RESUMO
PURPOSE: Expression of the receptor tyrosine kinase c-Met and its ligand scatter factor/hepatocyte growth factor (SF/HGF) are strongly increased in glioblastomas, where they promote tumor proliferation, migration, invasion, and angiogenesis. We used a novel one-armed anti-c-Met antibody to inhibit glioblastoma growth in vivo. EXPERIMENTAL DESIGN: U87 glioblastoma cells (c-Met and SF/HGF positive) or G55 glioblastoma cells (c-Met positive and SF/HGF negative) were used to generate intracranial orthotopic xenografts in nude mice. The one-armed 5D5 (OA-5D5) anti-c-Met antibody was infused intratumorally using osmotic minipumps. Following treatment, tumor volumes were measured and tumors were analyzed histologically for extracellular matrix (ECM) components and proteases relevant to tumor invasion. Microarray analyses were done to determine the effect of the antibody on invasion-related genes. RESULTS: U87 tumor growth, strongly driven by SF/HGF, was inhibited > 95% with OA-5D5 treatment. In contrast, G55 tumors, which are not SF/HGF driven, did not respond to OA-5D5, suggesting that the antibody can have efficacy in SF/HGF-activated tumors. In OA-5D5-treated U87 tumors, cell proliferation was reduced > 75%, microvessel density was reduced > 90%, and apoptosis was increased > 60%. Furthermore, OA-5D5 treatment decreased tumor cell density > 2-fold, with a consequent increase in ECM deposition and increased immunoreactivity for laminin, fibronectin, and tenascin. Microarray studies showed no increase in these ECM factors, rather down-regulation of urokinase-type plasminogen activator and matrix metalloproteinase 16 in glioblastoma cells treated with OA-5D5. CONCLUSIONS: Local treatment with OA-5D5 can almost completely inhibit intracerebral glioblastoma growth when SF/HGF is driving tumor growth. The mechanisms of tumor inhibition include antiproliferative, antiangiogenic, and proapoptotic effects.
Assuntos
Anticorpos/farmacologia , Divisão Celular/efeitos dos fármacos , Glioblastoma/patologia , Proteínas Proto-Oncogênicas c-met/imunologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Transplante HeterólogoRESUMO
PURPOSE: Inhibition of angiogenesis can influence tumor cell invasion and metastasis. We previously showed that blockade of vascular endothelial growth factor receptor-2 (VEGFR-2) with the monoclonal antibody DC101 inhibited intracerebral glioblastoma growth but caused increased tumor cell invasion along the preexistent vasculature. In the present study, we attempted to inhibit glioma cell invasion using a monoclonal antibody against the epidermal growth factor receptor (EGFR), which in the context of human glioblastomas, has been implicated in tumor cell invasion. In addition, we analyzed whether blockade of vascular endothelial (VE)-cadherin as a different antiangiogenic target could also inhibit glioblastoma angiogenesis and growth. EXPERIMENTAL DESIGNS: Nude mice who received intracerebral glioblastoma xenografts were treated using monoclonal antibodies against VEGFR-2 (DC101), EGFR (C225), and VE-cadherin (E4G10) either alone or in different combinations. RESULTS: Increased tumor cell invasion provoked by DC101 monotherapy was inhibited by 50% to 66% by combined treatment with C225 and DC101. C225 inhibited glioblastoma cell migration in vitro, but had no effect on the volume of the main tumor mass or on tumor cell proliferation or apoptosis in vivo, either alone or in combination with DC101. The anti-VE-cadherin monoclonal antibody E4G10 was a weaker inhibitor of tumor angiogenesis and growth than DC101, and also caused a weaker increase in tumor cell invasion. CONCLUSIONS: Inhibition of angiogenesis achieved by blocking either VEGFR-2 or VE-cadherin can cause increased glioma cell invasion in an orthotopic model. Increased tumor cell invasion induced by potent inhibition of angiogenesis with DC101 could be inhibited by simultaneous blockade of EGFR.
Assuntos
Anticorpos Monoclonais/farmacologia , Glioblastoma/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Antígenos CD , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Caderinas/imunologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Receptores ErbB/imunologia , Feminino , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Humanos , Camundongos , Camundongos SCID , Invasividade Neoplásica , Neovascularização Patológica/patologia , Fator de Crescimento Transformador alfa/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
PURPOSE: Scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor MET are strongly up-regulated in malignant gliomas. The SF/HGF-MET system contributes to glioma invasion and angiogenesis via autocrine and paracrine mechanisms. We analyzed whether local treatment with NK4, an antagonistic fragment of SF/HGF, could inhibit glioma growth in vivo. EXPERIMENTAL DESIGN: A guide-screw system was used to implant tumor cells intracerebrally and to perform therapeutic injections. Mice received daily intratumoral injections of NK4 or buffer as of day 1 or 7 after tumor cell injection until day 20. Functional effects of NK4 on glioma and endothelial cells were analyzed in vitro. RESULTS: Tumor volume was reduced by 61.1% in mice treated with NK4 compared with controls when treatment was initiated on day 1 (P < 0.05) and by 61.4% when treatment was initiated on day 7 (P < 0.001). Intratumoral microvessel density was reduced by 64.9% when treatment started on day 1 and by 36.7% when it started on day 7. The proliferative activity of the tumor cells was reduced by >30% regardless of when NK4-treatment was initiated. The apoptotic fraction of tumor cells was increased 2-fold and 1.5-fold when animals were treated with NK4 as of day 1 or day 7, respectively. In vitro, NK4 inhibited SF/HGF-induced glioblastoma, and endothelial cell migration and proliferation in a dose-dependent fashion. CONCLUSION: NK4 inhibits glioblastoma growth in vivo, most likely via antimitogenic, antimotogenic, proapoptotic, and antiangiogenic mechanisms. Given the strong up-regulation of SF/HGF and MET in human malignant gliomas, NK4 holds promise as a direct interstitial therapeutic agent for these fatal tumors.
Assuntos
Neoplasias Encefálicas/prevenção & controle , Movimento Celular/efeitos dos fármacos , Glioblastoma/prevenção & controle , Fator de Crescimento de Hepatócito/uso terapêutico , Mitógenos , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Injeções Intralesionais , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neovascularização Patológica , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
PURPOSE: Vascular endothelial growth factor (VEGF)-A isa key mediator of angiogenesis in malignant gliomas. Soluble VEGF receptor 1 (sVEGFR-1) can complex VEGF-A and reduce its bioavailability. In several animal models sVEGFR-1 inhibited angiogenesis and tumor growth. We analyzed the levels of endogenous sVEGFR-1 in gliomas of different malignancy grades in relation to tumor vascularity and VEGF-A. EXPERIMENTAL DESIGN: The concentration of sVEGFR-1 was determined by ELISA in 104 gliomas and normal brain. Levels of sVEGFR-1 were compared with malignancy grade, microvessel density, and VEGF-A concentration. Effects of sVEGFR-1 on glioma extract-induced endothelial cell chemotaxis were analyzed in vitro. RESULTS: The concentration of sVEGFR-1 correlated with the malignancy grade and was 12-fold higher in glioblastomas than in diffuse astrocytomas (P < 0.001), with intermediate levels for anaplastic astrocytomas. VEGF-A levels were 30-fold higher (P < 0.001) in glioblastomas than in diffuse astrocytomas. The sVEGFR-1:VEGF-A ratio was 0.27 in glioblastomas and 0.70 in diffuse astrocytomas. Both sVEGFR-1 and VEGF-A correlated with microvessel density (P < 0.001) and with each other (P < 0.001); sVEGFR-1 and VEGF-A also correlated with each other when only glioblastomas were analyzed (P = 0.001). In vitro, recombinant sVEGFR-1 inhibited endothelial cell chemotaxis induced by tumor extracts. CONCLUSIONS: Although absolute levels of sVEGFR-1 are increased in the more malignant gliomas, the sVEGFR-1:VEGF-A ratio is decreased 2.6-fold in glioblastomas compared with diffuse astrocytomas, suggesting that the ensuing increased bioavailability of VEGF-A favors angiogenesis. The inhibition of tumor extract-induced endothelial chemotaxis by sVEGFR-1 suggests that sVEGFR-1 could be useful as an angiogenesis inhibitor in the specific context of human gliomas.
Assuntos
Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Glioma/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Células Cultivadas , Quimiotaxia , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Glioblastoma/metabolismo , Humanos , Imuno-Histoquímica , Fatores de Tempo , Veias Umbilicais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Using subtractive cloning combined with cDNA array analysis, we previously identified the genes encoding for the protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta (PTPzeta/RPTPbeta) and its ligand pleiotrophin (PTN) as overexpressed in human glioblastomas compared to normal brain. Both molecules have been implicated in neuronal migration during central nervous system development, and PTN is known to be involved in tumor growth and angiogenesis. We confirm overexpression of both molecules at the protein level in astrocytic gliomas of different malignancy grades. PTPzeta/RPTPbeta immunoreactivity was associated with increasing malignancy grade and localized predominantly to the tumor cells. PTN immunoreactivity as determined by ELISA and immunohistochemistry analysis was increased in low-grade astrocytomas compared to normal brain. Further increase in malignant gliomas was marginal, and thus no correlation with malignancy grade or microvessel density was present. However, PTN levels were significantly associated with those of fibroblast growth factor-2, suggesting co-regulation of both factors. Functionally, PTN induced weak chemotactic and strong haptotactic migration of glioblastoma and cerebral microvascular endothelial cells. Haptotaxis of glioblastoma cells towards PTN was specifically inhibited by an anti-PTPzeta/RPTPbeta antibody. Our findings suggest that upregulated expression of PTN and PTPzeta/RPTPbeta in human astrocytic tumor cells can create an autocrine loop that is important for glioma cell migration. Although PTN is a secreted growth factor, it appears to exert its mitogenic effects mostly in a matrix-immobilized form, serving as a substrate for migrating tumor cells.
Assuntos
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Transporte/biossíntese , Citocinas/biossíntese , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/fisiologia , Astrocitoma/genética , Neoplasias Encefálicas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Humanos , Ligantes , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a ReceptoresRESUMO
OBJECTIVE: Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another. METHODS: Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles. RESULTS: SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold). CONCLUSION: The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.
Assuntos
Neoplasias Encefálicas/fisiopatologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Fatores Quimiotáticos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Glioblastoma/fisiopatologia , Substâncias de Crescimento/farmacologia , Indutores da Angiogênese/farmacologia , Proteínas de Transporte/farmacologia , Citocinas/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Técnicas In Vitro , Midkina , Mitógenos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Somatomedinas/farmacologia , Fatores de Crescimento Transformadores/farmacologiaRESUMO
The c-Met receptor and its ligand scatter factor/hepatocyte growth factor (SF/HGF) are strongly overexpressed in malignant gliomas. Signaling through c-Met as well as exposure to hypoxia can stimulate glioma cell migration and invasion. In several cancer cell types, hypoxia was shown to activate the c-met promoter, which contains hypoxia inducible factor-1 (HIF-1) binding sites. We hypothesized that hypoxia might upregulate c-Met also in glioma cells. Analyzing 18 different glioblastoma cell lines and 10 glioblastoma primary cultures, we found that in 50% of both the cell lines and the primary cultures c-Met protein levels were increased following exposure to hypoxia. Upregulation of c-met in response to hypoxia was also detected at the transcriptional level. In all primary cultures and in 16 of the 18 cell lines (89%), HIF-1 alpha levels were increased by hypoxia. Transfection of siRNA against HIF-1 alpha abgrogated the hypoxic induction of c-Met, suggesting that c-Met expression is upregulated by a HIF-1 alpha-dependent mechanism. Hypoxia sensitized glioblastoma cell lines which showed hypoxic induction of c-Met to the motogenic effects of SF/HGF. These findings suggest that approximately half of all human glioblastomas respond to hypoxia with an induction of c-Met, which can enhance the stimulating effect of SF/HGF on tumor cell migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Western Blotting , Hipóxia Celular , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
The protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta (PTPzeta/RPTPbeta) and its ligand pleiotrophin (PTN) are overexpressed in human glioblastomas. Both molecules are involved in neuronal cell migration during CNS development. In addition, PTN can induce glioma cell migration which is at least in part mediated through binding to PTPzeta/RPTPbeta. To study the relevance of this ligand-receptor pair for glioma growth in vitro and in vivo, we transfected the human glioblastoma cell line U251-MG with small interfering RNA (siRNA) directed against PTPzeta/RPTPbeta. Stable siRNA transfection resulted in strong down-regulation of PTPzeta/RPTPbeta expression. When injected subcutaneously into nude mice, clones that expressed normal levels of PTPzeta/RPTPbeta (PTPzeta + clones) formed exponentially growing tumours, whereas tumour growth was almost completely abrogated for clones that expressed reduced PTPzeta/RPTPbeta levels (PTPzeta - clones). Similar results were obtained using an orthotopic intracerebral model. Proliferation of PTPzeta - cells in vitro was significantly reduced compared with that of control clones. Matrix-immobilized PTN stimulated the proliferation of PTPzeta + cells but not of PTPzeta - cells. Haptotactic migration induced by PTN was reduced for PTPzeta - clones compared with control clones. Our findings suggest that antagonization of PTPzeta/RPTPbeta expression can inhibit glioma growth in vivo and may thus represent a potentially promising treatment strategy.
Assuntos
Glioblastoma/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/fisiologia , Animais , Western Blotting/métodos , Contagem de Células/métodos , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Regulação para Baixo/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Nus , Transplante de Neoplasias/métodos , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Fosfatases/genética , RNA Interferente Pequeno/farmacologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Estatísticas não Paramétricas , Fatores de Tempo , Transfecção/métodosRESUMO
Contactin is a cell surface adhesion molecule that is normally expressed by neurons and oligodendrocytes. Particularly high levels of contactin are present during brain development. Using subtractive cloning, we identified contactin transcripts as overexpressed in glioblastomas compared with normal brain. We confirmed contactin overexpression in glioblastomas at the protein level, and localized contactin to the surface of glial fibrillary acidic protein (GFAP)-expressing glioblastoma cells. In contrast, normal astrocytes did not express contactin. Analyzing different types of astrocytic tumors, we detected an association between increasing malignancy grade and contactin expression. Functionally, contactin had repellent effects on glioma cells in vitro, as demonstrated by adhesion and migration assays. Overexpression of contactin by transfection into glioblastoma cells did not alter the proliferation rate or adhesion to various extracellular matrix proteins as well as adhesion to cells expressing the specific contactin ligand the protein tyrosine phosphatase zeta (PTPzeta). Our findings suggest that contactin has repellent effects on glioma cells to which it is presented as a ligand, but it does not alter the proliferative or adhesive capacities of cells that overexpress the molecule. The repulsive properties of contactin may be a key factor in glioma disaggregation, and may contribute to the diffuse infiltration pattern characteristic of glioma cells in human brain.