RESUMO
A series of analogues of trypsin inhibitor SFTI-1 were designed and synthesized to monitor peptide splicing. In the middle part of the SFTI-1 analogues, which is released upon incubation with proteinase, the RGD sequence or an acceptor of fluorescence for FRET was introduced. The results of studies with trypsin confirmed that the designed analogues underwent peptide splicing. Furthermore, we showed that a FRET displaying SFTI-1 analogue was internalized into the HaCaT keratinocytes, where it was degraded. Therefore, both proteolysis and the reduction of the disulfide bridge of the peptide took place. As a result, such analogues are a convenient tool to trace the proteolytic activity inside the cell. However, the cytotoxicity of SFTI-1 analogues grafted with the RGD sequence did not correlate with their susceptibility to peptide splicing. Nevertheless, these peptides were slightly more active than the reference peptide (GRGDNP). Interestingly, one of the analogues assigned as [desSer6 ]VI, under experimental conditions, appeared significantly more cytotoxic towards cancer cells U87-MG in contrast to the reference peptide.
Assuntos
Queratinócitos/metabolismo , Peptídeos/metabolismo , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Queratinócitos/citologia , Espectrometria de Massas , Microscopia de Fluorescência , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteólise , Tripsina/química , Inibidores da Tripsina/químicaRESUMO
The use of pegylated interferon-α (pegIFN-α) has replaced unmodified recombinant IFN-α for the treatment of chronic viral hepatitis. While the superior antiviral efficacy of pegIFN-α is generally attributed to improved pharmacokinetic properties, the pharmacodynamic effects of pegIFN-α in the liver have not been studied. Here, we analyzed pegIFN-α-induced signaling and gene regulation in paired liver biopsies obtained prior to treatment and during the first week following pegIFN-α injection in 18 patients with chronic hepatitis C. Despite sustained high concentrations of pegIFN-α in serum, the Jak/STAT pathway was activated in hepatocytes only on the first day after pegIFN-α administration. Evaluation of liver biopsies revealed that pegIFN-α induces hundreds of genes that can be classified into four clusters based on different temporal expression profiles. In all clusters, gene transcription was mainly driven by IFN-stimulated gene factor 3 (ISGF3). Compared with conventional IFN-α therapy, pegIFN-α induced a broader spectrum of gene expression, including many genes involved in cellular immunity. IFN-induced secondary transcription factors did not result in additional waves of gene expression. Our data indicate that the superior antiviral efficacy of pegIFN-α is not the result of prolonged Jak/STAT pathway activation in hepatocytes, but rather is due to induction of additional genes that are involved in cellular immune responses.
Assuntos
Interferon-alfa/farmacologia , Janus Quinases/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Polietilenoglicóis/farmacologia , Fatores de Transcrição STAT/metabolismo , Adulto , Idoso , Antivirais/farmacologia , Endopeptidases/genética , Endopeptidases/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/genética , Hepatite C Crônica/metabolismo , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/genética , Interferon alfa-2 , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Janus Quinases/genética , Cinética , Fígado/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia , Fatores de Transcrição STAT/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ubiquitina TiolesteraseRESUMO
BACKGROUND & AIMS: Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response to treatment with pegylated interferon (pegIFN)-α and ribavirin. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with sustained virologic response rates greater than 90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy. METHODS: We analyzed IFN signaling and ISG expression in liver samples from patients with AHC, patients with CHC, and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets. RESULTS: Expression levels of hundreds of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ-stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α-stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of suppresor of cytokine signaling 1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC. CONCLUSIONS: Differences in expression of ISGs might account for the greater response of patients with AHC, compared with those with CHC, to treatment with pegIFN-α and ribavirin. Specifically, USP18 is up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in patients with AHC.
Assuntos
Antivirais/uso terapêutico , Endopeptidases/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , Interferon gama/metabolismo , Fígado/efeitos dos fármacos , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Doença Aguda , Adolescente , Adulto , Biópsia , Western Blotting , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Farmacorresistência Viral/genética , Quimioterapia Combinada , Endopeptidases/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Hepacivirus/patogenicidade , Hepatite C/diagnóstico , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/genética , Hepatite C Crônica/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Recombinantes/uso terapêutico , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Suíça , Fatores de Tempo , Transcrição Gênica , Resultado do Tratamento , Ubiquitina TiolesteraseRESUMO
BACKGROUND/AIMS: Treatment of chronic hepatitis C (CHC) with pegylated interferon α (pegIFNα) and ribavirin results in a sustained response in approximately half of patients. Viral interference with IFNα signal transduction through the Jak-STAT pathway might be an important factor underlying treatment failure. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFNα signaling in cultured cells that express hepatitis C virus (HCV) proteins, and enhance the inhibitory effect of IFNα on HCV replicons. We have performed a clinical study with the aim to evaluate efficacy and safety of the addition of SAMe and betaine to treatment of CHC with pegIFNα/ribavirin. METHODS: In this open-label pilot study, 29 patients with CHC who failed previous therapy with (peg)IFNα/ribavirin were treated with SAMe, betaine, pegIFNα2b and ribavirin. Treatment duration was 6 or 12 months, depending on genotype, and the protocol comprised a stopping rule at week 12 if early virological response (EVR) was not achieved. Virological and biochemical response and safety were assessed throughout the treatment. RESULTS: 29 patients were enrolled and treated according to the study protocol. 79% of the patients were infected with genotype 1, 72% had advanced fibrosis, 76% had previously received pegIFNα/ribavirin, and only 14% achieved EVR to the previous treatment. When treated with the study medications, 17 patients (59%) showed an EVR, only 3 (10%) however achieved a sustained virological response (SVR). SAMe and betaine were found to be safe when used with pegIFNα/ribavirin. CONCLUSION: The addition of SAMe and betaine to pegIFNα/ribavirin improves early virological response in CHC. TRIAL REGISTRATION: ClinicalTrials.gov NCT00310336.
Assuntos
Betaína/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , S-Adenosilmetionina/uso terapêutico , Adulto , Antivirais/efeitos adversos , Antivirais/uso terapêutico , Artralgia/induzido quimicamente , Betaína/efeitos adversos , Diarreia/induzido quimicamente , Quimioterapia Combinada , Fadiga/induzido quimicamente , Feminino , Fármacos Gastrointestinais/efeitos adversos , Fármacos Gastrointestinais/uso terapêutico , Cefaleia/induzido quimicamente , Hepatite C Crônica/virologia , Humanos , Interferon alfa-2 , Interferon-alfa/efeitos adversos , Interferon-alfa/uso terapêutico , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/uso terapêutico , Estudos Prospectivos , Proteínas Recombinantes , Ribavirina/efeitos adversos , Ribavirina/uso terapêutico , S-Adenosilmetionina/efeitos adversos , Fatores de Tempo , Resultado do TratamentoRESUMO
Hepatitis C virus (HCV) infection induces the endogenous interferon (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-alpha. Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-beta. The HCV NS3-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and 3 than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hepatite C Crônica/metabolismo , Interferons/metabolismo , Fígado/metabolismo , Linhagem Celular , Hepatite C Crônica/virologia , Humanos , Carga ViralRESUMO
Hepatitis C virus (HCV) infection induces a state of oxidative stress by affecting mitochondrial-respiratory-chain activity. By using cell lines inducibly expressing different HCV constructs, we showed previously that viral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation and to major reliance on nonoxidative glucose metabolism. However, the bioenergetic competence of the induced cells was not compromised, indicating an efficient prosurvival adaptive response. Here, we show that HCV protein expression activates hypoxia-inducible factor 1 (HIF-1) by normoxic stabilization of its alpha subunit. In consequence, expression of HIF-controlled genes, including those coding for glycolytic enzymes, was significantly upregulated. Similar expression of HIF-controlled genes was observed in cell lines inducibly expressing subgenomic HCV constructs encoding either structural or nonstructural viral proteins. Stabilization and transcriptional activation of HIF-1alpha was confirmed in Huh-7.5 cells harboring cell culture-derived infectious HCV and in liver biopsy specimens from patients with chronic hepatitis C. The HCV-related HIF-1alpha stabilization was insensitive to antioxidant treatment. Mimicking an impairment of mitochondrial oxidative phosphorylation by treatment of inducible cell lines with oligomycin resulted in stabilization of HIF-1alpha. Similar results were obtained by treatment with pyruvate, indicating that accumulation of intermediate metabolites is sufficient to stabilize HIF-1alpha. These observations provide new insights into the pathogenesis of chronic hepatitis C and, possibly, the HCV-related development of hepatocellular carcinoma.
Assuntos
Adaptação Fisiológica , Glicólise , Hepacivirus/patogenicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/virologia , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Fígado/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estabilidade Proteica , Proteínas ViraisRESUMO
20 years have passed since the discovery of the hepatitis C virus (HCV), and yet therapeutic options remain limited. Current standard treatment of chronic hepatitis C (CHC) consists of pegylated interferon alpha (pegIFN) and ribavirin, and leads to a sustained virological response in approximately half of treated patients. Understanding non-responsiveness to pegIFN, by analysing the molecular mechanisms underlying treatment failure, is important for future therapeutic improvements. In the following review the current status of knowledge on the crosstalk between HCV and IFNs, as well as on the molecular events occurring in liver tissue of HCV-infected patients in response to pegIFN, is discussed. Furthermore, the review focuses on the prospect of developing a prognostic test that might direct treatment to those patients who will benefit from it. The outlook on novel therapeutics, including small molecule inhibitors of HCV proteins and immune modulators, is broadened by a glance at the exciting field of micro-RNAs that are likely to be implicated in viral replication and pathogenesis of CHC, thus representing a new therapeutic target.
Assuntos
Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/tratamento farmacológico , Interferons/uso terapêutico , Antivirais/uso terapêutico , Humanos , Resultado do TratamentoRESUMO
Recombinant alpha interferon (IFN-alpha) is used for the treatment of viral hepatitis and some forms of cancer. During these therapies IFN-alpha is injected once daily or every second day for several months. Recently, the long-acting pegylated IFN-alpha (pegIFN-alpha) has replaced standard IFN-alpha in therapies of chronic hepatitis C because it is more effective, supposedly by inducing a long-lasting activation of IFN signaling pathways. IFN signaling in cultured cells, however, becomes refractory within hours, and little is known about the pharmacodynamic effects of continuously high IFN-alpha serum concentrations. To investigate the behavior of the IFN system in vivo, we repeatedly injected mice with IFN-alpha and analyzed its effects in the liver. Within hours after the first injection, IFN-alpha signaling became refractory to further stimulation. The negative regulator SOCS1 was rapidly upregulated and likely responsible for early termination of IFN-alpha signaling. For long-lasting refractoriness, neither SOCS1 nor SOCS3 were instrumental. Instead, we identified the inhibitor USP18/UBP43 as the key mediator. Our results indicate that the current therapeutic practice using long-lasting pegIFN-alpha is not well adapted to the intrinsic properties of the IFN system. Targeting USP18 expression may allow to exploit the full therapeutic potential of recombinant IFN-alpha.
Assuntos
Endopeptidases/metabolismo , Interferon Tipo I/farmacologia , Janus Quinases/metabolismo , Fígado , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Endopeptidases/genética , Humanos , Interleucina-10/metabolismo , Janus Quinases/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Recombinantes , Fatores de Transcrição STAT/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ubiquitina TiolesteraseRESUMO
Several microRNAs (miRNAs), including liver-specific miR-122, have been implicated in the control of hepatitis C virus (HCV) RNA replication and its response to interferon (IFN) in human hepatoma cells. Our analysis of liver biopsies from subjects with chronic hepatitis C (CHC) undergoing IFN therapy revealed no correlation of miR-122 expression with viral load and markedly decreased pretreatment miR-122 levels in subjects who had no virological response during later IFN therapy; other investigated miRNAs showed only limited changes. These data have implications for the prospect of targeting miRNAs for CHC therapy.
Assuntos
Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , MicroRNAs/genética , Antivirais/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Células Cultivadas , Regulação para Baixo/genética , Farmacorresistência Viral/genética , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , MicroRNAs/metabolismo , Falha de Tratamento , Carga ViralRESUMO
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNalpha) and ribavirin. It achieves a sustained viral clearance in only 50-60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNalpha. In patients with a rapid virological response to treatment, pegIFNalpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNalpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.
Assuntos
Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Transdução de Sinais , Adulto , Biópsia , Feminino , Regulação da Expressão Gênica , Genótipo , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon alfa-2 , Janus Quinases/metabolismo , Leucócitos Mononucleares/metabolismo , Fígado/enzimologia , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição STAT/metabolismo , Resultado do TratamentoRESUMO
Hepatitis C virus (HCV) infection is an important cause of chronic liver disease. Standard therapy, pegylated interferon alpha (pegIFNalpha) combined with ribavirin, results in a sustained response rate in approximately half of patients. The cause of treatment failure in the other half of the patients is unknown, but viral interference with IFNalpha signal transduction through the Jak-STAT pathway might be an important factor. We have shown previously that the expression of HCV proteins leads to an impairment of Jak-STAT signaling because of an inhibition of STAT1 methylation. Unmethylated STAT1 is less active because it can be bound and inactivated by its inhibitor, protein inhibitor of activated STAT1 (PIAS1). We show that treating cells with S-adenosyl-L-methionine (AdoMet) and betaine could restore STAT1 methylation and improve IFNalpha signaling. Furthermore, the antiviral effect of IFNalpha in cell culture could be significantly enhanced by the addition of AdoMet and betaine. In conclusion, we propose that the addition of these drugs to the standard therapy of patients with chronic hepatitis C could overcome treatment resistance.
Assuntos
Betaína/farmacologia , Hepacivirus/fisiologia , Interferon-alfa/metabolismo , S-Adenosilmetionina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Antivirais/farmacologia , Linhagem Celular , DNA/antagonistas & inibidores , DNA/metabolismo , Hepatite C Crônica/enzimologia , Hepatite C Crônica/patologia , Humanos , Interferon-alfa/farmacologia , Fígado/enzimologia , Fígado/patologia , Metilação/efeitos dos fármacos , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/farmacologia , Proteína Fosfatase 2C , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: To establish a chronic infection, hepatitis C virus (HCV) has to evade the host defense. Expression of HCV proteins in cultured cells and in hepatocytes of transgenic mice inhibits interferon-alpha-induced intracellular signaling through the Jak-STAT (signal transducer and activator of transcription) pathway. It is not known if interferon-alpha signaling is inhibited in patients with chronic hepatitis C as well, and the molecular mechanisms are not well defined. METHODS: Interferon-alpha-induced signaling was investigated in liver biopsies from patients with chronic hepatitis C. The molecular mechanisms of HCV interference with Jak-STAT signaling were analyzed in cultured cells, HCV transgenic mice, and liver biopsies. RESULTS: Interferon-alpha-induced DNA binding of STAT1 was significantly impaired in liver biopsies from patients with chronic hepatitis C compared with controls. Tyrosine and serine phosphorylation of STAT1 were intact, but methylation of STAT1 on arginine 31 was reduced. Hypomethylated STAT1 associated with PIAS1, an inhibitor of STAT DNA binding. Increased expression levels of Protein Phosphatase 2A were found in liver extracts from HCV transgenic mice and in liver biopsies of patients with chronic hepatitis C. Overexpression of PP2Ac in Huh7 cells resulted in hypomethylation of STAT1, increased binding to PIAS1, and reduced interferon-alpha-induced DNA binding of STAT1. CONCLUSIONS: We conclude that HCV interferes with interferon-alpha signaling via up-regulation of PP2Ac, hypomethylation of STAT1, and increased STAT1-PIAS1 association, resulting in reduced transcriptional activation of interferon-alpha-stimulated genes.