RESUMO
SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/metabolismo , Linfocinas/fisiologia , Osteonectina/biossíntese , Células Cultivadas , Humanos , Osteonectina/genética , RNA Mensageiro/biossíntese , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/patologia , Neovascularização Patológica/patologia , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Adenoviridae/genética , Angiostatinas , Animais , Divisão Celular , Regulação para Baixo , Fatores de Crescimento Endotelial/genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Técnicas de Transferência de Genes , Linfocinas/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Plasminogênio/genética , Plasminogênio/fisiologia , Ratos , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performed in vivo and in vitro demonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.
Assuntos
Apoptose , Endotélio Vascular/patologia , Neoplasias/patologia , Adenocarcinoma , Mama/citologia , Mama/fisiologia , Linhagem Celular Transformada , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Feminino , Fibroblastos/fisiologia , Fibrossarcoma , Células HL-60 , Humanos , Células Jurkat , Células Tumorais CultivadasRESUMO
Co-injection of fibroblasts with human epithelial breast-tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor-promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over-production of TIMP-2 was induced in MCF7 cells by in vitro retroviral-mediated gene transfer. TIMP-2-producing MCF7 cells were then co-injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co-inoculated with fibroblasts into nude mice. Both physiological (TIMP-2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor-promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low-molecular-weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Comunicação Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Metaloendopeptidases/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Adenocarcinoma/genética , Animais , Neoplasias da Mama/genética , Colágeno , Combinação de Medicamentos , Fibroblastos/efeitos dos fármacos , Técnicas de Transferência de Genes , Humanos , Laminina , Metaloendopeptidases/fisiologia , Camundongos , Camundongos Nus , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Proteoglicanas , Células Estromais/enzimologia , Tiofenos/farmacologia , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Células Tumorais CultivadasRESUMO
Tumor cell extravasation is a determinant step in the process of hematogenous metastasis. The signal transduction pathways involved in the interactions between tumor cells and the vascular endothelium during transendothelial migration are still undefined. In the present study, we have investigated the influence of human breast adenocarcinoma cells (MCF7) on human umbilical vein endothelial cell (HUVEC) intracellular Ca2+ concentration ([Ca2+]i). We show that the contact between MCF7 cells and a confluent HUVEC monolayer induces an immediate and transient increase in HUVEC [Ca2+]i. This [Ca2+]i rise could not be elicited by tumor cell-conditioned medium, isolated tumor cell membranes, inert beads or normal breast epithelial cells, demonstrating the involvement of specific recognition mechanisms between MCF7 cells and HUVEC. Depletion of HUVEC intracellular Ca2+ stores by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin as well as the selective depletion of inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+ stores by prior activation of HUVEC using histamine resulted in a complete inhibition of tumor cell-induced [Ca2+]i elevation. Similar results were obtained when HUVEC monolayers were treated with the tyrosine kinase inhibitor herbimycin A, suggesting a role for tyrosine kinase-associated cell surface receptors in tumor cell-endothelial cell interactions. The depletion of HUVEC intracellular Ca2+ stores by thapsigargin was also shown to delay MCF7-induced endothelial cell disjunction, to prevent their spreading on the subendothelial extracellular matrix and transendothelial migration in vitro. These results suggest that transient changes in endothelial [Ca2+]i may govern multiple steps of tumor cell extravasation.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Endotélio Vascular/metabolismo , Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Comunicação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Proteínas Tirosina Quinases/metabolismo , Tapsigargina/farmacologia , Células Tumorais Cultivadas , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
Metalloproteinases and serine proteinases have been associated with tumor invasion and formation of metastasis which represent the major obstacles to cancer cure. The contribution of proteinases in these processes was initially thought to be the destruction of extracellular matrices. However, recent evidence suggests that they mainly affect tumor growth rather than invasion. Proteinases can indeed generate active matrix protein fragments, influence the release, the activation and the bioavailability of growth factors, and consequently modulate tumor cell growth, apoptosis and angiogenesis. Additionally, proteinases, their receptors and/or inhibitors can be directly involved in cell migration and in the processing or shedding of cell surface proteins. Further elucidation of the functions of proteinases is essential for the development of novel anticancer strategies.
Assuntos
Endopeptidases/fisiologia , Substâncias de Crescimento/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Animais , Movimento Celular , Matriz Extracelular/metabolismo , Gelatinases/metabolismo , Humanos , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
The integrity of the vascular endothelium is mainly dependent upon the organization of interendothelial adherens junctions (AJ). These junctions are formed by the homotypic interaction of a transmembrane protein, vascular endothelial cadherin (VE-cadherin), which is complexed to an intracellular protein network including alpha-, beta-, and gamma-catenin. Additional proteins such as vinculin and alpha-actinin have been suggested to link the VE-cadherin/catenin complex to the actin-based cytoskeleton. During the process of hematogenous metastasis, circulating tumor cells must disrupt these intercellular junctions in order to extravasate. In the present study, we have investigated the influence of tumor cell-endothelial cell interaction upon interendothelial AJ. We show that human breast adenocarcinoma cells (MCF-7), but not normal human mammary epithelial cells, induce a rapid endothelial cell (EC) dissociation which correlates with the loss of VE-cadherin expression at the site of tumor cell-EC contact and with profound changes in vinculin distribution and organization. This process could not be inhibited by metalloproteinase nor serine protease inhibitors. Immunoprecipitations and Western blot analysis demonstrate that the overall expression of VE-cadherin and vinculin as well as the composition of the VE-cadherin/catenins complex are not affected by tumor cells while the tyrosine phosphorylation status of proteins within the complex is significantly altered. Our data suggest that tumor cells modulate AJ protein distribution and phosphorylation in EC and may, thereby, facilitate EC dissociation.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Caderinas/fisiologia , Endotélio Vascular/ultraestrutura , Junções Intercelulares/ultraestrutura , Transativadores , Adesão Celular , Proteínas do Citoesqueleto/metabolismo , Desmoplaquinas , Feminino , Humanos , Microscopia Eletrônica de Varredura , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Células Tumorais Cultivadas , Vinculina/metabolismo , alfa Catenina , beta Catenina , gama CateninaRESUMO
The initiation of the angiogenic process requires a locally confined and time-limited proteolysis of the basement membrane (BM) components at the site of new vessel sprout. Gelatinase A, a member of the matrix metalloproteinase family, degrades BM type IV collagen and is involved in the BM breakdown by migrating tumor cells and endothelial cells (EC). Gelatinase A is synthesized as latent proenzyme and must be activated in order to express its proteolytic activity. A plasma membrane-dependent mechanism of activation has been described for several tumor and transformed cells lines. In the present study, we show that latent (72 kD) and mature (62-59 kD) forms of gelatinase A are present in EC membrane fraction from Triton X-114 extract while only latent form is found in the cytosolic fraction. The incubation of EC membrane fraction with exogenous latent gelatinase A resulted in a significant activation giving rise to 62-59 kD mature forms. 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong potentiator of angiogenesis in vitro and in vivo, increases the amount of both latent and activated forms of gelatinase A in EC membrane fraction as well as the ability of this latter fraction to activate exogenous latent gelatinase A. We show that the mRNA transcript coding for the membrane-integrated MMP, the MT-MMP, previously described as a potential gelatinase A activator in invasive tumor cells is also expressed in vascular EC and is regulated through a TPA sensitive process. This enzyme may be responsible for membrane-dependent gelatinase A activation in normal vascular EC and may therefore be a determinant in the control of BM proteolysis during angiogenesis.
Assuntos
Endotélio Vascular/enzimologia , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Sequência de Bases , Fracionamento Celular , Membrana Celular/enzimologia , Meios de Cultivo Condicionados , Endotélio Vascular/ultraestrutura , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/genética , Dados de Sequência Molecular , Inibidores de Proteases/farmacologia , Proteínas/farmacologia , RNA Mensageiro/análise , Inibidor Tecidual de Metaloproteinase-2 , Veias Umbilicais/citologiaRESUMO
Thrombospondin (TSP), a M(r) 450,000 cytoadhesive glycoprotein, has been shown to potentiate tumor cell metastasis in mice by a mechanism that involves the hemostatic system of the host. In this study, the potential involvement of TSP in the interaction of human mammary adenocarcinoma MCF-7 cells with human umbilical vein endothelial cells (HUVECs) in culture was investigated. Using an ELISA, preconfluent HUVECs synthesized 100-fold more TSP than did MCF-7 cells during 24 h of culture (20 versus 0.2 microgram/10(6) cells). Confocal microscopy localized TSP within intercellular junctions between aggregated MCF-7 cells in suspension. On adherent cells, TSP exhibited a patchy distribution both on the cell surface and in the cytosol. In HUVECs, TSP strongly stained the perinuclear space and was also found in association with cytoskeletal microfibrils. Flow cytometric analysis indicated the presence of a large number of unoccupied receptors for TSP on MCF-7 cells. Binding studies using [125I]TSP demonstrated the presence of 1.6 x 10(6) sites/cell with an apparent Kd of 28 nM. Attachment of radiolabeled MCF-7 cells to a TSP-coated substrate and to HUVEC monolayers was inhibited in the presence of a polyclonal antibody to TSP (10 micrograms/ml) or increasing concentrations (1-10 micrograms/ml) of soluble TSP. Neither nonimmune IgG nor the cell adhesion peptide Gly-Arg-Gly-Asp-Ser (100 micrograms/ml) inhibited these interactions. Inhibition was also observed with heparin (10 micrograms/ml), suggesting the participation of TSP heparin-binding domain(s) and heparin-like molecules. In the presence of an excess of soluble TSP or anti-TSP antibody, MCF-7 cells did not form aggregates in suspension and preformed aggregates were readily dissociated by the addition of soluble TSP. These results indicate that mammary adenocarcinoma cells use TSP to form aggregates and to attach to human endothelial cells. These interactions may have physiological implications during the hematogenous spread of tumor cells.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Endotélio Vascular/patologia , Glicoproteínas de Membrana/fisiologia , Adesão Celular , Moléculas de Adesão Celular/farmacologia , Células Cultivadas , Humanos , TrombospondinasRESUMO
Gelatinases/type-IV collagenases are metalloproteinases involved in some carcinoma invasion and metastatic processes. The exact cellular source of the 72-kDa gelatinase A is controversial. We have analyzed the expression of mRNA coding for gelatinase A in vivo by in situ hybridization on breast-cancer tissues. The mRNA for gelatinase A was present in fibroblasts. We have therefore evaluated the gelatinase-A activity in vitro, in co-cultures of different breast adenocarcinoma cell lines and human fibroblasts. In monoculture, none of the tumor cells tested produced detectable amounts of gelatinase A. The gelatinase-A activity was enhanced in cultures of fibroblasts maintained in the presence of MDA-MB 231 or SKBR3 cells, or their conditioned medium. This increased enzymatic activity was evidenced both in the culture medium and in the membrane fraction and was paralleled by enhancement of the steady-state levels of mRNA. These results are an in vitro demonstration of a regulation of fibroblasts gelatinase-A production by soluble factors secreted by breast-tumor cells.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias da Mama/enzimologia , Mama/enzimologia , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , RNA Mensageiro/metabolismo , Pele/enzimologia , Adenocarcinoma/fisiopatologia , Northern Blotting , Mama/fisiologia , Neoplasias da Mama/fisiopatologia , Comunicação Celular , Linhagem Celular , Membrana Celular/enzimologia , Células Cultivadas , Meios de Cultivo Condicionados , Precursores Enzimáticos/análise , Precursores Enzimáticos/metabolismo , Feminino , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Gelatinases/análise , Gelatinases/biossíntese , Expressão Gênica , Humanos , Hibridização In Situ , Metaloproteinase 2 da Matriz , Metaloendopeptidases/análise , Metaloendopeptidases/biossíntese , RNA Mensageiro/biossíntese , Fenômenos Fisiológicos da Pele , Células Tumorais CultivadasRESUMO
Cell interactions with the extracellular matrix are consistently modified in neoplasia. Malignant transformation has been correlated with modifications in the synthesis and distribution of matrix components and with alterations of cell adhesive properties to these components. A particular class of genes, able to suppress the transformed phenotype in normal cells, may be involved in those phenotypic changes. By studying somatic cell hybrids between mouse hepatoma (BWTG3) cells and normal rat skin fibroblasts (RSF), Islam and co-workers were able to localize a gene or a group of genes controlling anchorage dependence and cell growth in vitro. This (or these) gene(s) was (were) assigned to the q22-23 fragment of rat chromosome 5. In the present study, we compare the morphology and the interactions with the extracellular matrix proteins (laminin, fibronectin, and collagen IV) and the synthesis of these proteins by RSF X BWTG3 hybrid cells that had either retained (BS181p10) or lost (BS181a5) the q22-23 region of rat chromosome 5. Our results suggest that the rat 5q22-23 fragment controls a part of the cell differentiation program including morphology, attachment to extracellular matrix, and synthesis of some matrix proteins, particularly alpha 1 and alpha 2 chains of collagen IV.
Assuntos
Mapeamento Cromossômico , Matriz Extracelular/fisiologia , Células Híbridas/ultraestrutura , Neoplasias Hepáticas Experimentais/ultraestrutura , Animais , Northern Blotting , Linhagem Celular , Colágeno/análise , Sondas de DNA , Fibroblastos/ultraestrutura , Fibronectinas/análise , Células Híbridas/fisiologia , Laminina/análise , Camundongos , Microscopia Eletrônica de Varredura , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/análise , Ratos , Pele/ultraestruturaRESUMO
The success of blood-born metastatic spread depends upon a key event: the tumor cell arrest and attachment to the host organ vasculature. In the present study, we have investigated interactions between several normal and cancer cell lines and vascular endothelium in a model of ex vivo perfusion of human umbilical vein. In this system, hydrodynamic parameters are monitored and endothelial cells are kept in their original environment known to modulate their phenotype. Metastatic tumor cell adhesion to the perfused endothelium was found to be significantly higher than that of normal cells tested. Platelets and soluble plasma factors including fibronectin promoted tumor cell arrest and adhesion to endothelium. Altogether our results indicate that the ex vivo perfusion of human umbilical vein allows the study of the interactions between malignant tumor cells, circulating plasma or blood cells and the endothelium during blood-born metastatic spread.