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1.
Respir Res ; 15: 51, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24762191

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U.S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-ß1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition. METHODS: To identify the mechanism of Arsenic trioxide's (ATO)'s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-ß1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as α-smooth muscle actin (α-SMA) and α-1 type I collagen. RESULTS: Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-ß1-induced α-smooth muscle actin (α-SMA) and α-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-ß1-mediated contractile response in NHLFs. ATO's down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-ß1-induced H2O2 and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-ß1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung α-1 type I collagen mRNA and protein expression. CONCLUSIONS: In summary, these data indicate that low concentrations of ATO inhibit TGF-ß1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis.


Assuntos
Arsenicais/farmacologia , Bleomicina/toxicidade , Fibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Óxidos/farmacologia , Fibrose Pulmonar/prevenção & controle , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Trióxido de Arsênio , Arsenicais/uso terapêutico , Bleomicina/antagonistas & inibidores , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/fisiologia , Células Cultivadas , Fibroblastos/patologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/efeitos dos fármacos , Óxidos/uso terapêutico , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fator de Crescimento Transformador beta1/farmacologia
2.
Virol J ; 10: 152, 2013 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-23680002

RESUMO

We have previously shown that disruption of promyelocytic leukemia nuclear bodies (PML NBs) is sufficient to activate the EBV lytic cycle thus making infected cells susceptible to ganciclovir (GCV) mediated killing in vitro. Here we show that co-administration of GCV and arsenic trioxide (ATO), a PML NB disruptor, reduces tumor volume in a xenograft model of nasopharyngeal carcinoma utilizing CNE1 cells. When administered at pharmacologic levels, both GCV and ATO reduced tumor growth while co-treatment with GCV + ATO resulted in a diminution of tumor volume. Treatment with GCV or ATO individually resulted in an increased number of apoptotic cells while co-treatment with GCV + ATO synergistically induced apoptosis. Treatment with ATO or co-treatment with GCV + ATO resulted in expression of EBV lytic proteins. These data suggest that co-treatment with GCV + ATO may provide an effective treatment for nasopharyngeal carcinoma patients.


Assuntos
Antineoplásicos/administração & dosagem , Antivirais/administração & dosagem , Arsenicais/administração & dosagem , Ganciclovir/administração & dosagem , Xenoenxertos/efeitos dos fármacos , Neoplasias Nasofaríngeas/tratamento farmacológico , Óxidos/administração & dosagem , Animais , Trióxido de Arsênio , Carcinoma , Modelos Animais de Doenças , Quimioterapia Combinada/métodos , Humanos , Camundongos , Carcinoma Nasofaríngeo , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos
3.
Glycobiology ; 23(6): 709-19, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23418199

RESUMO

Previously, we have shown that heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) is a transforming growth factor-ß1 (TGF-ß1)-responsive gene in normal human lung fibroblasts and functions as a negative feedback regulator of TGF-ß1 and that TGF-ß1 induces the expression of Sulf1 as well as that of the closely related Sulf2 in a murine model of pulmonary fibrosis. In this study, we focused on the role of Sulf2 in modulating TGF-ß1 function and the development of pulmonary fibrosis. We found that Sulf2 mRNA was overexpressed in lung samples from human patients with idiopathic pulmonary fibrosis (IPF), and Sulf2 protein was specifically localized to the hyperplastic type II alveolar epithelial cells (AECs). In vitro, TGF-ß1 induced the expression of Sulf2 with accompanied HS 6-O-desulfation in A549 cells, adenocarcinoma cells derived from the type II alveolar epithelium. Using small interference RNA to block Sulf2 expression, we observed a biphasic TGF-ß1 response with early enhanced Smad activation, but eventually reduced TGF-ß1 target gene expression in Sulf2 knockdown A549 cells compared with the control cells. To study the role of Sulf2 in normal type II AECs, we isolated primary type II cells from wild-type and Sulf2 knockout mice. We observed enhanced Smad activation as well as enhanced TGF-ß1 target gene expression in Sulf2 knockout type II AECs compared with wild-type type II AECs. In conclusion, Sulf2 is overexpressed in IPF and may play a role in regulating TGF-ß1 signaling in type II AECs.


Assuntos
Fibrose Pulmonar Idiopática/enzimologia , Sulfotransferases/metabolismo , Idoso , Células Epiteliais Alveolares/enzimologia , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Linhagem Celular Tumoral , Indução Enzimática , Feminino , Expressão Gênica/efeitos dos fármacos , Heparitina Sulfato/metabolismo , Humanos , Fibrose Pulmonar Idiopática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Transdução de Sinais , Proteínas Smad/metabolismo , Sulfatases , Sulfotransferases/genética , Fator de Crescimento Transformador beta1/fisiologia
4.
Virol J ; 8: 461, 2011 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-21975125

RESUMO

The Epstein-Barr virus (EBV) encoded Latent Membrane Protein 1 (LMP1) has been shown to increase the expression of promyelocytic leukemia protein (PML) and the immunofluorescent intensity of promyelocytic leukemia nuclear bodies (PML NBs). PML NBs have been implicated in the modulation of transcription and the association of reporter plasmids with PML NBs has been implicated in repression of reporter activity. Additionally, repression of various reporters in the presence of LMP1 has been noted. This study demonstrates that LMP1 suppresses expression of reporter activity in a dose responsive manner and corresponds with the LMP1 induced increase in PML NB intensity. Disruption of PML NBs with arsenic trioxide or a PML siRNA restores reporter activity. These data offer an explanation for previously conflicting data on LMP1 signaling and calls attention to the possibility of false-positives and false-negatives when using reporter assays as a research tool in cells expressing LMP1.


Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Plasmídeos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas da Matriz Viral/metabolismo , Trióxido de Arsênio , Arsenicais/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Inativação Gênica/efeitos dos fármacos , Genes Reporter , Herpesvirus Humano 4/genética , Humanos , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/metabolismo , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/virologia , Luciferases/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Óxidos/farmacologia , Plasmídeos/genética , Proteína da Leucemia Promielocítica , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas da Matriz Viral/genética , beta-Galactosidase/análise
5.
Virology ; 416(1-2): 86-97, 2011 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-21605886

RESUMO

Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies.


Assuntos
Arsenicais/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Ganciclovir/farmacologia , Herpesvirus Humano 4/fisiologia , Proteínas Nucleares/metabolismo , Óxidos/farmacologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Antivirais/farmacologia , Trióxido de Arsênio , Linhagem Celular Tumoral , Farmacorresistência Viral , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Proteína da Leucemia Promielocítica , Regulação para Cima , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
6.
Am J Respir Cell Mol Biol ; 44(6): 852-62, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20693406

RESUMO

The histopathology of idiopathic pulmonary fibrosis (IPF) includes the presence of myofibroblasts within so-called fibroblastic foci, and studies suggest that lung myofibroblasts may be derived from epithelial cells through epithelial--mesenchymal transition (EMT). Transforming growth factor (TGF)-ß1 is expressed and/or activated in fibrogenesis, and induces EMT in lung epithelial cells in a dose-dependent manner. A higher occurrence of Epstein-Barr virus (EBV) has been reported in the lung tissue of patients with IPF. EBV expresses latent membrane protein (LMP) 1 during the latent phase of infection, and may play a role in the pathogenesis of pulmonary fibrosis inasmuch as LMP-1 may act as a constitutively active TNF-α receptor. Our data show a remarkable increase in mesenchymal cell markers, along with a concurrent reduction in the expression of epithelial cell markers in lung epithelial cells cotreated with LMP-1, and very low doses of TGF-ß1. This effect was mirrored in lung epithelial cells infected with EBV expressing LMP1 and cotreated with TGF-ß1. LMP1 pro-EMT signaling was identified, and occurs primarily through the nuclear factor-κB pathway and secondarily through the extracellular signal--regulated kinase (ERK) pathway. Activation of the ERK pathway was shown to be critical for aspects of TGF-ß1-induced EMT. LMP1 accentuates the TGF-ß1 activation of ERK. Together, these data demonstrate that the presence of EBV-LMP1 in lung epithelial cells synergizes with TGF-ß1 to induce EMT. Our in vitro data may help to explain the observation that patients with IPF demonstrating positive staining for LMP1 in lung epithelial cells have a more rapid demise than patients in whom LMP1 is not detected.


Assuntos
Células Epiteliais/citologia , Pulmão/citologia , Fator de Crescimento Transformador beta1/metabolismo , Proteínas da Matriz Viral/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Fibrose/patologia , Herpesvirus Humano 4/metabolismo , Humanos , Pulmão/patologia , Mesoderma/citologia , Modelos Biológicos , NF-kappa B/metabolismo , Transdução de Sinais
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