RESUMO
AIM: To investigate the mechanism by which hepatitis C virus (HCV) core protein-induced miR-93-5p up-regulation regulates the interferon (IFN) signaling pathway. METHODS: HCV-1b core protein was exogenously expressed in Huh7 cells using pcDNA3.1 (+) vector. The expression of miR-93-5p and interferon receptor 1 (IFNAR1) was measured using quantitative reverse transcription-polymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of miR-93-5p and IFNAR1 were performed using miR-93-5p agomir and antagomir, and pcDNA3.1-IFNAR1 and IFNAR1 siRNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of miR-93-5p. Cellular experiments were also conducted. RESULTS: Serum miR-93-5p level was increased in patients with HCV-1b infection and decreased to normal level after HCV-1b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum miR-93-5p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1b core protein increased miR-93-5p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of miR-93-5p, and IFNAR1 restore could rescue miR-93-5p-reduced STAT1 phosphorylation, suggesting that the miR-93-5p-IFNAR1 axis regulates the IFN signaling pathway. CONCLUSION: HCV-1b core protein-induced miR-93-5p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the miR-93-5p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1b infection.
Assuntos
Hepacivirus/metabolismo , Hepatite C/metabolismo , Hepatócitos/metabolismo , MicroRNAs/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Proteínas do Core Viral/metabolismo , Adulto , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Farmacorresistência Viral , Feminino , Células HEK293 , Hepacivirus/efeitos dos fármacos , Hepacivirus/patogenicidade , Hepatite C/sangue , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferon-alfa/uso terapêutico , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Fosforilação , Receptor de Interferon alfa e beta/efeitos dos fármacos , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Proteínas do Core Viral/genéticaRESUMO
The present study assessed the direct effects of IFNs on human astrocytes. Human astrocytes were exposed to human recombinant IFNs, and the proliferation of cells was measured. Type I IFN receptor mRNA and protein expression, the phosphoprotein levels of signaling molecules including JNK, ERK1/2, IκB, p38MAPK, Stat3, and the expression of cytokines were determined respectively. In addition, cellular glucose consumption was measured as well as Glut-1 protein and activation of GSK-3ß/mTOR signal were determined. The expression of Type I IFN receptor was detected in cultured human astrocytes. 2 IU/ml IFNα2a and IFNα2b significantly decreased the proliferation of human astrocytes respectively, compared to control. IFNß had no significant effect on the proliferation of the cells. The phosphorylation of JNK stimulated by all IFNs detected was more pronounced and sustained than ERK1/2 and IκB. No effects were observed on the activation of p38MAPK and Stat3. Moreover, Treatment with IFNα, especially with IFNα2b, decreased glucose consumption and stimulated phosphorylation of GSK-3ß and mTOR, but decreased the expression of Glut-1. In contrast, IFNß had no significant effect on either glucose consumption or activation of GSK-3ß/mTOR signals. INFα2b significantly decreased the levels of IL-8 whereas the levels of GM-CSF were increased. The present study demonstrates direct inhibitory effects of IFNα on cell proliferation, cell signaling and glucose utilization in human astrocytes.
Assuntos
Astrócitos/efeitos dos fármacos , Glucose/metabolismo , Interferon-alfa/farmacologia , Astrócitos/metabolismo , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Interferon beta/farmacologia , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Interferon alfa e beta/efeitos dos fármacos , Receptor de Interferon alfa e beta/metabolismo , Serina-Treonina Quinases TOR/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The antitumor activities of type III interferon (IFN) (interleukin [IL]-28 and IL-29) and the combination of type III IFN and type I IFN (IFN-α) were evaluated using human non-small cell lung cancer (NSCLC). The expression of type III and type I receptor complexes was detected in NSCLC lines. IL-29 significantly inhibited the in vitro growth of a wide range of NSCLC lines in a dose-dependent fashion. To a lesser degree, IL-28A also displayed growth inhibitory activity. Antitumor activity of type III IFN is associated with cell cycle arrest at the G1 phase and apoptosis. IL-29 upregulated cyclin-dependent kinase inhibitor p21Waf1/Cip1 in cells sensitive, but not insensitive, to antiproliferative activity, and knockdown of p21 with small interfering RNA largely attenuated the antiproliferative effect. Intratumoral and systemic administration of IL-29 inhibited OBA-LK1 and LK-1, but not A549, tumor growth in severe combined immunodeficiency mice. Immunohistochemical analyses demonstrated marked upregulated p21 and downregulated Ki-67 expression in tumors treated with IL-29. The interferon combination of IL-29 and IFN-α displayed a more effective antiproliferative effect and a more intense p21 expression than each reagent alone in vitro. Furthermore, interferon combination therapy suppressed in vivo NSCLC growth more effectively than interferon monotherapy. These findings demonstrate that type III IFN can mediate direct antitumor activities via increased p21 expression and induction of apoptosis and cooperate with type I IFN to elicit more efficient direct antitumor activities, and suggest the possibility that type III IFN might improve the efficacy and reduce the side-effects of type I IFN cancer therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Interferon-alfa/uso terapêutico , Interleucinas/uso terapêutico , Neoplasias Pulmonares/patologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Interferons , Interleucinas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma/patologia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , Receptor de Interferon alfa e beta/análise , Receptor de Interferon alfa e beta/efeitos dos fármacos , Receptores de Citocinas/análise , Receptores de Citocinas/biossíntese , Receptores de Citocinas/efeitos dos fármacos , Receptores de Citocinas/genética , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Antineoplásicos/farmacologia , Interferon-alfa/farmacologia , Proteínas de Neoplasias/farmacologia , Receptor de Interferon alfa e beta/metabolismo , Meios de Cultivo Condicionados/química , Regulação para Baixo , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Receptor de Interferon alfa e beta/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , UbiquitinaçãoRESUMO
OBJECTIVES: To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. METHODS: Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. RESULTS: According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. CONCLUSIONS: Our findings might support the establishment of an optimal schedule for IFN-alpha administration.
Assuntos
Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/tratamento farmacológico , Ritmo Circadiano , Regulação para Baixo , Fatores Imunológicos/uso terapêutico , Interferon-alfa/uso terapêutico , Neoplasias Renais/sangue , Neoplasias Renais/tratamento farmacológico , Leucócitos Mononucleares/metabolismo , Receptor de Interferon alfa e beta/biossíntese , Humanos , Fatores Imunológicos/farmacologia , Interferon-alfa/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Receptor de Interferon alfa e beta/efeitos dos fármacosAssuntos
Resistência a Medicamentos/genética , Interferon beta/farmacologia , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/imunologia , Receptor de Interferon alfa e beta/genética , Processamento Alternativo/genética , Resistência a Medicamentos/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Ligantes , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Isoformas de Proteínas/química , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , Receptor de Interferon alfa e beta/química , Receptor de Interferon alfa e beta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: The cytokine interferon beta (IFNbeta) is successfully used in the treatment of multiple sclerosis (MS), although there is a high degree of variability in the response. A common mechanism involved in the modulation of responsiveness to cytokines is represented by regulation of their receptor expression through autocrine ligand-mediated loops. The present study is aimed at investigating the regulation of IFNalpha/beta receptor (IFNAR) during IFNbeta therapy in patients with MS and at correlating it with the biologic responsiveness to the cytokine. METHODS: Quantitative PCR measurements of IFNAR-1 and the three IFNAR-2 isoforms were performed in 141 patients after short-term and long-term treatment. Patients were also regularly screened for anti-IFNbeta neutralizing antibodies (NAbs). IFN-inducible myxovirus resistance protein A messenger RNA was used as an indicator of bioactivity. RESULTS: Pretreatment levels of IFNAR-2 in patients were lower overall than in controls (p = 0.038), and high levels correlated with greater bioactivity. Upon prolonged treatment, NAb-negative patients displayed a state of decreased transmembrane IFNAR-2 expression (p < or = 0.025), whereas levels of soluble IFNAR-2 were slightly increased (p < 0.0001). The presence of NAbs reversed these effects (p < or = 0.0056). In NAb-positive patients, pretreatment expression levels of both transmembrane IFNAR-2 isoforms were significantly lower than in NAb-negative patients (p < or = 0.0089). CONCLUSIONS: Findings show that interferon-alpha/beta receptor (IFNAR)-2 isoforms are important regulators of the responsiveness to endogenous and systemically administered interferon beta (IFNbeta). They show a dual action, agonistic and antagonistic, that influences both the magnitude and the nature of the biologic response to IFNbeta. Levels of IFNAR-2 are regulated with the aim of keeping the body in a state of equilibrium, even when nonphysiologic stimuli are present.
Assuntos
Resistência a Medicamentos/genética , Interferon beta/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Receptor de Interferon alfa e beta/efeitos dos fármacos , Receptor de Interferon alfa e beta/genética , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Processamento Alternativo/imunologia , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Resistência a Medicamentos/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Homeostase/efeitos dos fármacos , Homeostase/genética , Homeostase/imunologia , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Interferon beta/uso terapêutico , Masculino , Esclerose Múltipla/fisiopatologia , Reação em Cadeia da Polimerase , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta/imunologia , Estudos Retrospectivos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Merkel cell carcinoma is a tumor with aggressive biological behavior and limited response to chemotherapy. The present study investigated the effect of interferon (IFN)-alpha on growth and apoptosis of Merkel carcinoma cells in vitro. Proliferation of MCC-1 cell line was reduced dose-dependently by IFN-alpha and diminished when higher IFN-alpha concentrations were used. Additionally, IFN-alpha potently decreased DNA-synthesis and Ki67/MIB-1 proliferation index of MCC-1 cultures. Furthermore, IFN-alpha induced dose-dependently apoptosis of MCC-1 cells as shown by caspase-3 activation, and detection of apoptotic DNA strand breaks and fragmented nuclei. These findings suggest that IFN-alpha may have antitumor activity against Merkel cell carcinoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Célula de Merkel/patologia , Proliferação de Células/efeitos dos fármacos , Interferon-alfa/farmacologia , Células de Merkel/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Carcinoma de Célula de Merkel/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Quebras de DNA , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Interferon alfa-2 , Interferon-alfa/metabolismo , Antígeno Ki-67/metabolismo , Células de Merkel/metabolismo , Células de Merkel/patologia , Receptor de Interferon alfa e beta/efeitos dos fármacos , Receptor de Interferon alfa e beta/metabolismo , Proteínas Recombinantes , Neoplasias Cutâneas/metabolismo , Fatores de TempoAssuntos
Antivirais/efeitos adversos , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/efeitos adversos , Pancitopenia/induzido quimicamente , Polietilenoglicóis/efeitos adversos , Ribavirina/uso terapêutico , Adulto , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Darbepoetina alfa , Quimioterapia Combinada , Eritropoetina/análogos & derivados , Eritropoetina/uso terapêutico , Filgrastim , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Hepatite C Crônica/complicações , Humanos , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Ferro/uso terapêutico , Masculino , Pancitopenia/fisiopatologia , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Receptor de Interferon alfa e beta/efeitos dos fármacos , Receptor de Interferon alfa e beta/fisiologia , Proteínas Recombinantes , Ribavirina/administração & dosagem , Transdução de SinaisRESUMO
Interferon (IFN) is a promising drug for prevention and treatment of hepatocellular carcinoma (HCC) in combination with chemotherapeutic agents. We previously reported that the spectra of antiproliferative activity and synergistic effect of IFN-beta when combined with anticancer drugs are more potent than those of IFN-alpha in HCC cells. However, the mechanism of the diverse antitumor effects of the IFNs is not understood yet. We studied the expression of IFN alpha receptor 2 (IFNAR2), STATs, and IFN-alpha, IFN-beta's growth-inhibitory effect, signal transduction and binding to IFNAR2 on three HCC cell lines and a tumor xenografted mouse model (12 animals/group). From the results, IFN-beta showed a significantly stronger growth-inhibitory effect than IFN-alpha on the HuH7 cell line (expressing low IFNAR2), however it was similarly high on PLC/PRF/5 and weak on HLE. In the nude mouse tumor xenograft model, IFN-beta injection significantly suppressed tumor volume relative to vehicle injection, while IFN-alpha showed weaker growth-inhibition. IFN signal transduction (phosphorylated-STAT1, 3) induced by IFN-beta was higher than that by IFN-alpha in HuH7 and tumor xenografts. Pretreatment of hepatoma cells with anti-IFNAR2 antibody blocked the IFN signaling, more for IFN-alpha. IFN-alpha's antiproliferative effect was reduced by the antibody in lower concentrations compared to that of IFN-beta. Taken together, the HCC cells that express low IFNAR2 and are resistant to IFN-alpha were sensitive to the growth-inhibitory effect of IFN-beta, which might be mediated by stronger IFN signal transduction and distinct binding to IFNAR compared to IFN-alpha.