RESUMO
Irisin is a recently discovered myokine reported as protective protein released from exercising skeletal muscles. Although myokines were recently reported to possess the antioxidizing properties, the impact of irisin on the functions of macrophages with respect to its anti-inflammatory potential has not been fully elucidated. Here, we determined the ability of irisin to interact with reactive oxygen species (ROS) in RAW 264.7 murine macrophages. The macrophages were pre-incubated with irisin (0 - 50 nM), some of which had undergone additional co-incubation with bacterial lipopolysaccharide (LPS) (100 ng/ml). Cell viability, the reactive oxygen species scavenging potential as well as the mRNA and protein expression of key oxidative stress factors such as superoxide dismutase 1 (SOD-1), superoxide dismutase 2 (SOD-2), glutathione peroxidase (GSH-Px), catalase 9 (Cat-9), nuclear factor (erythroid-derived 2)-like 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) were evaluated. We found that irisin applied in a concentration of 50 nM significantly attenuated the production of harmful H2O2 and this effect appears to be mediated by a significant increase in the expression of key enzymes involved with antioxidative stress pathways including SOD, GSH-Px and Cat-9 predominantly observed after stimulation of these cells with LPS. We conclude that 1) irisin exhibits a potent antioxidant and anti-inflammatory activities in non-stimulated and LPS-stimulated isolated murine macrophages in vitro, and 2) this protective and antioxidative activity of irisin in vitro might be considered as an important component of protective action of this peptide in vivo, especially under condition of exercise.
Assuntos
Fibronectinas/metabolismo , Macrófagos/metabolismo , Estresse Oxidativo/fisiologia , Animais , Catalase/genética , Catalase/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
A biological activity of myokine irisin, has been intensively investigated in the context of a browning process occurring in white adipose tissue, but its role as a modulator of immune response has been little studied. The aim of our study was to determine the impact of irisin (0 - 100 nM) on pro-inflammatory activation of adipocyte 3T3 L1 cell line. Irisin reduced in a concentration-dependent manner the expression and activity of major proinflammatory cytokines, e.g. tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) expression and their secretion into cell medium. Moreover, irisin enhanced adiponectin synthesis reversing the effect of the lipopolysaccharide (LPS)-induced attenuation of this adipokine expression. The opposite effect was observed for leptin whose expression increased by LPS and this effect was suppressed by irisin application. A decreased phosphorylation and activation of nuclear factor kappa B (NFκB) in the presence of irisin suggests that mechanism of action irisin involves the inhibition of an inflammatory transcription factor. Irisin exerts also an inhibitory effect on macrophage migration toward chemoattractants present in adipocyte supernatants. Among the specific molecules secreted by adipocytes was monocyte chemotactic protein 1 (MCP-1) whose expression was suppressed by irisn. In majority of experiments irisin was effective in 100 nM concentration but in some of them the inhibitory effects occurred already in a concentration of 50 nM of this peptide. This study for the first time showed that adipocytes are directly affected by irisin and provides an evidence on anti-inflammatory action of irisin on fat cells.
Assuntos
Adipócitos/metabolismo , Fibronectinas/metabolismo , Inflamação/metabolismo , Células 3T3-L1 , Animais , Sobrevivência Celular , Quimiotaxia , Citocinas/genética , Citocinas/metabolismo , Exercício Físico , Humanos , Inflamação/genética , Lipopolissacarídeos , Camundongos , NF-kappa B/metabolismo , Obesidade , PPAR gama/genética , PPAR gama/metabolismo , Células RAW 264.7RESUMO
Cell surface integrins, especially those binding to fibronectin (FN), participate in processes of tumor cell invasion and metastasis. Changes in glycosylation of cell surface adhesion proteins are often associated with malignant transformation of cells. In this study we examined the influence of swainsonine (SW) on adhesion, wound healing and haptotactic migration on FN, comparing the responses of different human melanoma cell lines: primary WM35 and metastatic WM9, WM239 and A375. We also examined the role of alpha subunits in adhesion to FN. All of the antibodies inhibited adhesion to FN but with different efficiencies depending on the cell line. Adhesion was mediated mainly by integrin alpha(5)beta(1) (WM9, A375), alpha(3)beta(1) (WM35, A375, WM239). Scratch wound repair was significantly faster on FN-coated wells than on plastic for all cells except for WM9. A375 and WM9 had the greatest migration ability, both expressing the highest level of alpha(5)beta(1) integrin. It seems very likely that adhesion to FN can be accomplished by many different integrins, but for effective migration alpha(5)beta(1) integrin is responsible. Only A375 and WM239 cell lines reacted to SW treatment. In the presence of SW WM239 and A375 cells had 70% and 40% increased adhesion to FN, and their migration was decreased 40% and 50%, respectively. Interestingly, although most of the cell lines share a common profile of integrins, each line interacted with FN differently. They differed mainly in the repertoire of integrins used for adhesion, and in the manner in which glycosylation affected these processes. The influence of SW was observed in two metastatic cell lines indicating the contribution of glycosylation status to the progression of melanoma. The lack of reaction to SW in WM9 cells may suggest that there is a threshold in the expression level of the highly branched N-glycans that may influence the adhesion and migration properties of the cell.
Assuntos
Fibronectinas/metabolismo , Melanoma/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Integrinas/metabolismo , Melanoma/patologia , Ligação Proteica/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Swainsonina/farmacologiaRESUMO
In tumour cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. This study used cell lines having very different behaviour in vivo: HCV29 non-malignant transitional epithelium and T24 bladder transitional cell carcinoma. These differences in behaviour might be due in part to differences in cellular glycosylation patterns. Glycan chain analysis of their glycoproteins was performed with the use of specific lectins. The functional role of carbohydrates was studied by treating these cells with swainsonine, an inhibitor of Golgi alpha-mannosidase II, and in vitro adhesion and migration assays. The adhesion of swainsonine-treated HCV29 and T24 cells was increased on fibronectin and type IV collagen by 1.5- and 2-fold, respectively, whereas adhesion on laminin was virtually unchanged after swainsonine-treatment in HCV29 cells and was increased in T24 cells. Swainsonine treatment reduced the rate of T24 cell migration by 20%. We concluded that beta1-6 branched tri- and tetraantennary complex-type glycans have an important function in adhesion and migration in the studied cell lines. These data support the view that oligosaccharides are involved in several steps of the metastatic process.
Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Polissacarídeos/biossíntese , Ureter/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/química , Feminino , Glicosilação/efeitos dos fármacos , Humanos , Masculino , Polissacarídeos/química , Especificidade da Espécie , Swainsonina/farmacologia , Ureter/química , Ureter/citologia , Neoplasias da Bexiga Urinária/química , Neoplasias da Bexiga Urinária/patologiaRESUMO
Ariboflavinosis, that is, vitamin B2 deficiency, is a common problem affecting the populations of both developing and affluent countries. Teenagers, elderly people, pregnant women, and alcohol abusers represent groups that are particularly susceptible to this condition. This study was aimed to determine the effect of different riboflavin concentrations (deficiency and supplementation) on macrophages response induced by bacteria or yeast-derived factors i.e. lipopolysaccharide (LPS) and zymosan, respectively. Mouse macrophage RAW 264.7 cells were cultured for 5 days in a medium with a riboflavin concentration corresponding to moderate riboflavin deficiency (3.1 nM), physiological state (10.4 nM), or vitamin pill supplementation (300 nM). On the third or fourth day of deprivation, the medium in some groups was supplemented with riboflavin (300 nM). Macrophages activation were assessed after LPS or zymosan stimulation. Short-term (5 days) riboflavin deprivation resulted in the pathological macrophages activation, manifested especially in a reduction of cell viability and excess release of tumor necrosis factor-α (TNF-α) and high-mobility group box 1 (HMGB1) protein. Moreover, the levels of inducible nitric oxide synthase (iNOS), nitric oxide (NO), heat shock protein (Hsp72), interleukin 1ß (IL-1ß), monocyte chemoattractant protein-1 (MCP-1), and interleukin 10 (IL-10) decreased after riboflavin deprivation, but medium enrichment with riboflavin (300 nM) on the third or fourth day reversed this effect. In the riboflavin-supplemented group, LPS-stimulated macrophages showed lower mortality accompanied by higher Hsp72 expression, reduction of Toll-like receptor 4 (TLR4) and TNF-α, and elevation of NO, IL-6, and IL-10. Moreover, the TLR6, NO, iNOS, IL-1ß, MCP-1, and the keratinocyte chemoattractant (KC) levels significantly decreased in the zymosan-stimulated groups maintained in riboflavin-enriched medium. We conclude that short-term riboflavin deficiency significantly impairs the ability of macrophages to induce proper immune response, while riboflavin enrichment decreases the proinflammatory activation of macrophages.
Assuntos
Macrófagos/imunologia , Deficiência de Riboflavina/imunologia , Animais , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Proteínas de Choque Térmico HSP72/imunologia , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Camundongos , NF-kappa B/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Nitritos/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , ZimosanRESUMO
Glycosylation is generally altered in tumour cells in comparison with their normal counterparts. These alterations are thought to be important because they contribute to the abnormal behaviour of cancer cells. Therefore, we have comparatively analysed the glycoproteins in cell extracts from human melanoma (primary site--WM35; metastatic sites-- WM239, WM9 and A375) cell lines using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and lectin staining. The glycoprotein pattern of the WM35 line differed from that of the other cell lines in having less proteins that reacted with Sambucus nigra, Maackia amurensis and Phaseolus vulgaris agglutinins. A glycoprotein of about 70 kDa had a significantly increased reaction with Sambucus nigra agglutinin in all the cell lines from metastatic sites. In the WM9, WM239 and A375 cell lines, additional bands (160-100 kDa) were stained with Phaseolus vulgaris agglutinin, suggesting that cells from metastatic sites contain more glycoproteins with beta1-6 branches. On the other hand, only minor changes in the reaction with Galanthus nivalis agglutinin, a mannose-specific lectin, were detected. Among the proteins showing different lectin staining, one, with an apparent molecular weight of 133 kDa, was recognized by antibodies as N-cadherin. The present results suggest that in human melanoma the expression of branched and sialylated complex type N-oligosaccharides consistently increased in cells from metastatic sites, and support the view that carbohydrates are associated with the acquisition of the metastatic potential of tumour cells.