RESUMO
Rat hepatoma cells H-35 cultured in serum-free medium were exposed to interleukin-6 (IL-6), interleukin-1 (IL-1), hepatocyte growth factor (HGF), retinoic acid (RA), or a mixture of these factors. Production of acute phase proteins, responding to IL-6 alone (type 2) or to the mixture of IL-6 and IL-1, was assessed by electroimmunoassay and the corresponding mRNAs were compared by Northern blot analysis. HGF enhanced IL-6-induced synthesis of alpha-2-macroglobulin but reduced synthesis of C3 complement and alpha-1-acid glycoprotein. Retinoic acid reduced the response to IL-6 of alpha-2-macroglobulin but enhanced that of alpha-1-acid glycoprotein and especially of C3 complement. In general, changes in protein secretion were correlated with the contents of their corresponding cellular mRNAs. These results indicate that hepatocyte growth factor can enhance basal or IL-6-induced gene expression of type 2 and reduce the expression of type 1 acute phase proteins, whereas the action of retinoic acid is opposite. The modulation of acute phase response by HGF and RA likely involves transcriptional factors and regulatory sequences in the genes coding for these two types of acute phase proteins.
Assuntos
Proteínas de Fase Aguda/biossíntese , Citocinas/farmacologia , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Tretinoína/farmacologia , Animais , Northern Blotting , Linhagem Celular , Complemento C3/biossíntese , Interleucina-6/farmacologia , Neoplasias Hepáticas Experimentais , Orosomucoide/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Células Tumorais Cultivadas , alfa 1-Antitripsina/biossíntese , alfa-Macroglobulinas/biossínteseRESUMO
Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.
Assuntos
Carboidratos , alfa 1-Antitripsina/metabolismo , 1-Desoxinojirimicina , Alcaloides/farmacologia , Linhagem Celular , Estabilidade de Medicamentos , Glucosamina/análogos & derivados , Glucosamina/farmacologia , Glicosilação , Temperatura Alta , Humanos , Elastase de Leucócito , Fígado/efeitos dos fármacos , Fígado/metabolismo , Elastase Pancreática/metabolismo , Relação Estrutura-Atividade , Swainsonina , Tunicamicina/farmacologia , alfa 1-Antitripsina/químicaRESUMO
The ability of dicatechol rooperol and esters to inhibit the production of cytokines in endotoxin-stimulated human alveolar macrophages, human blood monocyte/macrophages, histiocytic cell line U937, and rat alveolar macrophages was examined in vitro. Rooperol derivatives inhibited the production of tumour necrosis factor-alpha, interleukin-1 beta and interleukin-6. Of the esters tested on human cells, rooperol diacetate and tetraacetate were more potent inhibitors of cytokine production (IC50 in the range of 10-20 microM) than rooperol disulphate (IC50 in the range of 25-75 microM). The acetate esters also inhibited cytokine production in rat alveolar macrophages, whereas the sulphate had little effect. Rooperol and acetate esters, in the same concentration range, decreased the production of nitric oxide by rat alveolar macrophages stimulated by endotoxin. These concentrations of rooperol had no effect on cell viability, as indicated by incorporation of 14C-labelled leucine into macrophage proteins and their content of lactate dehydrogenase. The results obtained suggest that rooperol esters are potentially useful antiinflammatory agents.
Assuntos
Catecóis/farmacologia , Citocinas/metabolismo , Ésteres/farmacologia , Inibidores de Lipoxigenase/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Relação Dose-Resposta a Droga , Humanos , Ratos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Dexamethasone and insulin stimulate production of several plasma proteins in primary cultures of adult rat hepatocytes but inhibit their production in primary cultures of Morris hepatoma cell line 7777W. The acute phase response elicited in cultured cells by crude cytokines from activated rat peritoneal macrophages is considerably higher in hepatocytes in the presence of hormones, and especially of dexamethasone. In hepatoma cells the hormones enhance the cytokine-induced formation of fibrinogen and cysteine proteinase inhibitor but are without significant effect on suppression of albumin and alpha-fetoprotein synthesis by macrophage supernatants.
Assuntos
Proteínas de Fase Aguda/biossíntese , Dexametasona/farmacologia , Insulina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Fatores Biológicos/farmacologia , Células Cultivadas , Citocinas , Fígado/citologia , Neoplasias Hepáticas Experimentais/patologia , Ratos , Ratos EndogâmicosRESUMO
Long-term treatment of rats with insulin does not influence the yield of the Golgi membrane-rich fraction obtained by Fleischers' method or the activity of galactosyl transferase (EC 2.1.1.38) in present in this fraction. Exogenous insulin increases the total levels of the seromucoid and serum middle-weight molecular glycopeptide fraction, but does not alter the proportions of neutral sugars bound with the peptide residues of both fractions.
Assuntos
Galactosiltransferases/metabolismo , Glicopeptídeos/metabolismo , Complexo de Golgi/metabolismo , Insulina/farmacologia , Fígado/metabolismo , Orosomucoide/metabolismo , Animais , Fracionamento Celular , Feminino , Glicopeptídeos/sangue , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Membranas/efeitos dos fármacos , Membranas/metabolismo , Tamanho do Órgão , RatosRESUMO
Recombinant preparations of human anti-inflammatory cytokines: IL-4, IL-13 and IL-10, inhibited LPS-induced synthesis of TNFalpha and IL-6 in the whole human blood tested in vitro. These cytokines also inhibited LPS-induced IL-6 and TNF mRNA accumulation in isolated human blood monocytes/macrophages. On the other hand, similar concentrations of IL-4 and IL-13 (but not IL-10) enhanced synthesis of IL-6 in cultured human umbilical vein endothelial cells (HUVEC). In human hepatoma HepG2 cells IL-4 and IL-13 (but not IL-10) inhibited IL-6-induced synthesis of haptoglobin. These differential responses to the tested anti-inflammatory cytokines were observed at mRNA and protein levels and may reflect cell specificities in signalling pathways and gene expression. When HUVEC and HepG2 cells were cultured together and stimulated with LPS the addition of IL-4 or IL-13 resulted in the reduction of LPS-induced and IL-6-mediated haptoglobin synthesis. Thus in co-culture the inhibitory effects of IL-4 or IL-13 on HepG2 cells prevail over stimulation of IL-6 synthesis in HUVEC.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Interleucina-10/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Interleucina-6/biossíntese , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Human peripheral blood monocytes isolated by centrifugation with Mono-Poly resolving medium, and human alveolar macrophages obtained by lung lavage during fiberoscopic bronchoscopy, were cultured in RPMI containing 2% foetal calf serum. The cultures were exposed to modified human proteins: alpha-1-antitrypsin cleaved with papain, fibrinogen degradation products (fraction D) purified from plasmin digest, and non-enzymatically glycosylated (glycated) serum albumin. Conditioned macrophage media were tested for the contents of acute phase cytokines by bioassay with hepatoma cells, and the concentration of interleukin-6 was determined with ELISA. Modified proteins stimulated macrophages to produce acute phase cytokines and the response was not abrogated by polymyxin B in distinction to stimulation of macrophages by endotoxin. Our data indicate that some proteolytically damaged proteins or the end glycosylation products formed in pathological states (acute inflammation, diabetes) may be responsible for the appearance of cytokines in the circulation.
Assuntos
Interleucina-6/biossíntese , Macrófagos Alveolares/metabolismo , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Fibrinogênio/farmacologia , Humanos , Técnicas In Vitro , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Neoplasias Hepáticas Experimentais/metabolismo , Dados de Sequência Molecular , Monócitos/metabolismo , Polimixina B/farmacologia , Ratos , Albumina Sérica/química , Albumina Sérica/farmacologia , Células Tumorais Cultivadas , alfa 1-Antitripsina/farmacologiaAssuntos
Proteínas de Fase Aguda/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias Hepáticas Experimentais/metabolismo , Tretinoína/farmacologia , Animais , Complemento C3/genética , Fibrinogênio/genética , Expressão Gênica , Interleucina-1/farmacologia , Interleucina-6/farmacologia , RNA Mensageiro/genética , Ratos , Células Tumorais Cultivadas , alfa-Macroglobulinas/genéticaAssuntos
Reação de Fase Aguda , Proteínas Sanguíneas/química , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Complemento C3/biossíntese , Fibrinogênio/biossíntese , Humanos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Paclitaxel/farmacologia , Fagocitose/efeitos dos fármacos , Polimixina B/farmacologia , Transdução de SinaisAssuntos
Citocinas/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Tirosina Transaminase/metabolismo , Animais , Células Cultivadas , Dexametasona/farmacologia , Técnicas In Vitro , Insulina/farmacologia , RatosAssuntos
Proteínas de Fase Aguda/biossíntese , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , DNA/biossíntese , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Interleucina-6/farmacologia , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Ratos , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador alfa/farmacologia , Tretinoína/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoAssuntos
Glicoproteínas/biossíntese , Proteínas Sanguíneas/biossíntese , Fenômenos Químicos , Química , Colágeno/análogos & derivados , Colágeno/biossíntese , Membrana Eritrocítica/metabolismo , Glucose/metabolismo , Hemoglobinas Glicadas/biossíntese , Glicoproteínas/análise , Glicosilação , Humanos , Técnicas In Vitro , Proteínas Séricas GlicadasRESUMO
Recent investigations suggest that proinflammatory cytokines such as IL-6 and IL-8 are involved in the development of colorectal cancer (CRC), whereas statins, primarily used to decrease high levels of blood cholesterol, exhibit pleiotropic effects on carcinogenesis. In the present study we compared the expression of IL-8 and IL-6 in tissue samples of tumor and adjacent normal colon mucosa obtained from patients with advanced colorectal cancer (CRC). The analysis of mRNAs expression for these proinflammatory cytokines determined by RT-PCR showed a higher level of IL-8-mRNA in tumor tissue than in normal mucosa, while IL-6 was similarly expressed in tumor and normal tissue. The mean values of serum levels of both IL-6 and IL-8 were significantly higher in CRC patients than in healthy volunteers. Surgical removal of the tumor resulted in a prompt decrease of serum level of IL-8 already on the third day, whereas IL-6 level was transiently increased to become lower only after 7-10 days. Treatment of CRC with simvastatin (80 mg/day for 14 days) led to a significant decrease of serum IL-6, while the IL-8 level was less affected. The in vitro experiments on colorectal cancer-derived cell lines (HT-29 and Caco-2) demonstrated that application of simvastatin decreased generation of both IL-6 and IL-8. The differences in response of serum levels of IL-6 and IL-8 after tumor removal and treatment with simvastatin are novel observations suggesting distinct pathological roles of the two cytokines in CRC development. We conclude that 1) colorectal carcinogenesis is accompanied by increased synthesis and release of proinflammatory cytokines such as IL-6 and IL-8; 2) simvastatin therapy results in a decrease in serum level of proinflammatory cytokines, especially IL-6 in CRC and 3) simvastatin inhibits release of IL-8 and IL-6 from colorectal cell lines.
Assuntos
Neoplasias Colorretais/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Sinvastatina/farmacologia , Células CACO-2 , Neoplasias Colorretais/sangue , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/cirurgia , Células HT29 , Humanos , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-8/sangue , Interleucina-8/genética , Mucosa Intestinal/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinvastatina/uso terapêutico , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
During the inflammatory response at least 2 transcription factors, NF-kappaB and AP-1, are involved in the altered profile of gene expression. We used human hepatoma (HepG2) and human umbilical vein endothelial cells (HUVEC) as a model system: NF-kappaB and AP-1 were activated by the proinflammatory cytokine IL-1 in the absence or presence of 21 selected plant extracts and the effect was evaluated by the electrophoretic mobility shift assay (EMSA). In both types of cells activation of NF-kappaB by IL-1 was significantly inhibited by extracts from Scandix australis and Artemisia alba, whereas extracts from Amaranthus sp., Eryngium campestre, Thymus pulegioides and Reichardia picroides elicited cell-type dependent response. The IL-1-induced AP-1 activation was diminished by extracts from Scandix australis, Amaranthus sp. and Artemisia alba more potently in HUVEC, while extracts from Urospermum picroides and Scandix pecten-veneris in HepG2 cells. Inhibitory activities of plant extracts towards cytokine activated NF-kappaB and AP-1 depend to some extent on the order of addition of IL-1 and plant extract to the cell culture, but the mechanism of action of extract components is not clear: although plant polyphenols may participate they are unlikely to be the only mediators, and MAP kinases were found generally not involved in down-regulation of transcription factors activity by plant extracts.