RESUMO
Canine oral mucosal melanomas (OMM) are the most common oral malignancy in dogs and few treatments are available. Thus, new treatment modalities are needed for this disease. Bacillus anthracis (anthrax) toxin has been reengineered to target tumor cells that express urokinase plasminogen activator (uPA) and metalloproteinases (MMP-2), and has shown antineoplastic effects both, in vitro and in vivo. This study aimed to evaluate the effects of a reengineered anthrax toxin on canine OMM. Five dogs bearing OMM without lung metastasis were included in the clinical study. Tumor tissue was analyzed by immunohistochemistry for expression of uPA, uPA receptor, MMP-2, MT1-MMP and TIMP-2. Animals received either three or six intratumoral injections of the reengineered anthrax toxin prior to surgical tumor excision. OMM samples from the five dogs were positive for all antibodies. After intratumoral treatment, all dogs showed stable disease according to the canine Response Evaluation Criteria in Solid Tumors (cRECIST), and tumors had decreased bleeding. Histopathology has shown necrosis of tumor cells and blood vessel walls after treatment. No significant systemic side effects were noted. In conclusion, the reengineered anthrax toxin exerted inhibitory effects when administered intratumorally, and systemic administration of this toxin is a promising therapy for canine OMM.
Assuntos
Antígenos de Bactérias/uso terapêutico , Antineoplásicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Doenças do Cão/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Feminino , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Melanoma/veterinária , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/veterinária , Engenharia de Proteínas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND: In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis, and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). RESULTS: The kinetics of fibrin formation on HMEC-1 with 3 and 10% fibrinogen γ/γ' were similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3 to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. CONCLUSION: A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.
Assuntos
Células Endoteliais/metabolismo , Fibrinogênios Anormais/metabolismo , Fragmentos de Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Trombose , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Coagulação Sanguínea , Linhagem Celular , Fibrina/química , Fibrinogênio/química , Fibrinólise/fisiologia , Humanos , Trombina/metabolismo , Trombose/metabolismoRESUMO
SummaryThe mammalian oviduct plays a pivotal role in the success of early reproductive events. The urokinase plasminogen activator system (uPAS) is present in the bovine oviduct and is involved in extracellular matrix remodelling through plasmin generation. This system can be regulated by several members of the vascular endothelial growth factors (VEGF) and their receptors. In this study, the VEGF-D effect on the regulation of uPAS was evaluated. First, RT-polymerase chain reaction (PCR) analyses were used to evidence the expression of VEGF-D and its receptors in oviductal epithelial cells (BOEC). VEGF-D, VEGFR2 and VEGFR3 transcripts were found in ex vivo and in vitro BOEC, while only VEGFR2 mRNA was present after in vitro conditions. VEGF-D showed a regulatory effect on uPAS gene expression in a dose-dependent manner, inducing an increase in the expression of both uPA and its receptor (uPAR) at 24 h post-induction and decreases in the expression of its inhibitor (PAI-1). In addition, the regulation of cell migration induced by VEGF-D and uPA in BOEC monolayer cultures was analyzed. The wound areas of monolayer cultures incubated with VEGF-D 10 ng/ml or uPA 10 nM were modified and significant differences were found at 24 h for both stimulations. These results indicated that uPAS and VEGF-D systems can modify the arrangement of the bovine oviductal epithelium and contribute to the correct maintenance of the oviductal microenvironment.
Assuntos
Tubas Uterinas/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Células Cultivadas , Células Epiteliais/fisiologia , Tubas Uterinas/citologia , Tubas Uterinas/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Inibidor 1 de Ativador de Plasminogênio/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective ß1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin-Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5ß1integrin binding. Under subconfluent conditions, Gal-8-overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased ß-catenin activity. Changes related to migration/invasion included higher expression of α5ß1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8-stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and ß1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties.
Assuntos
Transição Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Galectinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Animais , Caderinas/metabolismo , Carcinogênese , Cães , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Neoplasias Experimentais , Proteínas Recombinantes/metabolismo , Transfecção , Regulação para Cima , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The extracellular matrix (ECM) is composed of several types of proteins, which interact and form dynamic networks. These components can modulate cell behaviour and actively influence the growth and differentiation of tissues. ECM is also important in several pathological processes, such as cancer invasion and metastasis, by creating favourable microenvironments. Proteolysis in neoplastic tissues is mediated by proteinases, whose regulation involves complex interactions between neoplastic cells and non-neoplastic stromal cells. In this review, we discuss aspects of proteinase expression and tumor behaviour in humans and dogs. Different classes of proteases are summarized, with special emphasis being placed on molecules that have been shown to correlate with prognosis, reinforcing the need for a better understanding of the regulation of this microenvironment and its influences in tumor progression and metastasis, which should significantly aid the development of improved prognosis and treatment.
Assuntos
Neoplasias/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Catepsinas/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias/diagnóstico , Neoplasias/veterinária , Prognóstico , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Urokinase-type plasminogen activator (uPA) is a serine protease involved in extracellular matrix remodeling through plasmin generation. uPA usually binds to its receptor, uPAR, which is anchored to the plasma membrane through a glycosylphosphatidylinositol anchor. uPA/uPAR binding increases proteolytic activity in the neighborhood of the cells containing uPAR and activates intracellular signaling pathways involved in extracellular matrix remodeling, cell migration and proliferation. The aim of this work was to study the expression of uPA, uPAR and plasminogen activator inhibitor-1 (PAI-1) in immature and in vitro matured bovine cumulus-oocyte complexes (COCs). uPA is only expressed in the cumulus cells of immature and in vitro matured COCs, while uPAR and PAI-1 are expressed in both the cumulus cells and the immature and in vitro matured oocytes. In addition, uPAR protein was localized by confocal microscopy in the plasma membrane of oocytes and cumulus cells of immature COCs. Results from this research led us to hypothesize that the uPA/uPAR interaction could cause the local production of uPA-mediated plasmin over oocyte and cumulus cell surface; plasmin formation could also be regulated by PAI-1.
Assuntos
Células do Cúmulo/metabolismo , Oócitos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Bovinos , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Células Cultivadas , Células do Cúmulo/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Microscopia Confocal , Oócitos/citologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Recent studies have demonstrated that carbon nanotubes (CNTs) induce platelet aggregation, endothelial dysfunction and vascular thrombosis. However, there is little information on the effects of CNTs on fibrinolysis. We investigated the role of pristine-commercial single-walled carbon nanotubes (SWCNTs) with <3% Co content in fibrinolysis and their contribution to the induction of pro-thrombotic processes in human vein endothelial cells (HUVEC). SWCNTs alone produced concentration-dependent oxidation, as measured by a dithiothreitol oxidation assay. Internalized SWCNTs were located in HUVEC treated with 25 µg/ml using transmission electron microscopy, whereas treatment with 50 µg/ml compromised cell viability, and oxidative stress increased significantly at 5 µg/ml. The study showed that in HUVEC treated with 25 µg SWCNT/ml, fibrinolysis-related gene expression and protein levels had increased by 3-12 h after treatment (serpine-1: 13-fold; PLAT: 11-fold and PLAU: 2-fold), but only the PAI-1 protein was increased (1.5-fold), whereas tissue and urokinase plasminogen activator proteins (tPA and uPA, respectively) tended to decrease. In summary, pristine SWCNTs treatment resulted in evident HUVEC damage caused by cell fiber contact, internalization, and oxidative stress due to contaminant metals. The generation of endothelial dysfunction, as shown by the altered expression of genes and proteins involved in fibrinolysis, suggest that SWCNTs display pro-thrombotic effects.
Assuntos
Fibrinólise/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Urokinase type plasminogen activator (uPA) is an oviductal fluid component whose activity is regulated by binding to urokinase type plasminogen activator receptor (uPAR). In this study uPAR and uPA gene expression in bovine oviduct were evaluated and similar expression patterns for both uPAR and uPA mRNAs were observed during the estrous cycle. Immunolocalization of uPAR at the apical zone of epithelial cells suggests that uPA action would be focalized in the oviductal lumen, triggering intracellular signaling pathways. As uPAR expression was also observed in in vitro cultures of oviductal epithelial cells, the effect of uPA was explored using this culture model. Real-time RT-PCR demonstrated that c-fos expression in oviductal cell cultures increases under uPA stimulation. These results suggest that uPA/uPAR binding would be involved in signaling pathways that activate transcription factors and would regulate the synthesis of molecules concerned with the arrangement of a particular oviductal microenvironment.
Assuntos
Microambiente Celular , Células Epiteliais/metabolismo , Ciclo Estral/metabolismo , Tubas Uterinas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Análise de Variância , Animais , Bovinos , Células Cultivadas , Tubas Uterinas/citologia , Feminino , Imuno-Histoquímica , Oligonucleotídeos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase-type plasminogen activator (uPA)-in association with its receptor-triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA-uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. RESULTS: In response to uPA, neuronal migration and neuritogenesis are promoted in a dose-dependent manner. After stimulation, uPAR interacts with α5- and ß1-integrin subunits, which may constitute an αß-heterodimer that acts as a uPA-uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA-promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. CONCLUSIONS: uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks.
Assuntos
Proteínas Aviárias/metabolismo , Movimento Celular/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Embrião de Galinha , Galinhas , Neurônios/citologiaRESUMO
BACKGROUND: The effect of retinoic acid (RA) on breast cancer progression is controversial. Our objective was to obtain information about breast cancer progression, taking advantage of the ER-negative murine mammary adenocarcinoma model LM38 (LM38-LP constituted by luminal (LEP) and myoepithelial-like cells (MEP), LM38-HP mainly composed of spindle-shaped epithelial cells, and LM38-D2 containing only large myoepithelial cells), and to validate the role of the retinoic acid receptors (RARs) in each cell-type compartment. MATERIALS AND METHODS: We studied the expression and functionality of the RARs in LM38 cell lines. We analyzed cell growth and cell cycle distribution, apoptosis, the activity of proteases, motility properties, and expression of the molecules involved in these pathways. We also evaluated tumor growth and dissemination in vivo under retinoid treatment. RESULTS: LM38 cell lines expressed most retinoic receptor isotypes that were functional. However, only the bi-cellular LM38-LP cells responded to retinoids by increasing RARß2 and CRBP1 expression. The growth of LM38 cell sublines was inhibited by retinoids, first by inducing arrest in MEP cells, then apoptosis in LEP cells. Retinoids induced inhibitory effects on motility, invasiveness, and activity of proteolytic enzymes, mainly in the LM38-LP cell line. In in-vivo assays with the LM38-LP cell line, RA treatment impaired both primary tumor growth and lung metastases dissemination. CONCLUSION: These in-vivo and in-vitro results show that to achieve maximum effects of RA on tumor progression both the LEP and MEP cell compartments have to be present, suggesting that the interaction between the LEP and MEP cells is crucial to full activation of the RARs.
Assuntos
Adenocarcinoma/tratamento farmacológico , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Neoplasias Mamárias Animais/tratamento farmacológico , Receptores do Ácido Retinoico/metabolismo , Retinoides/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Citometria de Fluxo , Imunofluorescência , Técnicas Imunoenzimáticas , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitose/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Plasminogen activator activities have previously been reported in oviductal fluid. At present the question was whether the source of these activities is molecules come from blood plasma or if these activators are synthesized by the oviduct. Gene expression and protein synthesis of urokinase type (u-PA) and tissue type (t-PA) occur in different regions of the pig oviduct. Their relative concentrations do not vary between the ampulla and isthmus regions and are similar throughout the estrous cycle. However, while relative amounts of t-PA mRNA were not different between the different stages of the estrous cycle, u-PA mRNA was greater after ovulation (P<0.05). Regarding the function of u-PA, its receptor (u-PAR) was distinguished by immunohistochemistry at the apical region of the epithelial cells and was more noticeable in the isthmus. Expression of u-PA, t-PA, u-PAR and PAI-1 genes in primary oviductal epithelial cell cultures was studied under 17-ß-estradiol (100 pg/ml) and progesterone (100 ng/ml). u-PA mRNA increased in the presence of progesterone (P<0.05), but not by action of 17-ß-estradiol. t-PA, PAI-1 and u-PAR were similar when cultured with the hormones. These results suggest that u-PA could be regulated by progesterone at a transcriptional level, by the balance of their activity for PAI-1 or at the epithelial surface through the binding of u-PAR. In conclusion, plasminogen activation system components might cooperate in the oviductal lumen to control plasmin generation.
Assuntos
Oviductos/enzimologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Suínos/fisiologia , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Estradiol/farmacologia , Ciclo Estral/fisiologia , Feminino , Oviductos/efeitos dos fármacos , Oviductos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide. The objective of this study was to examine whether carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., would inhibit the cell viability of three CRC cell lines: Caco-2, HT29 and LoVo in a dose-dependent manner, with IC50 values in the range of 24-96 µM. CA induced cell death by apoptosis in Caco-2 line after 24 h of treatment and inhibited cell adhesion and migration, possibly by reducing the activity of secreted proteases such as urokinase plasminogen activator (uPA) and metalloproteinases (MMPs). These effects may be associated through a mechanism involving the inhibition of the COX-2 pathway, because we have determined that CA downregulates the expression of COX-2 in Caco-2 cells at both the mRNA and protein levels. Therefore, CA modulates different targets involved in the development of CRC. These findings indicate that carnosic acid may have anticancer activity and may be useful as a novel chemotherapeutic agent.
Assuntos
Abietanos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Extratos Vegetais/farmacologia , Inibidores de Proteases/farmacologia , Apoptose/efeitos dos fármacos , Células CACO-2 , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Células HT29 , Humanos , Concentração Inibidora 50 , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , RNA Mensageiro/metabolismo , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Toxoplasmosis is a world wide spread zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells for dispersal throughout the body. However, the molecular mechanisms or outcomes of the subversion of the host cell are largely unknown. Recently our group established that metalloproteinases are involved in migration of infected macrophages. Herein, we evaluated the recruitment of host invasive machinery components in T. gondii infected murine macrophages. We showed by immunoprecipitation assays that MMP-9, CD44 TIMP-1 and uPAR were secreted as a multi-protein complex by infected macrophages. Zymographic analysis revealed that MMP-9 was present in its pro- and active form. Moreover, inhibition of uPA/uPAR pathway by PAI-1 decreased secretion of MMP-9 active forms, as well those associated to uPAR and TIMP-1, but not to CD44. Data presented here suggest that MMP-9 is secreted as a multiprotein complex by T. gondii infected macrophages, similar to that observed in metastatic cells. We further speculate that uPA/uPAR system is involved in the expression/secretion of complexes containing active MMP-9 forms.
Assuntos
Macrófagos/metabolismo , Macrófagos/parasitologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Toxoplasma/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
BACKGROUND: Liver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by accumulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals. METHODS: Wistar rats (80 g) underwent chronic intoxication with CCl4: mineral oil for 8 weeks. Cirrhotic animals were injected with a combined dose of Ad-delta-huPA plus Ad-MMP8 (3 x 10(11) and 1.5 x 10(11) vp/Kg, respectively) or with Ad-beta-Gal (4.5 x 10(11)) and were killed after 2, 4, 6, 8 and 10 days. Then, liver and serum were collected. An additional set of cirrhotic animals injected with combined gene therapy was also monitored for their probability of survival. RESULTS: Only the cirrhotic animals treated with therapeutical genes (Ad-delta-huPA+Ad-MMP-8) showed improvement in liver fibrosis. These results correlated with hydroxyproline determinations. A significant decrement in alpha-SMA and TGF-beta1 gene expression was also observed. Cirrhotic rats treated with Ad-delta-huPA plus Ad-MMP8 had a higher probability of survival at 60 days with respect to Ad-beta-Gal-injected animals. CONCLUSION: A single administration of Ad-delta-huPA plus Ad-MMP-8 is efficient to induce fibrosis regression and increase survival in experimental liver fibrosis.
Assuntos
Terapia Genética , Cirrose Hepática Experimental/terapia , Adenoviridae/genética , Animais , Sequência de Bases , Tetracloreto de Carbono/toxicidade , Primers do DNA/genética , Expressão Gênica , Vetores Genéticos , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
TGF-beta1 has been postulated as a pro-oncogenic factor in the late step of the tumoral progression. In transformed cells, TGF-beta1 enhances the capacity to degrade the extracellular matrix, cell invasiveness and epithelial-mesenchymal transition, which are crucial steps for metastasis. Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) are critical components in cell migration and invasion induced by TGF-beta1, however, the exact mechanism by which TGF-beta1 regulates uPA and MMP-9 is not well elucidated so far. In the present study, we analyzed the role of ROS-NFkappaB, signal as mediator in the cell malignity enhancement by TGF-beta1. We found that TGF-beta1 activates NFkappaB, through Rac1-NOXs-ROS-dependent mechanism. Our results shows that TGF-beta1 stimulation of uPA and MMP-9 expression involve NOXs-dependent ROS and NFkappaB, activation, demonstrated by using DPI, NOXs inhibitor, ROS scavenger N-acetylcysteine and SN50, an NFkb inhibitor. Furthermore, we found that the inhibition of ROS and NFkappaB, abrogates TGF-beta1 stimulation of EMT, cell motility and invasion. Thus, ROS-NFkappaB acts as the crucial signal in TGF-beta1-induced uPA and MMP-9 expression thereby mediating the enhancement of cellular malignity by TGF-beta1.
Assuntos
Movimento Celular , Queratinócitos/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Transdiferenciação Celular , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Genes Reporter , Queratinócitos/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Camundongos , Mutação , NADPH Oxidases/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição RelA/genética , Transfecção , Regulação para Cima , Ativador de Plasminogênio Tipo Uroquinase/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Plasmin role in transforming growth factor-beta (TGF-beta)-responsive gene regulation remains to be elucidated. Also, plasmin action on co-repressor Ski-related novel protein N (SnoN) and differential activation of matrix metalloproteinases (MMPs) are unknown. Thus, the role of plasmin on profibrogenic molecule expression, SnoN transcriptional kinetics and gelatinase activation was investigated. METHODS: Hepatic stellate cells (HSC) were transduced with adenovirus-mediated human urokinase plasminogen activator (Ad-huPA) (4 x 10(9) viral particles/ml). Overexpression of urokinase plasminogen activator and therefore of plasmin, was blocked by tranexamic acid (TA) in transduced HSC. Gene expression was monitored by reverse transcriptase polymerase chain reaction. HSC-free supernatants were used to evaluate MMP-2 and MMP-9 by zymography. SnoN, TGF-beta and tissue inhibitor of metalloproteinase (TIMP)-1 were analysed by Western blot. Plasmin and SnoN expression kinetics were evaluated in bile duct-ligated (BDL) rats. RESULTS: Plasmin overexpression in Ad-huPA-transduced HSC significantly decreased gene expression of profibrogenic molecules [alpha1(I)collagen 66%, TIMP-1 59%, alpha-smooth muscle actin 90% and TGF-beta 55%]. Interestingly, both SnoN gene and protein expression increased prominently. Plasmin inhibition by TA upregulated the profibrogenic genes, which respond to TGF-beta-intracellular signalling. In contrast, SnoN mRNA and protein dropped importantly. Plasmin-activated MMP-9 and MMP-2 in HSC supernatants. Taken together, these findings indicate that MMP-9 activation is totally plasmin dependent. SnoN levels significantly decreased in cholestatic-BDL rats (82%) as compared with control animals. Interestingly, hepatic plasmin levels dropped 46% in BDL rats as compared with control. CONCLUSION: Plasmin plays a key role in regulating TGF-beta-responding genes. In particular, regulation of TGF-beta-co-repressor (SnoN) is greatly affected, which suggests SnoN as a cardinal player in cholestasis-induced fibrogenesis.
Assuntos
Fibrinolisina/fisiologia , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Proteínas do Tecido Nervoso/metabolismo , Ratos , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Ácido Tranexâmico/farmacologia , Fatores de Transcrição/metabolismo , Transdução Genética , Fator de Crescimento Transformador beta/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
TGF-beta1 is a potent inductor of malignance in cancer cells. TGF-beta1 stimulates the expression of extracellular matrix degrading proteases, cell migration and it is also involved in the epithelial-mesenchymal transition (EMT). In the present work, we analyzed the role of Spred2 in the urokinase-type plasminogen activator (uPA) stimulation, EMT and cell migration by TGF-beta1. We found that both the expression of mRNA and the protein level of Spred2 were lower in transformed keratinocytes PDV compared with immortalized keratinocytes MCA-3D. The transient ectopic expression of Spred2 in PDV cells inhibited the TGF-beta1-transactivated SRE-Luc reporter which is related with the ERK1,2 signal. The stable ectopic expression of Spred2 in PDV cells (SP cells) led to the loss of ERK 1,2 activation by TGF-beta1, although Smad2 activation was not affected, and the knockdown of Spred2 enhanced the activation of ERK1,2 signal by TGF-beta1. The increment of uPA expression induced by TGF-beta1 was suppressed in SP cells. In contrast, the stimulus on PAI-1 expression was not affected and comparable to parental PDV cells. SP cells under TGF-beta1 treatment were unable to display the EMT, since the overexpression of Spred2 abolished the TGF-beta1-induced disruption of the E-cadherin cell to cell interactions, reorganization of the actin cytoskeleton and upregulation of the mesenchymal marker vimentin. Finally, SP cells could not respond to the TGF-beta1 stimulus on cell migration. Taken together, the data in the present study suggests that Spred2 is a regulator of TGF-beta1-induced malignance in transformed keratinocytes.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/citologia , Mesoderma/citologia , Proteínas Repressoras/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Bases , Primers do DNA , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.
Assuntos
Proteínas de Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Dobramento de Proteína , Estrutura Terciária de Proteína , Inibidores de Serina Proteinase/metabolismo , Tripsina/química , Sequência de Aminoácidos , Animais , Células Cultivadas , Escherichia coli/metabolismo , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Tripsina/genética , Tripsina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Myofibroblasts are frequent in the stroma of neoplasm and by the expression of proteinases they can influence tumor infiltration and progression. In the present study, presence of myofibroblasts and expression of matrix metalloproteinase-2 (MMP-2) and urokinase plasminogen activator (uPA) were examined in intra-osseous solid multicystic ameloblastomas to determine their roles in the clinicopathological features of the tumors. Fifty seven ameloblastomas were analyzed immunohistochemically with antibodies against the isoform alpha of the smooth muscle actin (alpha-SMA), a specific marker of myofibroblasts, MMP-2 and uPA. Myofibroblasts were found in the stroma, in close contact with neoplastic cell islands, of approximately 58% (n = 33) of the ameloblastomas. MMP-2 and uPA were found in the cytoplasm of both neoplastic and stromal cells. A significant correlation between presence of myofibroblasts and MMP-2 expression was observed. Abundant presence of myofibroblast in the stroma of the tumors and expression of MMP-2 in the neoplastic or stromal cells were significantly correlated with rupture of the osseous cortical, which has been considered an important prognostic marker of ameloblastoma aggressiveness. Ours results suggest that abundant presence of myofibroblasts and expression of MMP-2 in solid ameloblastomas may be associated with a more aggressive infiltrative behavior.
Assuntos
Ameloblastoma/enzimologia , Neoplasias Ósseas/enzimologia , Fibroblastos/patologia , Metaloproteinase 2 da Matriz/metabolismo , Adolescente , Adulto , Idoso , Ameloblastoma/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Criança , Citoplasma/metabolismo , Progressão da Doença , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/enzimologia , Prognóstico , Células Estromais/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto JovemRESUMO
In previous studies we have determined that protein kinase C (PKC) delta, a widely expressed member of the novel PKC serine-threonine kinases, induces in vitro changes associated with the acquisition of a malignant phenotype in NMuMG murine mammary cells. In this study we show that PKCdelta overexpression significantly decreases urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) production, two proteases associated with migratory and invasive capacities. This effect is markedly enhanced by treatment with phorbol 12-myristate 13-acetate (PMA). On the other hand, depletion of PKCdelta using RNAi led to a marked increase in both uPA and MMP-9 secretion, suggesting a physiological role for PKCdelta in controlling protease secretion. The MEK-1 inhibitor PD98059 reverted the characteristic pattern of proteases secretion and phospho-ERK1/2 up-regulation observed in PKCdelta overexpressors, suggesting that the PKCdelta effect is mediated by the MEK/ERK pathway. Our results suggest a dual role for PKCdelta in murine mammary cell cancer progression. While this kinase clearly promotes mitogenesis and favors malignant transformation, it also down-modulates the secretion of proteases probably limiting metastatic dissemination.