RESUMO
Increasing consumer aversion to non-natural flavoring substances is prompting a heightened interest in enzymatic processes for flavor production. This includes methylation reactions, which are often performed by using hazardous chemicals. By correlation of aroma profile data and transcriptomic analysis, a novel O-methyltransferase (OMT) catalyzing a respective reaction within the formation of p-anisaldehyde was identified in the mushroom Pleurotus sapidus. Heterologous expression in E. coli followed by purification allowed for further characterization of the enzyme. Besides p-hydroxybenzaldehyde, the proposed precursor of p-anisaldehyde, the enzyme catalyzed the methylation of further hydroxylated aromatic compounds at the meta- and para-position. The Km values determined for p-hydroxybenzaldehyde and S-adenosyl-l-methionine were 80 and 107 µM, respectively. Surprisingly, the studied enzyme enabled the transmethylation of thiol-nucleophiles, as indicated by the formation of 2-methyl-3-(methylthio)furan from 2-methyl-3-furanthiol. Moreover, the enzyme was crystallized at a resolution of 2.0 Å, representing the first published crystal structure of a basidiomycetous OMT.
Assuntos
Benzaldeídos , Metiltransferases , Pleurotus , Metiltransferases/metabolismo , Escherichia coli/metabolismo , Pleurotus/metabolismoRESUMO
BACKGROUND & AIMS: In liver fibrosis, activated hepatic stellate cells (HSC) secrete excess extracellular matrix, thus, represent key targets for antifibrotic treatment strategies. Intermediate-conductance Ca(2) (+) -activated K(+) -channels (KCa3.1) are expressed in non-excitable tissues affecting proliferation, migration and vascular resistance rendering KCa3.1 potential targets in liver fibrosis. So far, no information about KCa3.1 expression and their role in HSC exists. Aim was to quantify the KCa3.1 expression in HSC depending on HSC activation and investigation of antifibrotic properties of the specific KCa3.1 inhibitor TRAM-34 in vitro and in vivo. METHODS: KCa3.1 expression and functionality were studied in TGF-ß1-activated HSC by quantitative real time PCR, western-blot and patch-clamp analysis respectively. Effects of TRAM-34 on HSC proliferation, cell cycle and fibrosis-related gene expression were assessed by [(3) H]-thymidine incorporation, FACS-analysis and RT-PCR respectively. In vivo, vascular resistance and KCa3.1 gene and protein expression were determined in bile duct ligated rats by in situ liver perfusion, Taqman PCR and immunohistochemistry respectively. RESULTS: Fibrotic tissues and TGF-ß1-activated HSC exhibited higher KCa3.1-expressions than normal tissue and untreated cells. KCa3.1 inhibition with TRAM-34 reduced HSC proliferation by induction of cell cycle arrest and reduced TGF-ß1-induced gene expression of collagen I, alpha-smooth muscle actin and TGF-ß1 itself. Furthermore, TRAM-34 blocked TGF-ß1-induced activation of TGF-ß signalling in HSC. In vivo, TRAM-34 reduced the thromboxane agonist-induced portal perfusion pressure. CONCLUSION: Inhibition of KCa3.1 with TRAM-34 downregulates fibrosis-associated gene expression in vitro, and reduces portal perfusion pressure in vivo. Thus, KCa3.1 may represent novel targets for the treatment of liver fibrosis.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Cirrose Hepática Experimental/tratamento farmacológico , Fígado/efeitos dos fármacos , Pressão na Veia Porta/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Cirrose Hepática Experimental/fisiopatologia , Masculino , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Resistência Vascular/efeitos dos fármacosRESUMO
The mucin MUC1 is a glycoprotein involved in fundamental biological processes, which can be found over-expressed and with a distinctly altered glycan pattern on epithelial tumor cells; thus it is a promising target structure in the quest for effective carbohydrate-based cancer vaccines and immunotherapeutics. Natural glycopeptide antigens indicate only a low immunogenicity and a T-cell independent immune response; however, this major drawback can be overcome by coupling of glycopeptide antigens multivalently to immunostimulating carrier platforms. In particular, gold nanoparticles are well suited as templates for the multivalent presentation of glycopeptide antigens, due to their remarkably high surface-to-volume ratio in combination with their high biostability. In this work the synthesis of novel MUC1-glycopeptide antigens and their coupling to gold nanoparticles of different sizes are presented. In addition, the development of a new dot-blot immunoassay to test the potential antigen-antibody binding is introduced.
Assuntos
Glicopeptídeos/química , Glicopeptídeos/síntese química , Coloide de Ouro/química , Mucina-1/química , Técnicas de Química Sintética , Glicopeptídeos/imunologia , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Imunoensaio , Mucina-1/imunologia , Técnicas de Microbalança de Cristal de QuartzoRESUMO
Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC50 of 10µM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and ß-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.
Assuntos
Adipogenia/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Dioxóis/farmacologia , Lignanas/farmacologia , Óxido Nítrico Sintase Tipo II/genética , PPAR gama/genética , Via de Sinalização Wnt/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Lindera/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/tratamento farmacológico , PPAR gama/metabolismo , Fosforilação , Extratos Vegetais/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is the most common liver malignancy still demanding for novel therapeutic options. Since the ion channel inhibitor TRAM-34 (1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole) was shown to block growth in various cancer cells, we investigated anti-tumor effects of TRAM-34 in human HCC cell lines. We found that TRAM-34 reduced HCC cell proliferation without induction of apoptosis. This was due to a decreased mRNA expression of estrogen receptor alpha (ESR1) and a reduced activation of NF-kappaB, which both are implicated in the development of HCC. Therefore, TRAM-34 might represent a novel therapeutic target for the treatment of HCC.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Pirazóis/farmacologia , Fator de Transcrição RelA/metabolismo , Western Blotting , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Células Tumorais CultivadasRESUMO
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Treatment options, especially in advanced tumor stages, are still limited. Inhibition of signaling cascades involved in the pathogenesis of HCC - such as NF-ĸB - offer a promising therapeutic approach. Aim of this study was to examine anti-neoplastic effects of (+)-episesamin which has been isolated from an anti-fibrotic extract of Lindera obtusiloba on human HCC cells with particular interest in activation of NF-κB. The human HCC cell lines HepG2, Huh-7 and SK-Hep1 were treated with (+)-episesamin. Beside measurement of proliferation, invasion and apoptosis, effects of (+)-episesamin on NF-κB-activity, VEGF secretion and enzymatic MMP-9 activity were determined. Anti-inflammatory effects were assessed by IL-6 ELISA using HCC cells and RAW264.7 macrophages. 10 µM (+)-episesamin reduced the proliferation of HCC cells by ~50%, suppressed invasion and induced apoptosis. DNA-binding ELISA experiments revealed that (+)-episesamin treated HCC cells showed a suppressed basal and TNFα-induced activation of NF-κB and a subsequent suppression of TNFα- and LPS-induced IL-6 production. Further, (+)-episesamin exhibited inhibitory effects on the enzymatic activity of recombinant MMP-9 and the secretion of MMP-9 and VEGF by HCC cells into their supernatants. Our findings show that anti-neoplastic effects of (+)-episesamin are mediated via suppressed activation of NF-κB which entails a decreased release of pro-inflammatory IL-6. In addition, (+)-episesamin inhibits MMP-9, which is strongly expressed in invasive HCC, and the production of proangiogenic VEGF. We conclude that (+)-episesamin has the potential to be further explored as a complementary treatment for HCC.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Dioxóis/farmacologia , Lignanas/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , NF-kappa B/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Camundongos , NF-kappa B/metabolismo , Invasividade Neoplásica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Development of end-stage liver and graft disease is suspected to be partially determined by the individual genetic background. Mannose-binding lectin (MBL) is an important immunomodulatory factor, which is supposed to be involved in complement activation and oncogenesis. Genetic polymorphisms of MBL-2 alter MBL functionality. The aim of our study was to determine the prevalence of MBL-2 polymorphism (rs7096206) in hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) based on histological analysis of explanted livers in patients undergoing liver transplantation (LT). METHODS: One hundred and seventy-seven patients, who underwent LT for HCV-induced liver disease, were genotyped for MBL-2 by TaqMan genotyping assay. Sixty-two patients with histologically confirmed HCC were compared with 115 patients without HCC. MBL-2 genotypes were corelated with the growth patern, tumour size and pretransplant α-fetoprotein (AFP) level of HCC patients. RESULTS: The prevalence of GG/GC genotypes was significantly higher among HCC patients compared with tumour-free explanted livers (P = 0.004; odds ratio 2.5; 1.3-4.8). GG/GC genotype group was significantly associated with the size of HCC (P = 0.022), higher pretransplant AFP level (P = 0.010) and bilobar tumour growth (P = 0.038). Furthermore, CC genotype was found to be significantly more frequent in AFP-negative HCCs (P = 0.002). CONCLUSION: Mannose-binding lectin-2 polymorphism seems to be involved in the development of pretransplant HCV-induced HCC and should be further investigated as potential risk factor for HCV-associated carcinogenesis.
Assuntos
Carcinoma Hepatocelular/genética , Hepatite C/complicações , Neoplasias Hepáticas/genética , Lectina de Ligação a Manose/genética , Polimorfismo Genético , Idoso , Carcinoma Hepatocelular/etiologia , Primers do DNA/genética , Feminino , Genótipo , Alemanha , Humanos , Neoplasias Hepáticas/etiologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , alfa-Fetoproteínas/metabolismoRESUMO
BACKGROUND: In traditional Chinese and Korean medicine, an aqueous extract derived from wood and bark of the Japanese spice bush Lindera obtusiloba (L.obtusiloba) is applied to treat inflammations and chronic liver diseases including hepatocellular carcinoma. We previously demonstrated anti-fibrotic effects of L.obtusiloba extract in hepatic stellate cells. Thus, we here consequently examine anti-neoplastic effects of L.obtusiloba extract on human hepatocellular carcinoma (HCC) cell lines and the signaling pathways involved. METHODS: Four human HCC cell lines representing diverse stages of differentiation were treated with L.obtusiloba extract, standardized according to its known suppressive effects on proliferation and TGF-ß-expression. Beside measurement of proliferation, invasion and apoptosis, effects on signal transduction and NF-κB-activity were determined. RESULTS: L.obtusiloba extract inhibited proliferation and induced apoptosis in all HCC cell lines and provoked a reduced basal and IGF-1-induced activation of the IGF-1R signaling cascade and a reduced transcriptional NF-κB-activity, particularly in the poorly differentiated SK-Hep1 cells. Pointing to anti-angiogenic effects, L.obtusiloba extract attenuated the basal and IGF-1-induced expression of hypoxia inducible factor-1α, vascular endothelial growth factor, peroxisome proliferator-activated receptor-γ, cyclooxygenase-2 and inducible nitric oxide synthase. CONCLUSIONS: The traditional application of the extract is confirmed by our experimental data. Due to its potential to inhibit critical receptor tyrosine kinases involved in HCC progression via the IGF-1 signaling pathway and NF-κB, the standardized L.obtusiloba extract should be further analysed for its active compounds and explored as (complementary) treatment option for HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Lindera , Neoplasias Hepáticas/tratamento farmacológico , NF-kappa B/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Receptor IGF Tipo 1/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Hepáticas/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fitoterapia , Casca de Planta , Extratos Vegetais/farmacologia , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , MadeiraRESUMO
Up to 30% of liver transplants will develop graft cirrhosis within 5 years after liver transplantation (LT) due to recurrent HCV-infection forwarding accelerated graft damage. Genetic variants of cytokines involved in the immune response may contribute to the degree of graft inflammation, fibrosis progression, and antiviral therapy outcome. The aim of our study was to analyze biochemical and histological inflammation extent based on protocol liver biopsies and to evaluate the role of genetic variants of IL-28b in HCV-related graft disease and antiviral treatment response. 183 patients, who underwent liver transplantation for HCV-induced liver disease, were genotyped for IL-28b (rs8099917, G ≥ T) by TaqMan Genotyping Assay. 56 of 159 patients have been successfully treated with interferon-based antiviral therapy. 605 protocol liver biopsies performed 0.5 to 10 and more than 10 years after transplantation were evaluated according to Desmet and Scheuer classification of inflammation and fibrosis. Prevalence of IL-28b-genotypes was correlated with histological severity of graft damage, levels of aminotransferases, occurrence of acute cellular rejection, pre-treatment viremia, and antiviral therapy outcome. Significant association of IL-28b-genotype distribution was observed to the median grade of inflammation (p < 0.001), mean levels of aminotransferases (ALT: p = 0.001, AST: p = 0.003), median pre-treatment viremia level within 1 year after LT (p = 0.046) and interferon-based antiviral therapy failure (p < 0.001). Among successfully treated patients, G-allele was significantly less frequent, and the genotype GG was not present at all. No differences were observed regarding acute cellular rejection (p = 0.798) and fibrosis stages (p = 0.586). IL-28b polymorphism seems to influence the degree of graft inflammation at biochemical and histological levels. G-allele might serve as a marker for graft inflammation and as a predictor for unfavorable antiviral therapy outcome in HCV-re-infected LT-population.
Assuntos
Antivirais/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/genética , Interleucinas/genética , Transplante de Fígado/imunologia , Polimorfismo Genético , Adulto , Idoso , Biomarcadores/sangue , Biópsia , Distribuição de Qui-Quadrado , Feminino , Frequência do Gene , Predisposição Genética para Doença , Alemanha , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/genética , Hepatite C/diagnóstico , Hepatite C/imunologia , Humanos , Interferons , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/imunologia , Masculino , Pessoa de Meia-Idade , Recidiva , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: Fibrolytic and profibrotic activities of the matrix metalloproteinases (MMPs)-2 and -9 play a central role in liver fibrosis. Since binding to the extracellular matrix influences the activity of both gelatinases, here the role of fibrillar collagens as the most abundant matrix components in fibrotic tissue was investigated. RESULTS: In situ zymography and immunohistology showed association of enzymatically inactive prodomain-containing proMMP-2 and proMMP-9 but not of their activated forms to fibrillar collagen structures, which are not substrates of these gelatinases. In solid-phase binding studies with human collagens and collagen fragments, up to 45% of [125I]-labeled proMMP-2 and proMMP-9 but not of active (act)MMP-2 and actMMP-9 were retained by natural collagenous molecules and by synthetic analogs containing repeated Gly-Pro-Hyp triplets (GPO). Surface plasmon resonance yielded binding constants for the interaction of collagen type I (CI) with proMMP-2 and proMMP-9 in a nanomolar range. Values for actMMP-2 and actMMP-9 were 30-40 times higher. Tenfold molar excesses of (GPO)10 reduced the interaction of CI with pro- and actMMP-2 by 22- or 380-fold and resulted in prodomain release accompanied by high enzymatic activation and activity. Pointing to gelatine substrate displacement, higher (GPO)10 concentrations blocked the enzymatic activity. The MMP-2 prodomain-derived collagen-binding domain peptide (P33-42) binds to the collagen-binding domain of MMP-2, thereby preserving enzymatic inactivity. Synthetic P33-42 peptide competed with proMMP-2 binding to CI and prevented (GPO)10-mediated proMMP-2 activation. In contrast to (GPO)10, P33-42 did not activate proMMP-2, making triple helical and hydroxyproline-containing (GPO)10 unique in modulating gelatinase availability and activity. CONCLUSIONS: These findings suggest novel strategies using collagen analogs for the resolution of liver fibrosis via fibrotic matrix-sequestered gelatinases.
RESUMO
Obesity, the related metabolic syndrome and associated liver diseases represent an epidemic problem and demand for effective therapeutic strategies. In this regard, natural compounds derived from Oriental medicine such as green tea polyphenols influencing adipogenesis attract growing attention. In Korea, an aqueous extract from the Japanese spice bush Lindera obtusiloba is traditionally used for treatment of inflammation and prevention of liver damage. We here investigated effects of the L. obtusiloba extract on cell growth, apoptosis, Wnt signaling and differentiation of (im)mature adipocytes using 3T3-L1, an established cell line for studying adipogenesis. L. obtusiloba extract reduced the de novo DNA synthesis of 3T3-L1 preadipocytes in a concentration dependent manner with an IC(50) of â¼135 µg/ml paralleled by induction of caspase 3/7 mediated apoptosis. Hormone-induced 3T3 L1 differentiation in the presence of L. obtusiloba extract resulted in a reduced accumulation of intracellular lipid droplets by 70%, in down-regulated expression of the adipogenesis-associated proteins glucose transporter-4 and vascular endothelial growth factor, in reduced secretion of the proadipogenic matrix metalloproteinase-2, and in dampened phosphorylation of the Wnt pathway effector protein ß-catenin with subsequent diminished expression of the peroxisome proliferator-activated receptor-γ. Treatment of mature adipocytes with L. obtusiloba extract also significantly reduced intracellular lipid droplets. In addition to this strong interference of L. obtusiloba extract with adipogenesis, L. obtusiloba extract exerted anti-inflammatory effects. L. obtusiloba extract significantly attenuated lipopolysaccharide- and tumor necrosis factor α-induced secretion of IL-6 by preadipocytes, thus influencing insulin resistance and inflammatory state characterizing obesity. In conclusion, extracts of L. obtusiloba should be evaluated as a potential complementary treatment option for obesity associated with the metabolic syndrome.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia , Lindera/química , Fitoterapia , Proteínas Wnt/metabolismo , Células 3T3-L1 , Animais , Anti-Inflamatórios/farmacologia , Apoptose , Diferenciação Celular , Proliferação de Células , Regulação para Baixo , Inflamação/tratamento farmacológico , Interleucina-6/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Obesidade/tratamento farmacológico , Extratos Vegetais/farmacologia , Transdução de Sinais , beta Catenina/metabolismoRESUMO
The extracellular matrix (ECM) attracts increasing attention as a store of biologically active molecules and as a reservoir of potent cell signalling molecules released by proteolytic action. Both, cytokines and proteases mediating such release are sequestered in the ECM. Here, we found matrix metalloproteinase (MMP) proforms closely associated with collagenous septae in fibrotic liver tissue, and we screened immobilized human placenta-derived collagen chains and other ECM proteins for MMP-binding activity. Following the establishment of a novel highly-efficient two-step chromatography procedure for the isolation of the purified alpha-chains of the pepsin-resistant triple-helical CVI fragment (CVI/PR) solid phase and surface plasmon resonance binding studies were performed. We identified the triple-helical domain of the alpha2 chain of microfilamentous CVI alpha2(VI) as having nanomolar affinity for the collagenases proMMP-1, -8 , -13 and stromelysin-1 (MMP-3), thus extending the repertoire of pericellular and substrate-based interactions of MMPs. Enzymatic activity assays enabled the correlation of MMP activity with CVI binding, in that alpha2(VI) chain-mediated inhibition of enzymatic activity is accompanied by increased binding. Similar results were shown for the gelatinase proMMP-9, whereas for proMMP-2, the alpha2(VI) chain at low concentrations seems to interfere with prodomain binding resulting in enhanced activity without scission of the prodomain. Stable complexes of proMMP-2 and alpha2(VI) chain competed with gelatinase binding to the preferred ligand, collagen type I. In conclusion, the alpha2(VI) chain modulates MMP availability by sequestering proMMPs in the ECM, blocking proteolytic activity. Therefore, CVI and especially its alpha2(VI) chain might serve as a lead structure for MMP-based therapeutics which modulates the action of these matrix components, e.g. in fibrosis and cancer.
Assuntos
Colágeno Tipo VI/metabolismo , Precursores Enzimáticos/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Subunidades Proteicas/metabolismo , Ligação Competitiva/fisiologia , Biocatálise/efeitos dos fármacos , Cromatografia Líquida/métodos , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo VI/isolamento & purificação , Colágeno Tipo VI/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Precursores Enzimáticos/antagonistas & inibidores , Feminino , Humanos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz Secretadas/antagonistas & inibidores , Placenta/química , Gravidez , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/farmacologia , Proteínas Recombinantes/metabolismoRESUMO
Liver fibrosis is characterized by high expression of the key profibrogenic cytokine transforming growth factor (TGF)-beta and the natural tissue inhibitor of metalloproteinases (TIMP)-1, leading to substantial accumulation of extracellular matrix. Liver fibrosis originates from various chronic liver diseases, such as chronic viral hepatitis that, to date, cannot be treated sufficiently. Thus, novel therapeutics, for example, those derived from Oriental medicine, have gained growing attention. In Korea, extracts prepared from Lindera obtusiloba are used for centuries for treatment of inflammation, improvement of blood circulation and prevention of liver damage, but experimental evidence of their efficacy is lacking. We studied direct antifibrotic effects in activated hepatic stellate cells (HSCs), the main target cell in the fibrotic liver. L. obtusiloba extract (135 mug/ml) reduced the de novo DNA synthesis of activated rat and human HSCs by about 90%, which was not accompanied by cytotoxicity of HSC, primary hepatocytes and HepG2 cells, pointing to induction of cellular quiescence. As determined by quantitative polymerase chain reaction, simultaneous treatment of HSCs with TGF-beta and L. obtusiloba extract resulted in reduction of TIMP-1 expression to baseline level, disruption of the autocrine loop of TGF-beta autoinduction and increased expression of fibrolytic matrix metalloproteinase (MMP)-3. In addition, L. obtusiloba reduced gelatinolytic activity of HSC by interfering with profibrogenic MMP-2 activity. Since L. obtusiloba extract prevented intracellular oxidative stress experimentally induced by tert-butylhydroperoxide, we concluded that the direct antifibrotic effect of L. obtusiloba extract might be mediated by antioxidant activity. Thus, L. obtusiloba, traditionally used in Oriental medicine, may complement treatment of chronic liver disease.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Lindera , Cirrose Hepática/tratamento farmacológico , Extratos Vegetais/farmacologia , Actinas/genética , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/genética , Relação Dose-Resposta a Droga , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Estresse Oxidativo/efeitos dos fármacos , Casca de Planta , Ratos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , MadeiraRESUMO
BACKGROUND AND AIM: It has been proven in various animal studies that recombinant human erythropoietin (rHuEPO) protects renal, cardiac and neuronal, as well as hepatic, tissue from ischemia, and promotes regeneration of adult central nervous system neurons. To date, no data are available as to whether rHuEPO has the ability to stimulate liver regeneration after liver resection. METHODS: Rats undergoing 70% or 90% hepatectomy received an intraportalvenous administration (i.p.) of rHuEPO prior to resection or a subcutaneous injection (s.c.) for 3 days postoperatively, control animals were treated with surgery and saline injection only. Regeneration capacity of remnant livers was studied over 7 days by histology and immunohistochemistry (Ki-67, proliferating cell nuclear antigen [PCNA]). Polymerase chain reaction was carried out to measure transforming growth factor beta (TGF-beta), hypoxia induced factor (HIF), signal transducing activator 3 and vascular endothelial growth factor. RESULTS: Ten-day survival in rats undergoing 90% hepatectomy significantly increased in i.p.-pretreated animals. After 70% hepatectomy the mitotic index was significantly increased in both rHuEPO-treated groups. These data were confirmed by PCNA and Ki-67 expression, which was significantly increased in the treated groups 24 h and 2 days after liver resection. TGF-beta and HIF mRNA both were upregulated in control animals 3 h after surgery. CONCLUSION: rHuEPO effectively increased liver regeneration in rats after 70% liver resection and enhanced survival after 90% hepatectomy. Thus, rHuEPO may increase the regenerative capacity after major hepatectomy.
Assuntos
Eritropoetina/farmacologia , Hepatectomia , Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Eritropoetina/administração & dosagem , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Antígeno Ki-67/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/cirurgia , Masculino , Índice Mitótico , Modelos Animais , Veia Porta , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fatores de Tempo , 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/metabolismoRESUMO
Collagen XIV (CXIV) is a fibril-associated collagen that is mainly expressed in well differentiated tissues and in late embryonic development. Because CXIV is almost absent in proliferating and/or dedifferentiated tissues, a functional role in maintaining cell differentiation is suspected. We demonstrate antiproliferative, quiescence- and differentiation-inducing effects of human CXIV and its recombinant fragments on mesenchymal cells. In primary human fibroblasts, in mouse 3T3 fibroblasts and in 3T3-L1 preadipocytes, CXIV reduced de novo DNA synthesis by 75%, whereas cell numbers and viability remained unaltered. Cells showed no signs of apoptosis, and maximal proliferation was restored when serum was supplemented, thus indicating that CXIV induced reversible cellular quiescence. Exposure of fibroblasts to CXIV in vitro led to cellular bundles and clusters. CXIV also triggered trans-differentiation of 3T3-L1 preadipocytes into adipocytes, as could be shown by lipid accumulation and by expression of the glucose transporter Glut4. These effects were also observed with the amino-terminal recombinant fragment Gln(29)-Pro(154) that harbors the first fibronectin type III domain and a 39-amino-acid extension, whereas no activity was found for all other recombinant CXIV fragments. Based on these finding the development of small molecular analogs that modulate fibroblast cell growth and differentiation, e.g. in wound healing and fibrosis, seems feasible.
Assuntos
Adipócitos/citologia , Fibroblastos/citologia , Fibronectinas/química , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Apoptose , Compostos Azo/farmacologia , Transporte Biológico , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno/química , Fibroblastos/metabolismo , Fibrose/patologia , Citometria de Fluxo , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glutamina/química , Glutationa Transferase/metabolismo , Glicoproteínas/química , Humanos , Lipídeos/química , Mesoderma/metabolismo , Camundongos , Placenta/metabolismo , Prolina/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Timidina/química , Fatores de Tempo , CicatrizaçãoRESUMO
BACKGROUND: Schizophrenia is frequently accompanied by hypometabolism and altered gene expression in the prefrontal cortex. Cellular metabolism regulates chromatin structure, including covalent histone modifications, which are epigenetic regulators of gene expression. OBJECTIVE: To test the hypothesis that down-regulated metabolic gene expression is associated with histone modification changes in the prefrontal cortex of subjects with schizophrenia. DESIGN AND SUBJECTS: Histones and gene transcripts were profiled in the postmortem prefrontal cortex of 41 subjects with schizophrenia and 41 matched controls. The phosphorylation, acetylation, and methylation of 6 lysine, serine, and arginine residues of histones H3 and H4 were examined together with 16 metabolic gene transcripts using serial immunoblotting, immunohistochemical analysis, custom-made complementary DNA arrays, and quantitative real-time reverse transcriptase-polymerase chain reaction. RESULTS: Subjects with schizophrenia, as a group, showed no significant alterations in histone profiles or gene expression. In a subgroup of 8 patients with schizophrenia, levels of H3-(methyl)arginine 17, H3meR17, exceeded control values by 30%, and this was associated with the decreased expression of 4 metabolic transcripts. CONCLUSIONS: High levels of H3-(methyl)arginine 17 are associated with down-regulated metabolic gene expression in the prefrontal cortex of a subset of subjects with schizophrenia. Histone modifications may contribute to the pathogenesis of prefrontal dysfunction in schizophrenia.
Assuntos
Cromatina/genética , Regulação para Baixo , Expressão Gênica/genética , Córtex Pré-Frontal/metabolismo , Esquizofrenia/genética , Cromatina/metabolismo , Epigênese Genética/genética , Perfilação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esquizofrenia/metabolismoRESUMO
The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that mesenchymal cell-derived keratinocyte growth factor (KGF), a key stimulator of epithelial cell proliferation during wound healing, preferentially binds to collagens I, III, and VI. Binding is inhibited in a dose-dependent manner by denatured single collagen chains and collagen cyanogen bromide peptides. This interaction is saturable with dissociation constants of approximately 10(-8) to 10(-9) m and estimated molar ratios of up to three molecules of KGF bound to one molecule of triple helical collagen. Furthermore, collagen-bound KGF stimulated the proliferation of transformed keratinocyte or HaCaT cells. Ligand blotting of collagen-derived peptides points to a limited set of collagenous consensus sequences that bind KGF. By using synthetic collagen peptides, we defined the consensus sequence (Gly-Pro-Hyp)(n) as the collagen binding motif. We conclude that the preferential binding of KGF to the abundant collagens leads to a spatial pattern of bioavailable KGF that is dictated by the local organization of the collagenous extracellular matrix. The defined collagenous consensus peptide or its analogue may be useful in wound healing by increasing KGF bioactivity and thus modulating local epithelial remodeling and regeneration.
Assuntos
Colágeno/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Motivos de Aminoácidos , Colágeno/química , Colódio/química , Brometo de Cianogênio/química , Relação Dose-Resposta a Droga , Fator 7 de Crescimento de Fibroblastos , Glicina/química , Humanos , Hidroxiprolina/química , Cinética , Ligantes , Peptídeos/metabolismo , Prolina/química , Ligação Proteica , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that oncostatin M (OSM), a profibrogenic cytokine and modulator of cancer cell proliferation, specifically binds to collagen types I, III, IV, and VI, immobilized on polystyrene or nitrocellulose. Single collagen chains inhibit these interactions in a dose-dependent manner. Cross-inhibition experiments of collagen-derived peptides point to a limited set of OSM-binding collagenous consensus sequences. Furthermore, this interaction is found for OSM but not for other interleukin-6 type cytokines. OSM binding to collagens is saturable, with dissociation constants around 10(-8) m and estimated molar ratios of 1-3 molecules of OSM bound to one molecule of triple helical collagen. Furthermore, collagen-bound OSM is biologically active and able to inhibit proliferation of A375 melanoma cells. We conclude that abundant interstitial collagens dictate the spatial pattern of bioavailable OSM. This interaction could be exploited for devising collagenous peptide-antagonists that modulate OSM bioactivity in tumor growth and fibrotic disorders like rheumatoid arthritis and hepatic fibrosis.