animais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Injeções Subcutâneas , Masculino , Fármacos Neuroprotetores , Obesidade/complicações , Obesidade/enzimologia , Estresse Oxidativo , Fosforilação , Ratos , Ratos Wistar
7.
Biochem J ; 474(20): 3421-3437, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28887384

RESUMO

Adipogenic differentiation plays a vital role in energy homeostasis and endocrine system. Several transcription factors, including peroxisome proliferator-activated receptor gamma 2 and CCAAT-enhancer-binding protein (C/EBP) α, ß, and δ, are important for the process, whereas the stage-specific intracellular signal transduction regulating the onset of adipogenesis remains enigmatic. Here, we explored the functional role of c-jun N-terminal kinases (JNKs) in adipogenic differentiation using in vitro differentiation models of 3T3-L1 cells and primary adipo-progenitor cells. JNK inactivation with either a pharmacological inhibitor or JNK2-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the down-regulation of Adiponectin, fatty acid protein 4 (Fabp4), Pparg2, and C/ebpa expressions. Conversely, increased adipogenesis was observed by the inducible overexpression of p46JNK2 (JNK2-1), whereas it was not observed by that of p54JNK2 (JNK2-2), indicating a distinct role of p46JNK2. The essential role of JNK appears restricted to the early stage of adipogenic differentiation, as JNK inhibition in the later stages did not influence adipogenesis. Indeed, JNK phosphorylation was significantly induced at the onset of adipogenic differentiation. As for the transcription factors involved in early adipogenesis, JNK inactivation significantly inhibited the induction of C/ebpd, but not C/ebpb, during the initial stage of adipogenic differentiation. JNK activation increased C/ebpd mRNA and protein expression through the induction and phosphorylation of activating transcription factor 2 (ATF2) that binds to a responsive element within the C/ebpd gene promoter region. Taken together, these data indicate that constitutive JNK activity is specifically required for the initial stage differentiation events of adipocytes.


Assuntos
Adipogenia/fisiologia , Proteína delta de Ligação ao Facilitador CCAAT/biossíntese , Diferenciação Celular/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Antracenos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores
8.
Anal Biochem ; 532: 26-28, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28552758

RESUMO

In order to evaluate the isoform selectivity of novel inhibitors within the c-Jun N-terminal kinase (JNK) family, a fluorescence polarization-based competition binding assay, previously developed for JNK3, was extended to the other isoforms JNK1 and JNK2. The assay is based on the displacement of a versatile fluorescent pyridinylimidazole-based probe and was validated by testing the precursor of the probe as well as standard JNK inhibitors.


Assuntos
Polarização de Fluorescência , Corantes Fluorescentes/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Ligação Competitiva , Humanos , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Ligação Proteica
9.
Antiviral Res ; 141: 7-18, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28188818

RESUMO

High viral load with liver injury is exhibited in severe dengue virus (DENV) infection. Mitogen activated protein kinases (MAPKs) including ERK1/2 and p38 MAPK were previously found to be involved in the animal models of DENV-induced liver injury. However, the role of JNK1/2 signaling in DENV-induced liver injury has never been investigated. JNK1/2 inhibitor, SP600125, was used to investigate the role of JNK1/2 signaling in the BALB/c mouse model of DENV-induced liver injury. SP600125-treated DENV-infected mice ameliorated leucopenia, thrombocytopenia, hemoconcentration, liver transaminases and liver histopathology. DENV-induced liver injury exhibited induced phosphorylation of JNK1/2, whereas SP600125 reduced this phosphorylation. An apoptotic real-time PCR array profiler was used to screen how SP600125 affects the expression of 84 cell death-associated genes to minimize DENV-induced liver injury. Modulation of caspase-3, caspase-8 and caspase-9 expressions by SP600125 in DENV-infected mice suggests its efficiency in restricting apoptosis via both extrinsic and intrinsic pathways. Reduced expressions of TNF-α and TRAIL are suggestive to modulate the extrinsic apoptotic signals, where reduced p53 phosphorylation and induced anti-apoptotic Bcl-2 expression indicate the involvement of the intrinsic apoptotic pathway. This study thus demonstrates the pivotal role of JNK1/2 signaling in DENV-induced liver injury and how SP600125 modulates this pathogenesis.


Assuntos
Antracenos/farmacologia , Fígado/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Dengue Grave/metabolismo , Dengue Grave/patologia , Animais , Antracenos/administração & dosagem , Antracenos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Vírus da Dengue/efeitos dos fármacos , Modelos Animais de Doenças , Leucopenia/tratamento farmacológico , Fígado/efeitos dos fármacos , Fígado/virologia , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Dengue Grave/tratamento farmacológico , Dengue Grave/virologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Necrose Tumoral alfa/genética , Carga Viral , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Biochem Biophys Res Commun ; 483(1): 64-68, 2017 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-28062184

RESUMO

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year worldwide. With no RSV antiviral or efficacious vaccine currently available, improved understanding of the host-RSV interaction is urgently required. Here we examine the contribution to RSV infection of the host stress-regulated c-Jun N-terminal kinase (JNK), for the first time. Peak JNK1/2 phosphoactivation is observed at ∼24 h post-infection, correlating with the time of virus assembly. The release of infectious RSV virions from infected cells was significantly reduced by either JNK1/2 siRNA knockdown or treatment with the JNK-specific inhibitor, JNK-IN-VIII. High resolution microscopy confirmed RSV accumulation in the host cell cytoplasm. The results implicate JNK1/2 as a key host factor for RSV virus production, raising the possibility of agents targeting JNK activity as potential anti-RSV therapeutics.


Assuntos
Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral/fisiologia , Células A549 , Ativação Enzimática , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/patogenicidade , Vírion/fisiologia , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologia
11.
Matrix Biol ; 60-61: 190-205, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-27913195

RESUMO

Adipose-derived stem cells (ASCs) are abundantly present in the mammary microenvironment and can promote breast cancer malignancy by differentiating into myofibroblasts. However, it remains largely unclear which role tumor-derived extracellular vesicles (TEVs) play in this process. Here, we used microfabricated, type I collagen-based 3-D tissue culture platforms to investigate the effect of breast cancer cell-derived TEVs on ASCs myofibroblast differentiation and consequential changes in extracellular matrix remodeling and vascular sprouting. TEVs collected from MDA MB-231 human metastatic breast cancer cells (MDAs) promoted ASC myofibroblast differentiation in both 2-D and 3-D cultures as indicated by increased alpha smooth muscle actin (α-SMA) and fibronectin (Fn) levels. Correspondingly, TEV-treated ASCs were more contractile, secreted more vascular endothelial growth factor (VEGF), and promoted angiogenic sprouting of human umbilical vein endothelial cells (HUVECs). These changes were dependent on transforming growth factor beta (TGF-ß)-related signaling and tumor cell glutaminase activity as their inhibition decreased TEV-related myofibroblastic differentiation of ASCs and related functional consequences. In summary, our data suggest that TEVs are important signaling factors that contribute to ASC desmoplastic reprogramming in the tumor microenvironment, and suggest that tumor cell glutamine metabolism may be used as a therapeutic target to interfere with this process.


Assuntos
Adipócitos/metabolismo , Matriz Extracelular/química , Vesículas Extracelulares/química , Miofibroblastos/metabolismo , Neovascularização Patológica/genética , Células-Tronco/metabolismo , Actinas/genética , Actinas/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Benzofenantridinas/farmacologia , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Fibronectinas , Regulação da Expressão Gênica , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Free Radic Biol Med ; 101: 272-283, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27989749

RESUMO

Accumulating evidence identifies autophagy as an inflammation-related defensive mechanism against diseases including liver fibrosis. Therefore, autophagy may represent a new pharmacologic target for drug development to treat liver fibrosis. In this study, we sought to investigate the effect of dihydroartemisinin (DHA) on autophagy, and to further examine the molecular mechanisms of DHA-induced anti-inflammatory effects. We found that DHA appeared to play an essential role in controlling excessive inflammation. DHA suppressed inflammation in rat liver fibrosis model and inhibited the expression of proinflammatory cytokines in activated hepatic stellate cells (HSCs). Interestingly, DHA increased the autophagosome generation and autophagic flux in activated HSCs, which is underlying mechanism for the anti-inflammatory activity of DHA. Autophagy depletion impaired the induction of anti-inflammatory effect of DHA, while autophagy induction showed a synergistic effect with DHA. Importantly, our study also identified a crucial role for reactive oxygen species (ROS) in the facilitation of DHA-induced autophagy. Antioxidants, such as glutathione and N-acetyl cysteine, significantly abrogated ROS production, and in turn, prevented DHA-induced autophagosome generation and autophagic flux. Besides, we found that c-Jun N-terminal kinase1/2 (JNK1/2) was a downstream signaling molecule of ROS that mediated the induction of autophagy by DHA. Down-regulation of JNK1/2 activity, using selective JNK1/2 inhibitor (SP600125) or siJNK1/2, led to an inhibition of DHA-induced autophagy. Overall, these results provide novel implications to reveal the molecular mechanism of DHA-induced anti-inflammatory effects, by which points to the possibility of using DHA based proautophagic drugs for the treatment of inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Artemisininas/farmacologia , Autofagia/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Acetilcisteína/farmacologia , Animais , Antracenos/farmacologia , Antioxidantes/farmacologia , Autofagia/genética , Tetracloreto de Carbono , Regulação da Expressão Gênica , Glutationa/farmacologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Fígado , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo
13.
Arterioscler Thromb Vasc Biol ; 36(6): 1122-31, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27102962

RESUMO

OBJECTIVE: The c-Jun NH2-terminal kinases (JNK) are regulated by a wide variety of cellular stresses and have been implicated in apoptotic signaling. Macrophages express 2 JNK isoforms, JNK1 and JNK2, which may have different effects on cell survival and atherosclerosis. APPROACH AND RESULTS: To dissect the effect of macrophage JNK1 and JNK2 on early atherosclerosis, Ldlr(-/-) mice were reconstituted with wild-type, Jnk1(-/-), and Jnk2(-/-) hematopoietic cells and fed a high cholesterol diet. Jnk1(-/-)→Ldlr(-/-) mice have larger atherosclerotic lesions with more macrophages and fewer apoptotic cells than mice transplanted with wild-type or Jnk2(-/-) cells. Moreover, genetic ablation of JNK to a single allele (Jnk1(+/-)/Jnk2(-/-) or Jnk1(-/-)/Jnk2(+/-)) in marrow of Ldlr(-/-) recipients further increased atherosclerosis compared with Jnk1(-/-)→Ldlr(-/-) and wild-type→Ldlr(-/-) mice. In mouse macrophages, anisomycin-mediated JNK signaling antagonized Akt activity, and loss of Jnk1 gene obliterated this effect. Similarly, pharmacological inhibition of JNK1, but not JNK2, markedly reduced the antagonizing effect of JNK on Akt activity. Prolonged JNK signaling in the setting of endoplasmic reticulum stress gradually extinguished Akt and Bad activity in wild-type cells with markedly less effects in Jnk1(-/-) macrophages, which were also more resistant to apoptosis. Consequently, anisomycin increased and JNK1 inhibitors suppressed endoplasmic reticulum stress-mediated apoptosis in macrophages. We also found that genetic and pharmacological inhibition of phosphatase and tensin homolog abolished the JNK-mediated effects on Akt activity, indicating that phosphatase and tensin homolog mediates crosstalk between these pathways. CONCLUSIONS: Loss of Jnk1, but not Jnk2, in macrophages protects them from apoptosis, increasing cell survival, and this accelerates early atherosclerosis.


Assuntos
Aorta/enzimologia , Doenças da Aorta/enzimologia , Apoptose , Aterosclerose/enzimologia , Células da Medula Óssea/enzimologia , Macrófagos/enzimologia , Proteína Quinase 8 Ativada por Mitógeno/deficiência , Receptores de LDL/deficiência , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apoptose/efeitos dos fármacos , Aterosclerose/genética , Aterosclerose/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Sobrevivência Celular , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Predisposição Genética para Doença , Hipercolesterolemia/enzimologia , Hipercolesterolemia/genética , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/deficiência , Proteína Quinase 9 Ativada por Mitógeno/genética , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Placa Aterosclerótica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de LDL/genética , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/metabolismo
14.
J Recept Signal Transduct Res ; 36(6): 558-571, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26906522

RESUMO

c-Jun-NH2 terminal kinases (JNKs) come under a class of serine/threonine protein kinases and are encoded by three genes, namely JNK1, JNK2 and JNK3. Human JNK1 is a cytosolic kinase belonging to mitogen-activated protein kinase (MAPK) family, which plays a major role in intracrinal signal transduction cascade mechanism. Overexpressed human JNK1, a key kinase interacts with other kinases involved in the etiology of many cancers, such as skin cancer, liver cancer, breast cancer, brain tumors, leukemia, multiple myeloma and lymphoma. Thus, to unveil a novel human JNK1 antagonist, receptor-based pharmacophore modeling was performed with the available eighteen cocrystal structures of JNK1 in the protein data bank. Eighteen e-pharmacophores were generated from the 18 cocrystal structures. Four common e-pharmacophores were developed from the 18 e-pharmacophores, which were used as three-dimensional (3D) query for shape-based similarity screening against more than one million small molecules to generate a JNK1 ligand library. Rigid receptor docking (RRD) performed using GLIDE v6.3 for the 1683 compounds from in-house library and 18 cocrystal ligands with human JNK1 from lower stringency to higher stringency revealed 17 leads. Further to derive the best leads, dock complexes obtained from RRD were studied further with quantum-polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area (MM-GBSA). Four leads have showed lesser binding free energy and better binding affinity towards JNK1 compared to 18 cocrystal ligands. Additionally, JNK1-lead1 complex interaction stability was reasserted using 50 ns MD simulations run and also compared with the best resolute cocrystal structure using Desmond v3.8. Thus, the results obtained from RRD, QPLD, IFD and MD simulations indicated that lead1 might be used as a potent antagonist toward human JNK1 in cancer therapeutics.


Assuntos
Inibidores Enzimáticos/química , Proteína Quinase 8 Ativada por Mitógeno/química , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Cristalografia por Raios X , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neoplasias/enzimologia , Fosforilação , Ligação Proteica , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico
15.
Gastroenterology ; 150(4): 968-81, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26708719

RESUMO

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) 1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated via phosphorylation in response to acetaminophen- or carbon tetrachloride (CCl4)-induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissue from patients and in mice with genetic deletion of JNK in hepatocytes. METHODS: We studied liver sections from patients with DILI (due to acetaminophen, phenprocoumon, nonsteroidal anti-inflammatory drugs, or autoimmune hepatitis) or patients without acute liver failure (controls) collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and Western blotting. Mice with hepatocyte-specific deletion of Jnk1 (Jnk1(Δhepa)) or combination of Jnk1 and Jnk2 (Jnk(Δhepa)), as well as Jnk1-floxed C57BL/6 (control) mice, were given injections of CCl4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. RESULTS: Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to Jnk(Δhepa) mice produced a greater level of liver injury than that observed in Jnk1(Δhepa) or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl4 also induced stronger hepatic injury in Jnk(Δhepa) mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared with Jnk1(Δhepa) or control mice. Hepatocytes from Jnk(Δhepa) mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of adenosine monophosphate-activated protein kinase, total protein adenosine monophosphate-activated protein kinase levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected Jnk(Δhepa) and control mice from liver injury. CONCLUSIONS: In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl4- and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Hepatócitos/enzimologia , Falência Hepática Aguda/prevenção & controle , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Acetaminofen , Animais , Tetracloreto de Carbono , Estudos de Casos e Controles , Morte Celular , Proliferação de Células , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/enzimologia , Falência Hepática Aguda/genética , Falência Hepática Aguda/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/deficiência , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/deficiência , Proteína Quinase 9 Ativada por Mitógeno/genética , Estresse Oxidativo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Fatores de Tempo , Adulto Jovem
16.
Acta Pharmacol Sin ; 36(9): 1099-106, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26235742

RESUMO

AIM: Many studies reveal an association between the acquired chemoresistant phenotype of cancer cells and tumor-initiating cell-like properties. The aim of this study was to determine the impact of c-Jun N-terminal kinase (JNK) on the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells. METHODS: Two well-established human acquired chemoresistant cancer cell lines K562/A02 and KB/VCR, as well as their respective parental counterparts K562 and KB were tested. The expression of relevant mRNAs and proteins was detected using qRT-PCR and Western blotting, respectively. Sphere formation and self-renewal assays were used to study the tumor-initiating cell-like properties. Soft agar and colony formation assays were used to investigate tumorigenic ability. RESULTS: We observed that suppressing JNK activity by its specific small molecule inhibitor SP600125 or by limiting JNK1/2 expression with JNK1/2 shRNA lentiviruses inhibited the expression of pluripotent stem cell markers such as Oct4, Sox2, and Nanog in KB/VCR cells and K562/A02 cells as well as sphere formation and self-renewal abilities of K562/A02 cells. Additionally, inhibition of JNK activity significantly inhibited the in vitro and in vivo tumor-initiating abilities of KB/VCR cells. Furthermore, our data suggest that blocking JNK activity abundantly inhibited the Hedgehog (Hh) pathway activity, as reflected by reduction of Hedgehog (Hh) pathway target genes Gli1 and ptch1 at the mRNA level as well as Gli-luciferase activity. CONCLUSION: JNK maintains the tumor-initiating cell-like properties of acquired chemoresistant K562/A02 and KB/VCR cells potentially through activating the Hedgehog pathway. Thus, disruption of tumor-initiating cell-like properties by targeting JNK may be a new approach to combating acquired chemoresistance.


Assuntos
Antracenos/farmacologia , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Células K562 , Células KB , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Interferência de RNA , RNA Interferente Pequeno/genética
17.
PLoS One ; 10(4): e0124007, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25875631

RESUMO

Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/genética , Fator de Transcrição AP-1/genética , Anticorpos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Gástrica/metabolismo , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Estômago/efeitos dos fármacos , Estômago/patologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismo
18.
Appl Biochem Biotechnol ; 175(2): 657-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25342257

RESUMO

Grateloupia lanceolata is a red alga native to coastal areas of East Asia. In this study, extract from G. lanceolata (EGL) was investigated for suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages. EGL was found to have anti-inflammatory properties with the inhibition of nitric oxide (NO), pro-inflammatory cytokine production, and MAPK signaling in LPS-induced RAW 264.7 macrophages. Moreover, treatment of RAW 264.7 macrophage with EGL inhibited LPS-induced IL-1ß production in a dose-dependent manner. These inhibitory effects were found with the blockage of p38 mitogen-activated protein kinases (MAPK), extracellular signal regulated kinases 1 and 2 (ERK1/2), and also c-Jun N-terminal kinases 1 and 2 (JNK1/2). These results indicated that anti-inflammatory actions of EGL in RAW 264.7 macrophages involved in the inhibition of LPS-induced p38MAPK/ERK/JNK signaling pathways. In addition, our findings suggest that EGL holds great promise for use in the treatment of various inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Extratos Vegetais/farmacologia , Rodófitas/química , Animais , Anti-Inflamatórios/química , Linhagem Celular , Regulação da Expressão Gênica , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Óxido Nítrico/biossíntese , Extratos Vegetais/química , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Biochem Biophys Res Commun ; 452(3): 845-51, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25218503

RESUMO

Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G2/M arrest, phosphorylation of Bcl-2, Mcl-1, and Bim, Δψm loss, and activation of Bak and caspase cascade. These JNKi-induced apoptotic events were abrogated by Bcl-2 overexpression, whereas G2/M arrest, cyclin B1 up-regulation, Cdk1 activation, and phosphorylation of Bcl-2 family proteins were sustained. In the concomitant presence of the G1/S blocking agent aphidicolin and JNKi, the cells underwent G1/S arrest and failed to induce all apoptotic events. The JNKi-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by the Cdk1 inhibitor. Immunofluorescence microscopic analysis revealed that mitotic spindle defect and prometaphase arrest were the underlying factors for the G2/M arrest. These results demonstrate that JNKi-induced mitochondrial apoptosis was caused by microtubule damage-mediated prometaphase arrest, prolonged Cdk1 activation, and phosphorylation of Bcl-2 family proteins in Jurkat T cells.


Assuntos
Mitocôndrias/efeitos dos fármacos , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Prometáfase/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Afidicolina/farmacologia , Apoptose/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais
20.
Oncotarget ; 5(13): 5100-12, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24947996

RESUMO

Cancer cells with self-renewal and tumor-initiating capacity, either quiescent (cancer stem cells, CSCs) or proliferating (cancer stem-like cells, CSLCs), are now deemed responsible for the pervasive therapy resistance of pancreatic cancer, one of the deadliest human cancers characterized by high prevalence of K-Ras mutation. However, to date, much remains unknown how pancreatic CSCs/CSLCs are regulated. Here we show that the K-Ras - JNK axis plays a pivotal role in the maintenance of pancreatic CSCs/CSLCs. In vitro inhibition of JNK, either pharmacological or genetic, caused loss of the self-renewal and tumor-initiating capacity of pancreatic CSLCs. Importantly, JNK inhibition in vivo via systemic JNK inhibitor administration, which had no discernible effect on the general health status of mice, efficiently depleted the CSC/CSLC population within pre-established pancreatic tumor xenografts. Furthermore, knockdown of K-Ras in pancreatic CSLCs with K-Ras mutation led to downregulation of the JNK pathway as well as in loss of self-renewal and tumor-initiating capacity. Together, our findings suggest that pancreatic CSCs/CSLCs are dependent on K-Ras activation of JNK and also suggest that the K-Ras - JNK axis could be a potential target in CSC/CSLC-directed therapies against pancreatic cancer.


Assuntos
Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Antígeno AC133 , Animais , Antracenos/farmacologia , Antígenos CD/genética , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Immunoblotting , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/prevenção & controle , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Proteínas ras/metabolismo
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