RESUMO
PURPOSE: Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-ß1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). METHODS: We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-ß1. RESULTS: The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-ß1, while the NEP downregulation decreased the synthesis of TGF-ß1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-ß1 production. CONCLUSIONS: The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-ß1 synthesis during paracrine interactions with NCFs.
Assuntos
Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fibroblastos , Neprilisina/fisiologia , Fator de Crescimento Transformador beta1/biossíntese , Células Cultivadas , Técnicas de Cocultura , Humanos , Células Tumorais CultivadasRESUMO
BACKGROUND: Methylchloroisothiazolinone (MCI) and methylisothiazolinone (MI) are the cause of an increasing number of contact allergies. Understanding the mechanisms by which MCI/MI induces proinflammatory and regulatory factors production is necessary to understand the outcome of allergic contact dermatitis (ACD). OBJECTIVES: To evaluate the dysfunction of proinflammatory cytokines and regulatory factors in the positive MCI/MI patch test at the transcriptional and protein expression levels. Moreover, to analyse the cytokines production induced by MI in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: The selected patients had positive MCI/MI patch test results. The expression of proinflammatory factors was evaluated by q-PCR and immunochemistry at 48 hours of positive MCI/MI patch test. The MCI/MI- or MI- induced secretion of IL-1ß, TNF and IL-6 by PBMC was analysed by flow cytometry. RESULTS: The results showed a decreased TLR4 expression with upregulated IL6, FOXP3, IL10 and TGFß mRNA expression as assessed by q-PCR at the site of the MCI/MI skin reaction. We detected increased protein levels of TLR4, FOXP3 and IL-10 in the dermis layer in the ACD reaction by immunocitochemistry. Moreover, MCI/MI induced proinflammatory cytokine production by PBMC through the NF-κB signalling pathway. CONCLUSION: Considering the altered innate immune response triggered by MCI/MI sensitization, these findings indicate that the regulatory process at the induction phase of ACD is a crucial mechanism. Given the increase in occupational and domestic exposure to MCI/MI, the underlying immunological mechanisms should be understood.
Assuntos
Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/fisiopatologia , Tiazóis/efeitos adversos , Adulto , Animais , Citocinas/metabolismo , Feminino , Fatores de Transcrição Forkhead/biossíntese , Humanos , Inflamação , Interleucina-10/biossíntese , Interleucina-1beta/metabolismo , Interleucina-6/biossíntese , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Masculino , Camundongos , Pessoa de Meia-Idade , Transdução de Sinais , Receptor 4 Toll-Like/biossíntese , Fator de Crescimento Transformador beta1/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that can infect almost all warm-blooded species and induce a chronic infection in human hosts. The aim of this work was to investigate Th1, Th2, Th17 and Treg polarization, induced by four important T. gondii antigens (SAG1, ROP1, GRA8 and MAG1) in acutely and chronically infected patients. For this purpose, SAG1, ROP1, GRA8 and MAG1 were expressed as recombinant proteins, purified, and used to evaluate the proinflammatory and regulatory immune response profiles in seropositive and seronegative individuals. Our results show that SAG1 and ROP1 elicited a proinflammatory profile (INF-γ, IL-12 and IL-17) in individuals in the acute phase, whereas MAG1 and GRA8 induced a regulatory pattern (Treg and TGF-ß) in chronically infected patients. These results reveal fundamental differences in T-cell polarization induced by T. gondii antigens, which could have important implications in the immunopathogenesis of the disease and in future proposals of therapeutic strategies.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Adulto , Animais , Feminino , Humanos , Interferon gama/biossíntese , Subunidade p35 da Interleucina-12/biossíntese , Interleucina-17/biossíntese , Masculino , Proteínas de Membrana/imunologia , Camundongos , Proteínas de Protozoários/imunologia , Toxoplasmose/parasitologia , Fator de Crescimento Transformador beta1/biossínteseRESUMO
Purpose: To evaluate the concentration of transforming growth factor beta 1 (TGFB1) levels in a rat pleural effusion obtained by inoculation of intrapleural bacteria or turpentine through thoracentesis.Methods: Thirty-Nine Wistar rats were divided into three groups: Staphylococcus aureus (SA, n = 17); Streptococcus pneumoniae (SP, n = 12); and turpentine (control, n = 10). Pleural fluid was collected through ultrasound-guided thoracentesis 12 h, 24 h, and 36 h after instillation of bacteria or turpentine. Levels of TGFB1 were measured in pleural fluid.Results: At 12 h, mean TGFB1concentrations were 5.3450 pg/mL in the SA group, 5.3449 pg/mL in the SP group, and 5.3450 pg/mL in controls. At 24 h, they were 4.6700 pg/mL in the SA group, 4.6700 pg/mL in the SP group, and 4.6700 pg/mL in controls. At 36 h, they were 4.6699 pg/mL in the SA group and in control. No difference was observed among the groups in mean TGFB1concentration (p = 0.12); however, a significant intragroup reduction in mean TGFB1 was observed between 12 and 24 h (p < 0.01).Conclusion: The transforming growth factor beta 1 concentrations were not useful as a diagnostic tool or an early marker of infected pleural effusion.(AU)
Assuntos
Animais , Ratos , Empiema Pleural/induzido quimicamente , Derrame Pleural/induzido quimicamente , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1 , Modelos Animais de Doenças , Ratos WistarRESUMO
Leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for its important role in cell signaling. The effects of Leu on several kinds of cells have been studied, altough little is known on its action upon bone cells and cell proliferation. Thus, the aim of this study is to investigate the effects of Leu supplementation on the proliferation of pre-osteoblasts from MC3T3-E1 lineage. MC3T3-E1 cells were kept in Alpha medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimitotic. Cells were treated during 48h by adding 50µM of Leu, which corresponds to a 12.5% increase of the amino acid in the culture medium. The evaluation of viability and proliferation of cultured cells was performed using Trypan Blue dye. In order to identify the mechanisms related to the decreased cellular proliferation, assays were performed to assess cytotoxicity, apotosis, oxidative stress, inflammation, autophagy, senescence and DNA damage. Results showed that Leu supplementation decreased cell proliferation by 40% through mechanisms not related to cell necrosis, apoptosis, oxidative stress, autophagy or inhibition of the mTORC1 pathway. On the other hand, Leu supplementation caused DNA damage. In conclusion, Leu caused a negative impact on bone cell proliferation by inducing cell senescence through DNA damage.
Assuntos
Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Leucina/farmacologia , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Camundongos , Osteoblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/biossínteseRESUMO
End-to-end anastomosis in the treatment for bile duct injury during laparoscopic cholecystectomy has been associated with stricture formation. The aim of this study was to experimentally investigate the effect of oral tamoxifen (tmx) treatment on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs. Twenty-six pigs were divided into three groups [sham (n = 8), control (n = 9) and tmx (n = 9)]. The common bile ducts were transected and anastomosed in the control and tmx groups. Tmx (40 mg/day) was administered orally to the tmx group, and the animals were euthanized after 60 days. Fibrosis was analysed by Masson's trichrome staining. Picrosirius red was used to quantify the total collagen content and collagen type I/III ratio. mRNA expression of transforming growth factor (TGF)-ß1, -ß2 and -ß3 was quantified using real-time polymerase chain reaction (qRT-PCR). The control and study groups exhibited higher fibrosis than the sham group, and the study group showed lower fibrosis than the control group (P = 0.011). The control and tmx groups had higher total collagen content than the sham group (P = 0.003). The collagen type I/III ratio was higher in the control group than in the sham and tmx groups (P = 0.015). There were no significant differences in the mRNA expression of TGF-ß1, -ß2 and -ß3 among the groups (P > 0.05). Tmx decreased fibrosis and prevented the change in collagen type I/III ratio caused by the procedure.
Assuntos
Anastomose Cirúrgica/efeitos adversos , Colágeno/metabolismo , Ducto Colédoco/patologia , Ducto Colédoco/cirurgia , Tamoxifeno/uso terapêutico , Fator de Crescimento Transformador beta/biossíntese , Animais , Ducto Colédoco/lesões , Ducto Colédoco/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fibrose , Masculino , RNA Mensageiro/genética , Sus scrofa , Tamoxifeno/farmacologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/biossíntese , Fator de Crescimento Transformador beta3/genética , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: Indoleamine 2, 3-dioxygenase (IDO) is an immunomodulatory molecule that has been implicated in several biological processes. Although IDO has been linked with some renal diseases, its role in renal fibrosis is still unclear. Because IDO may be modulated by TGF-ß1, a potent fibrogenic molecule, we hypothesized that IDO could be involved in renal fibrosis, especially acting in the TGF-ß1-induced tubular epithelial-mesenchymal transition (EMT). We analyzed the IDO expression and activity in a model of renal fibrogenesis, and the effect of the IDO inhibitor 1-methyl-tryptophan (MT) on TGF-ß1-induced EMT using tubular cell culture. METHODS: Male Wistar rats where submited to 7 days of UUO. Non-obstructed kidneys (CL) and kidneys from SHAM rats were used as controls. Masson's Tricrome and macrophages counting were used to chatacterize the tissue fibrosis. The EMT was analysed though immunohistochemistry and qRT-PCR. Immunohistochemestry in tissue has used to show IDO expression. MDCK cells were incubated with TGF- ß1 to analyse IDO expression. Additionally, effects of TGF- ß1 and the inhibition of IDO over the EMT process was acessed by immunoessays and scrath wound essay. RESULTS: IDO was markedly expressed in cortical and medular tubules of the UUO kidneys. Similarly to the immunolocalizaton of TGF- ß1, accompanied by loss of e-cadherin expression and an increase of mesenchymal markers. Results in vitro with MDCK cells, showed that IDO was increased after stimulus with TGF-ß1, and treatment with MT potentiated its expression. MDCK stimulated with TGF-ß1 had higher migratory activity (scratch-wound assay), which was exacerbated by MT treatment. CONCLUSIONS: IDO is constitutively expressed in tubular cells and increases during renal fibrogenesis. Although IDO is induced by TGF-ß1 in tubular cells, its chemical inhibitor acts as a profibrotic agent.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/biossíntese , Nefropatias/metabolismo , Nefropatias/patologia , Fator de Crescimento Transformador beta1/biossíntese , Animais , Cães , Fibrose/metabolismo , Fibrose/patologia , Células Madin Darby de Rim Canino , Masculino , Ratos , Ratos Wistar , Triptofano/análogos & derivados , Triptofano/farmacologiaRESUMO
Tracheal stenosis (TS) is a fibrosis originated by prolonged inflammation and increased transforming growth factor beta 1 (TGF-ß1) expression and collagen deposition (CD) in the tracheal wound. Several wound-healing modulators (WHMs) have been used to modulate the tracheal healing process and prevent TS, but they have failed, justifying the need to evaluate alternative WHM. The pirfenidone (PFD) and collagen-polyvinylpyrrolidone (Collagen-PVP) decrease inflammation and fibrosis. This study assessed the effect of PFD administration and Collagen-PVP topical application on macroscopic and microscopic changes, TGF-ß1 expression, and CD in an experimental model of tracheal wound healing. Forty Wistar rats underwent cervical tracheoplasty, were divided into 4 groups (n = 10), and were treated with different WHM: group I, saline solution (SS); group II, Collagen-PVP; group III, mitomycin C (MMC); and group IV, 40 mg/kg PFD. Four weeks after surgery, the macroscopic and microscopic changes, in situ TGF-ß1 expression, and CD in posttracheoplasty scars were evaluated. The animals treated with Collagen-PVP and PFD developed less inflammation and fibrosis than animals in the other study groups (p < 0.05, Kruskal-Wallis) and, moreover, showed lower TGF-ß1 expression and CD than animals in group I (p < 0.05, ANOVA and Tukey's test). In conclusion, PFD and Collagen-PVP decrease inflammation, fibrosis, TGFß-1 expression, and CD in the posttracheoplasty rats' scar.
Assuntos
Colágeno/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Povidona/farmacologia , Piridonas/farmacologia , Traqueia , Fator de Crescimento Transformador beta1/biossíntese , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Feminino , Masculino , Ratos , Ratos Wistar , Traqueia/lesões , Traqueia/metabolismo , Traqueia/patologiaRESUMO
Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms (LUTS) in older men. In this regard, recent studies have attempted to define the relationships between prostatic fibrosis, LUTS, and increased expression of transforming growth factor ß1 (TGF ß1) in BHP. Therapeutic approaches for BPH such as 5-α-reductase inhibitors and alpha-adrenergic blocking agents increase TGF ß1 expression in the prostatic tissue. Here, we investigated the effects of the 5-α-reductase inhibitor-finasteride-on rat ventral prostate tissue, especially with regard to the tissue distribution and gene expression of fibrillar collagens. Adult Wistar rats (n = 15) were treated with finasteride (25 mg/kg/day) by subcutaneous injection for 7 and 30 days. Age-matched, vehicle-treated (n = 15) adult Wistar rats were used as control. Finasteride treatment reduced prostate size and increased the area of types I and III collagen fibers in the prostatic stroma. As expected, TGF ß1 mRNA expression was upregulated by finasteride treatment. However, COL1A1 and COL3A1 mRNA expressions decreased after both 7 and 30 days of finasteride treatment, suggesting that finasteride treatment promotes prostate parenchyma and stroma changes, which lead to the observed types I and III collagen remodeling without de novo collagen synthesis. The upregulation of TGF ß1 mRNA and protein associated with the 5-α-reductase inhibitor is more closely related to epithelial and stromal cell death pathways than to prostatic fibrosis.
Assuntos
Colágenos Fibrilares/genética , Finasterida/farmacologia , Próstata/efeitos dos fármacos , Fator de Crescimento Transformador beta1/biossíntese , Animais , Colágenos Fibrilares/biossíntese , Expressão Gênica/efeitos dos fármacos , Masculino , Próstata/metabolismo , Próstata/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ativação Transcricional/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-ß1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-ß-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm2) were significantly higher in the chronic periapical disease tissues (P < 0.01) compared to that in the control tissue; in addition, the density of TGF-ß1-CD14 double positive cells was significantly higher in the periapical cyst group than in the periapical granuloma group (P < 0.01). The number of TGF-ß1 expressing macrophages varied with human chronic periapical diseases. The TGF-ß1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.
Assuntos
Macrófagos/metabolismo , Doenças Periapicais/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Adulto , Estudos de Casos e Controles , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Periapicais/genética , Doenças Periapicais/imunologia , Granuloma Periapical/genética , Granuloma Periapical/imunologia , Granuloma Periapical/metabolismo , Cisto Radicular/genética , Cisto Radicular/metabolismo , Fator de Crescimento Transformador beta1/genéticaRESUMO
Saliva is the first barrier to entry of bacteria and viruses into the body. The submandibular glands (SMG) contribute to the maintenance of oral health and regulation of immune/ inflam matory responses. Previous studies suggest that transforming growth factor beta 1 (TGFB1) may contribute to salivary gland fibrosis but the expression of the TGFB1 system in the SMG has not been elucidated. Thus, the aim of this study was to analyze in rat SMG the immunolocalization of TGFB1 and its specific receptors ALK5 (profibrotic) and ALK1 (proproliferative) and the coreceptor endoglin (EDG) in a bilateral experimental periodontitis (EP) model (cotton thread ligature around the neck of the first lower molars) for 1 and 6 weeks. Fixed SMG were embedded in paraffin and serially cut for routine hematoxylin-eosin staining for histological analysis or immunohistochemical techniques by diaminobenzidine detection. SMG histology from animals with EP showed timedependent structural changes involving marked reduction in the height of the contoured ducts, cell destruction, loss of secretory granules, periductal congestion and excess connective tissue surrounding these ducts indicative of a fibrotic process, compared to control SMG. TGFB1, ALK5 and ALK1 receptors and the coreceptor EDG were mainly immunolocalized in ductal cells and in the fibrotic areas in EP groups. The expression of the profibrotic ALK5 receptor was increased in areas of fibrosis in SMG of animals with EP. In SMG of rats with EP, the localization of the TGFB1 specific receptors in the ducts and cells from fibrotic areas, due to the expression of TGFB1 in the surrounding areas, might indicate paracrine and autocrine actions exerted by TGFB1 via its specific receptors. The results of this study suggest that TGFB1 promotes fibrosis, inducing cell proliferation via ALK1 and EDG receptors and stimulates fibrosis relatedprocesses via ALK5 receptor, which could lead to abnormal secretor activity of the SMG during periodontal disease.
La saliva es la primera barrera para la entrada de bacterias y virus en el cuerpo. Las glándulas submandibulares (GSM) contribuyen al mantenimiento de la salud oral y a la regulación de las respuestas inmunoinflamatorias. Estudios previos sugieren que el factor de crecimiento transformante beta 1 (TGFB1) puede contribuir a la fibrosis de las glándulas salivales, pero la expresión y localización del sistema TGFB1 en las GSM no ha sido dilucidada. El objetivo del presente trabajo fue analizar por inmunohistoquímica en las GSM de ratas la expresión de TGFB1 y sus receptores específicos ALK5 (profibrótico) y ALK1 (proproliferativo) y el coreceptor endoglina (EDG) en un modelo de periodontitis bilateral experimental (PE) (hilo de algodón alrededor del cuello de los primeros molares inferiores) durante 1 y 6 semanas. Las GSM fueron fijadas y embebidas en parafina para realizar cortes seriados los cuales se tiñeron con hematoxilinaeosina para analizar la histología o se procesaron para realizar la técnica de inmunohistoquímica mediante detección con diaminobenzidine. La histología de las GSM de animales con PE reveló cambios estructurales tiempo dependientes, con una marcada reducción de la altura de los conductos, destrucción celular, pérdida de gránulos secretores, congestión periductal y exceso de tejido conectivo que rodea los conductos, indicando un proceso de fibrosis respecto de las GSM de animales control. TGFB1, ALK5 y ALK1 y el coreceptor EDG fueron principalmente inmunolocalizados en las células que forman los ductos y en las áreas de fibrosis en los grupos con PE. La expresión del receptor profibrótico ALK5 se incrementó en las áreas de fibrosis en GSM de animales con PE. En GSM de ratas con PE, la localización de los receptores específicos de TGFB1 en las células de los conductos y áreas de fibrosis, junto con la expresión de TGFB1 en las áreas circundantes, podría indicar acciones paracrinas y autocrinas ejercidas por TGFB1 a través de sus receptores específicos. Los resultados de este estudio sugieren que TGFB1 podría inducir un proceso de fibrosis promoviendo la proliferación celular a través de los receptores ALK1 y EDG, y favoreciendo procesos relacionados con la fibrosis a través de su receptor ALK5, lo que conduciría a una actividad secretora anormal de la GSM durante la enfermedad periodontal.
Assuntos
Glândula Submandibular/patologia , Fator de Crescimento Transformador beta1/biossíntese , Animais , Fibrose/etiologia , Imuno-Histoquímica , Masculino , Periodontite/complicações , Ratos , Ratos Wistar , Glândula Submandibular/química , Fator de Crescimento Transformador beta1/análiseRESUMO
The aim of this study was to evaluate the mechanism involved in the stem cell factor (SCF)-induced production of fibroblast growth factor-2 (FGF-2), transforming growth factor-ß1 (TGF-ß1), and chemokine (C-C motif) ligand 3 (CCL3) in tracheal smooth muscle cells (tSMCs) and the signaling pathway involved in the process. tSMC primary cultures were stimulated with SCF and evaluated at 24 h. Cells treated with specific antibodies did not show any immunolabeling for cytokeratin or fibroblast activation protein, but were positive for α-smooth muscle actin, indicating the purity of the primary cell line. Western blot analysis showed constitutive phosphorylation of c-Kit, as well as increased total protein and phosphorylated c-Kit levels in tSMCs after SCF stimulation. Flow cytometry analysis also showed an increase in cell-surface c-Kit expression in the presence of SCF. SCF induced TGF-ß mRNA expression in tSMCs, as well as the production of TGF-ß1, CCL3, and FGF-2. Pretreatment with anti-CCL3 antibody blocked TGF-ß1 expression and partially inhibited FGF-2 production. On the other hand, anti-c-Kit antibody blocked TGF-ß1 expression and FGF-2 production. Thus, TGF-ß1 and FGF-2 production were mediated by CCL3 production through c-Kit. Pretreatment with mitogen-activated protein kinase kinase 1, p38, and Jun N-terminal kinase inhibitors showed that the effects mediated by SCF were involved with the modulation of mitogen-activated protein kinase (MAPK) pathways. Development of inhibitors targeting CCL3 through MAPK activation could thus be an attractive strategy to inhibit tSMC activation during asthma.
Assuntos
Quimiocina CCL3/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismo , Traqueia/citologia , Fator de Crescimento Transformador beta1/biossíntese , Animais , Quimiocina CCL3/genética , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Fator de Crescimento Transformador beta1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The participation of oxidative stress in the mechanism of metformin action in breast cancer remains unclear. We investigated the effects of clinical (6 and 30 µM) and experimental concentrations of metformin (1000 and 5000 µM) in MCF-7 and in MDA-MB-231 cells, verifying cytotoxicity, oxidative stress, DNA damage, and intracellular pathways related to cell growth and survival after 24 h of drug exposure. Clinical concentrations of metformin decreased metabolic activity of MCF-7 cells in the MTT assay, which showed increased oxidative stress and DNA damage, although cell death and impairment in the proliferative capacity were observed only at higher concentrations. The reduction in metabolic activity and proliferation in MDA-MB-231 cells was present only at experimental concentrations after 24 h of drug exposition. Oxidative stress and DNA damage were induced in this cell line at experimental concentrations. The drug decreased cytoplasmic extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT and increased nuclear p53 and cytoplasmic transforming growth factor ß1 (TGF-ß1) in both cell lines. These findings suggest that metformin reduces cell survival by increasing reactive oxygen species, which induce DNA damage and apoptosis. A relationship between the increase in TGF-ß1 and p53 levels and the decrease in ERK1/2 and AKT was also observed. These findings suggest the mechanism of action of metformin in both breast cancer cell lineages, whereas cell line specific undergoes redox changes in the cells in which proliferation and survival signaling are modified. Taken together, these results highlight the potential clinical utility of metformin as an adjuvant during the treatment of luminal and triple-negative breast cancer.
Assuntos
Metformina/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Fator de Crescimento Transformador beta1/biossíntese , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Galectin-1 (Gal1), a ß-galactoside-binding protein elevated in hepatocellular carcinoma (HCC), promotes epithelial-mesenchymal transition (EMT) and its expression correlates with HCC growth, invasiveness, and metastasis. During the early stages of HCC, transforming growth factor ß1 (TGF-ß1 ) acts as a tumor suppressor; however in advanced stages, HCC cells lose their cytostatic response to TGF-ß1 and undergo EMT. Here, we investigated the role of Gal1 on liver endothelial cell biology, and the interplay between Gal1 and TGF-ß1 in HCC progression. By Western blot and immunofluorescence, we analyzed Gal1 expression, secretion and localization in HepG2 and HuH-7 human HCC cells, and in SK-HEP-1 human liver sinusoidal endothelial cells (SECs). We used loss-of-function and gain-of-function experiments to down- or up-regulate Gal1 expression, respectively, in HepG2 cells. We cultured SK-HEP-1 cells with conditioned media from HCC cells secreting different levels of Gal1, and demonstrated that Gal1 derived from tumor hepatocytes induced its own expression in SECs. Colorimetric and scratch-wound assays revealed that secretion of Gal1 by HCC cells induced SEC proliferation and migration. Moreover, by fluorescence microscopy we demonstrated that Gal1 promoted glycan-dependent heterotypic adhesion of HepG2 cells to SK-HEP-1 SECs. Furthermore, TGF-ß1 induced Gal1 expression and secretion by HCC cells, and promoted HepG2 cell adhesion to SK-HEP-1 SECs through a Gal1-dependent mechanism. Finally, Gal1 modulated HepG2 cell proliferation and sensitivity to TGF-ß1 -induced growth inhibition. Our results suggest that Gal1 and TGF-ß1 might function coordinately within the HCC microenvironment to regulate tumor growth, invasion, metastasis, and angiogenesis.
Assuntos
Carcinoma Hepatocelular/genética , Galectina 1/genética , Neoplasias Hepáticas/genética , Fator de Crescimento Transformador beta1/genética , Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Proliferação de Células/genética , Células Endoteliais , Transição Epitelial-Mesenquimal , Galectina 1/biossíntese , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Neovascularização Patológica/genética , Fator de Crescimento Transformador beta1/biossíntese , Microambiente TumoralRESUMO
Saliva is the first barrier to entry of bacteria and viruses into the body. The submandibular glands (SMG) contribute to the maintenance of oral health and regulation of immune/ inflam matory responses. Previous studies suggest that transforming growth factor beta 1 (TGFB1) may contribute to salivary gland fibrosis but the expression of the TGFB1 system in the SMG has not been elucidated. Thus, the aim of this study was to analyze in rat SMG the immunolocalization of TGFB1 and its specific receptors ALK5 (profibrotic) and ALK1 (proproliferative) and the coreceptor endoglin (EDG) in a bilateral experimental periodontitis (EP) model (cotton thread ligature around the neck of the first lower molars) for 1 and 6 weeks. Fixed SMG were embedded in paraffin and serially cut for routine hematoxylin-eosin staining for histological analysis or immunohistochemical techniques by diaminobenzidine detection. SMG histology from animals with EP showed timedependent structural changes involving marked reduction in the height of the contoured ducts, cell destruction, loss of secretory granules, periductal congestion and excess connective tissue surrounding these ducts indicative of a fibrotic process, compared to control SMG. TGFB1, ALK5 and ALK1 receptors and the coreceptor EDG were mainly immunolocalized in ductal cells and in the fibrotic areas in EP groups. The expression of the profibrotic ALK5 receptor was increased in areas of fibrosis in SMG of animals with EP. In SMG of rats with EP, the localization of the TGFB1 specific receptors in the ducts and cells from fibrotic areas, due to the expression of TGFB1 in the surrounding areas, might indicate paracrine and autocrine actions exerted by TGFB1 via its specific receptors. The results of this study suggest that TGFB1 promotes fibrosis, inducing cell proliferation via ALK1 and EDG receptors and stimulates fibrosis relatedprocesses via ALK5 receptor, which could lead to abnormal secretor activity of the SMG during periodontal disease.
La saliva es la primera barrera para la entrada de bacterias y virus en el cuerpo. Las glándulas submandibulares (GSM) contribuyen al mantenimiento de la salud oral y a la regulación de las respuestas inmunoinflamatorias. Estudios previos sugieren que el factor de crecimiento transformante beta 1 (TGFB1) puede contribuir a la fibrosis de las glándulas salivales, pero la expresión y localización del sistema TGFB1 en las GSM no ha sido dilucidada. El objetivo del presente trabajo fue analizar por inmunohistoquímica en las GSM de ratas la expresión de TGFB1 y sus receptores específicos ALK5 (profibrótico) y ALK1 (proproliferativo) y el coreceptor endoglina (EDG) en un modelo de periodontitis bilateral experimental (PE) (hilo de algodón alrededor del cuello de los primeros molares inferiores) durante 1 y 6 semanas. Las GSM fueron fijadas y embebidas en parafina para realizar cortes seriados los cuales se tiñeron con hematoxilinaeosina para analizar la histología o se procesaron para realizar la técnica de inmunohistoquímica mediante detección con diaminobenzidine. La histología de las GSM de animales con PE reveló cambios estructurales tiempo dependientes, con una marcada reducción de la altura de los conductos, destrucción celular, pérdida de gránulos secretores, congestión periductal y exceso de tejido conectivo que rodea los conductos, indicando un proceso de fibrosis respecto de las GSM de animales control. TGFB1, ALK5 y ALK1 y el coreceptor EDG fueron principalmente inmunolocalizados en las células que forman los ductos y en las áreas de fibrosis en los grupos con PE. La expresión del receptor profibrótico ALK5 se incrementó en las áreas de fibrosis en GSM de animales con PE. En GSM de ratas con PE, la localización de los receptores específicos de TGFB1 en las células de los conductos y áreas de fibrosis, junto con la expresión de TGFB1 en las áreas circundantes, podría indicar acciones paracrinas y autocrinas ejercidas por TGFB1 a través de sus receptores específicos. Los resultados de este estudio sugieren que TGFB1 podría inducir un proceso de fibrosis promoviendo la proliferación celular a través de los receptores ALK1 y EDG, y favoreciendo procesos relacionados con la fibrosis a través de su receptor ALK5, lo que conduciría a una actividad secretora anormal de la GSM durante la enfermedad periodontal.
Assuntos
Animais , Masculino , Ratos , Glândula Submandibular/patologia , Fator de Crescimento Transformador beta1/biossíntese , Periodontite/complicações , Glândula Submandibular/química , Fibrose/etiologia , Imuno-Histoquímica , Ratos Wistar , Fator de Crescimento Transformador beta1/análiseRESUMO
Oleanolic (OA) and ursolic acids (UA) are triterpenes that are abundant in vegetables, fruits and medicinal plants. They have been described as active moieties in medicinal plants used for the treatment of tuberculosis. In this study, we analyzed the effects of these triterpenes on macrophages infected in vitro with Mycobacterium tuberculosis (MTB). We evaluated production of nitric oxide (NO), reactive oxygen species (ROS), and cytokines (TNF-α and TGF-ß) as well as expression of cell membrane receptors (TGR5 and CD36) in MTB-infected macrophages following treatment with OA and UA. Triterpenes caused reduced MTB growth in macrophages, stimulated production of NO and ROS in the early phase, stimulated TNF-α, suppressed TGF-ß and caused over-expression of CD36 and TGR5 receptors. Thus, our data suggest immunomodulatory properties of OA and UA on MTB infected macrophages. In conclusion, antimycobacterial effects induced by these triterpenes may be attributable to the conversion of macrophages from stage M2 (alternatively activated) to M1 (classically activated).
Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Triterpenos/farmacologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Mycobacterium tuberculosis/isolamento & purificação , Óxido Nítrico/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Ácido UrsólicoRESUMO
Leprosy is a chronic infectious disease caused by Mycobacterium leprae which affects the skin and peripheral nervous system. The immune response of the host determines the clinical course of the disease. The tuberculoid form is the result of high cell-mediated immunity characterized by a Th1 response, whereas the lepromatous form is characterized by low cell-mediated immunity and a Th2 humoral response. The neural damage established produces marked changes in the expression of growth factors such as nerve growth factor (NGF) and its receptors (NGF-R). The expression of NGF, associated with the expression of Th1 and Th2 cytokines, might be involved in the tissue damage caused by the bacillus. Therefore, the objective of this study was to correlate the immunoexpression patterns of NGF and NGF-R in the different clinical forms of leprosy, and to associate the findings with the in situ expression of TGF-ß and clinical classification of the disease. TGF-ß, NGF and NGF-R immunoexpression was analyzed by immunohistochemistry in paraffin-embedded material. Most patients were males with a mean age of 40.7 years. TGF-ß levels were significantly higher in the lepromatous forms. No significant difference in the immunoexpression of NGF or NGF-R was observed between the clinical forms, but expression tended to be higher at the lepromatous pole. There was a significant positive correlation between NGF and NGF-R in the different clinical forms of leprosy. A significant positive correlation was observed between NGF, NGF-R and TGF-ß. It can be concluded that, even existing evidence on the role of these molecules in the clinical spectrum of leprosy.
Assuntos
Citocinas/metabolismo , Hanseníase/microbiologia , Hanseníase/patologia , Mycobacterium leprae/imunologia , Fatores de Crescimento Neural/biossíntese , Humanos , Imuno-Histoquímica , Receptores de Fator de Crescimento Neural/biossíntese , Fator de Crescimento Transformador beta1/biossínteseRESUMO
The physiology of hepatic hematopoiesis is largely unknown, although studies have indicated that vasoactive intestinal polypeptide (VIP) is involved in this disease. To validate this hypothesis, we assessed the effects of VIP on human cord blood CD34+ cells. We also measured VIP levels and the capacity of vasoactive intestinal polypeptide receptor (VIPR) to bind to VIP in the rat liver during different developmental phases. VIP inhibited the proliferation of cord blood-derived CD34(+) cells from concentrations of 10-7-10-12 M. The highest suppression was achieved with 10-8 M VIP at day 10. Intracellular levels of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1 in CD34(+) cells treated with VIP were increased by 50.70 and 43.46%, respectively. Variations in VIP levels in the rat fetal liver generally increased rapidly with the stage of fetal development. In addition, the affinity of VIPR for VIP increased from relatively low levels in the rat fetal liver and peaked at birth, after which it gradually decreased. VIP had a suppressive effect on the proliferation of human cord blood-derived CD34(+) cells, partially by increasing the production of TNF-α and TGF-ß. Low VIP levels in the fetal liver and gradually increasing levels after birth may in part be responsible for suppressing hematopoietic stem cell and progenitor proliferation in the liver.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator de Crescimento Transformador beta1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Animais Recém-Nascidos , Antígenos CD34/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Sangue Fetal/citologia , Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Ratos Sprague-Dawley , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
This study presents a comprehensive view of the histological and functional status of the prostate of adult rat offspring of mothers subjected to gestational diabetes induced by alloxan. The ventral prostate of male adult offspring of diabetic (DP) or normal (CP) mothers was evaluated for collagen fibres, cell death, fibroblasts, smooth muscle cells, cell proliferation, matrix metalloproteinases (MMPs), androgen receptors (AR), transforming growth factor ß1 (TGFß-1), catalase and total antioxidant activity. The prostates of DP animals were lower in weight than those of the CP group. The DP group also exhibited hyperglycaemia and hypotestosteronemia, higher cell proliferation and AR expression, a reduction in α-actin (possibly interfering with the reproductive function of the prostate), and enhanced activity of MMP-2, although the absolute content of MMP-2 was lower in this group. These findings were associated with increased TGFß-1 and decreased collagen distribution. The prostates of DP rats additionally exhibited reductions in catalase and total antioxidant activity. Thus, rats developing in a diabetic intrauterine environment have glycaemic and hormonal changes that impact on the structure and physiology of the prostate in adulthood. The increased AR expression possibly leads to elevated cell proliferation. Stromal remodelling was characterized by enhanced activity of MMP-2 and collagen degradation, even with increased TGFß-1 activation. These changes associated with increased oxidative stress might interfere with tissue architecture and glandular homeostasis.
Assuntos
Diabetes Mellitus Experimental , Diabetes Gestacional , Metaloproteinase 2 da Matriz/biossíntese , Gravidez em Diabéticas , Efeitos Tardios da Exposição Pré-Natal/enzimologia , Próstata/enzimologia , Animais , Colágeno/metabolismo , Feminino , Regulação da Expressão Gênica , Hiperglicemia/enzimologia , Hiperglicemia/etiologia , Hiperglicemia/patologia , Masculino , Estresse Oxidativo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Próstata/patologia , Ratos , Ratos Wistar , Receptores Androgênicos/biossíntese , Fator de Crescimento Transformador beta1/biossínteseRESUMO
End-stage renal disease is a cause for death worldwide. Renal transplant is a therapeutic alternative, restricted by the scant number of donors. Function of the donor kidney is under risk of adverse circumstances such as fibrosis, where profibrotic effect of transforming growth factor beta 1 (TGF-ß1) plays a key role. Efforts to diminish risks of damage in the remnant kidney of the donor are required. Vitamin A represents one alternative. It has beneficial effects on some nephropathies, mainly those related to oxidative stress. It also participates in normal intrauterine renal development. We studied the effect of all-trans retinoic acid (ATRA), active form of vitamin A, on postnephrectomy compensatory growth, in male or female rats. Compensatory growth and renal function were evaluated on four experimental groups: Control without treatment (CTL), ATRA-treated intact rats (CTL + RA), nephrectomized rats (NFX), and ATRA-treated nephrectomized rats (NFX + RA). We evaluated glomerular function (inulin clearance), tubular function (fractional excretions of sodium and potassium), and urinary flow. Renal mass was also estimated. In ATRA-treated animals, compensatory growth was higher than in nephrectomized rats without treatment. Hyperfiltration after nephrectomy was less intense in ATRA-treated female than in male rats. In tubular functions, effect of ATRA was more evident in female than in male rats. Glomerular expression of TGF-ß1 was lower in ATRA-treated animals than in controls. ATRA reduced intensity and duration of compensatory changes after nephrectomy, improving recovery.