RESUMO
Human gliomas are highly invasive, and remain to be a major obstacle for any effective therapeutic remedy. Among many other factors, gliomas express elevated levels of matrix metalloproteinases (MMPs), which have been implicated to play an important role in tumor invasion as well as neovascularization. The tumor suppressor gene mutated in multiple advanced cancers/phosphatase and tensin homologue (MMAC/PTEN) has been shown to inhibit cell migration, spreading, and focal adhesion. In this study, we determined whether MMAC/PTEN inhibits tumor invasion by modulating MMP-2 activity. Our results showed that reintroduction of the MMAC/PTEN gene into human glioma U251 and U87 cells modified their phenotype and growth characteristics. The ability of MMAC/PTEN to induce anoikis in U251 cells was accompanied by a significant inhibition of in vitro invasion (70%). Expression of MMAC/PTEN in U251 and U87 cells inhibited MMP-2 enzymatic activity as determined by zymography. Furthermore, MMAC/PTEN expression strongly decreased MMP-2 mRNA levels, which correlated well with the inhibition of invasion capacity in these cells. Concomitant with MMP-2 expression and activity, MMP-2 promoter activity was also reduced in MMAC/PTEN expressing cells. Our observations suggest that MMAC/PTEN inhibits tumor cell invasion in part by regulating MMP-2 gene transcription and thereby its enzymatic activity. Further characterization of this regulation will facilitate the development of MMAC/PTEN based gene therapy for gliomas.
Assuntos
Glioma/enzimologia , Metaloproteinase 2 da Matriz/biossíntese , Inibidores de Metaloproteinases de Matriz , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Anoikis , Divisão Celular , Genes Reporter , Humanos , Immunoblotting , Luciferases/metabolismo , Microscopia de Contraste de Fase , Invasividade Neoplásica , PTEN Fosfo-Hidrolase , Fenótipo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Mensageiro/metabolismo , Retroviridae/genética , Fatores de Tempo , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-kappaB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eye's aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.
Assuntos
Olho/fisiopatologia , Glaucoma/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Primers do DNA , Selectina E/metabolismo , Olho/metabolismo , Feminino , Glaucoma/classificação , Glaucoma/metabolismo , Humanos , Interleucina-1/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Estresse Oxidativo , Transdução de SinaisRESUMO
Glioblastomas express more urokinase-type plasminogen activator receptor (uPAR) than do low-grade gliomas and normal brain tissue. We previously showed that downregulation of uPAR through the transfection of SNB19 cells with an antisense cDNA construct corresponding to 300 bp of the 5' end of the human uPAR gene inhibited tumor cell invasion in vitro and tumor formation in vivo. Here we sought to determine whether uPAR is necessary for cell survival and whether the inhibition of tumor formation in nude mice is due to apoptosis of intracerebrally injected SNB19 cells. Apoptosis measured by DNA fragmentation were higher in the brains of animals injected with the antisense stable transfectants than in those injected with the parental cells. Moreover, the increase in apoptotic cell death in vitro was associated with increased expression of apoptotic protein BAX in antisense clones compared to controls. To our knowledge, this is the first report of uPAR playing a novel role in cell survival in human gliomas.
Assuntos
Apoptose , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/fisiopatologia , Glioma/patologia , Glioma/fisiopatologia , Receptores de Superfície Celular/fisiologia , Animais , Neoplasias Encefálicas/prevenção & controle , DNA Antissenso/farmacologia , Feminino , Glioma/prevenção & controle , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Invasividade Neoplásica/prevenção & controle , Ativadores de Plasminogênio/fisiologia , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transfecção , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Increased expression of matrix metalloproteinases (MMPs) has been associated with human glioblastoma tumor progression. In this study, we sought to down-regulate MMP-9 expression by stably transfecting a high-grade glioblastoma cell line with a plasmid vector capable of expressing an antisense transcript complementary to a 528-bp segment at the 5' end of human MMP-9 cDNA. Stable transfectants were obtained through selection with G418. Of the clones transfected with vector, sense, and antisense constructs, Northern blotting, Western blotting, and gelatin zymography showed that MMP-9 expression was significantly reduced only in the antisense-transfected cells. A Matrigel invasion assay revealed marked reductions in invasiveness for the antisense clones relative to the parental, vector, and sense clones. Cocultures of tumor spheroids and fetal rat brain aggregates showed that the antisense-transfected stable clones showed no invasion of the rat brain aggregates; in contrast, 90% of the parental, vector, and sense clones invaded the rat brain aggregates. Intracerebral injection of antisense stable transfectants in nude mice produced no tumors or very small tumors, but intracerebral injection of parental or vector clones did produce tumors. These results suggest that MMP-9 expression is essential for the invasiveness of glioblastoma cells.
Assuntos
Técnicas de Transferência de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/genética , Metaloproteinase 9 da Matriz/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Colágeno/metabolismo , DNA Complementar/metabolismo , Regulação para Baixo , Combinação de Medicamentos , Humanos , Laminina/metabolismo , Camundongos , Camundongos Nus , Microscopia Confocal , Invasividade Neoplásica , Transplante de Neoplasias , Oligonucleotídeos/farmacologia , Plasmídeos/metabolismo , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Fatores de Tempo , TransfecçãoRESUMO
Cell-matrix interactions exert a profound influence on cell function and behavior. Our earlier observations suggested that disruption of the actin cytoskeleton results in the inhibition of phorbol ester-induced matrix metalloproteinase (MMP)-9 expression. In this study, to understand the role of protein tyrosine phosphatases in matrix metalloproteinase-9 expression, we treated glioblastoma cells with vanadate and phenylarsine oxide (PAO), which are inhibitors of protein tyrosine phosphatases. Vanadate and PAO inhibited expression of phorbol ester-induced MMP-9 as well as constitutive expression of matrix metalloproteinase-2 in a dose- and time-dependent fashion. An assay of the activity of phosphotyrosine phosphatase (PTPase) indicated that vanadate-treated cells had reduced PTPase activity compared with that of untreated controls. Vanadate and PAO also inhibited actin polymerization, cell spreading, migration, and invasion of glioma cells. Furthermore, elevated levels of protein tyrosine phosphorylation were observed in vanadate- and PAO-treated cells in both a concentration- and time-dependent fashion and were seen to have an inverse correlation with focal adhesion kinase protein expression. These results suggest that vanadate and PAO inhibited migration and invasion of glioma cells by their effect on the cytoskeleton and inhibition of MMP expression.
Assuntos
Actinas/efeitos dos fármacos , Arsenicais/farmacologia , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metaloproteinases da Matriz/genética , Invasividade Neoplásica/prevenção & controle , Proteínas Tirosina Fosfatases/metabolismo , Vanadatos/farmacologia , Moléculas de Adesão Celular/genética , Inibidores Enzimáticos/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glioma , Humanos , Fosfoproteínas/metabolismo , Fosfotirosina/análise , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Gliomas, a type of devastating primary brain tumors, are distinct from other solid, non-neural primary neoplasms, in that they display extensive infiltrative invasive behavior but seldom metastasize to distant organs. This invasiveness into the surrounding normal brain tissue makes gliomas a major challenge for clinical intervention. Total surgical resection of gliomas is not possible, and recurrence of tumor growth is common; mean survival time is 8-12 months. Although substantial progress has been made recently toward understanding the behavior of gliomas, the mechanisms that facilitate invasion are still poorly documented. Clues to the invasion process have been ascertained through clarification of the key roles played by the extracellular matrix (ECM), cell-adhesion molecules and matrix degrading proteases. Serine proteases and metalloproteinases have been implicated in glioma tumor cell-invasion. Matrix metalloproteinases (MMPs) in particular can degrade almost all known ECM components and seem to play important roles in mediating glioblastoma tumor cell invasion. This review focuses on recent developments concerning the role of MMPs in the invasiveness of human gliomas.
Assuntos
Neoplasias Encefálicas/enzimologia , Glioma/enzimologia , Metaloproteinases da Matriz/fisiologia , Neoplasias Encefálicas/patologia , Progressão da Doença , Gelatinases/fisiologia , Glioma/patologia , Humanos , Metaloproteinase 2 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/fisiologia , Invasividade NeoplásicaRESUMO
The urokinase-type plasminogen activator (uPA) and uPA receptor (UPAR) play important roles in the proteolytic cascade involved in the invasiveness of gliomas and other invasive tumors. High-level expression of uPAR has been correlated with high-grade glioma cell lines and tumors We report here that down-regulating uPAR levels by antisense strategy using an adenovirus construct (Ad-uPAR) inhibited glioma invasion in Matrigel and spheroid in vitro models. sc. (U87-MG) and intracranial (SNB19) injections of Ad-uPAR-infected glioma cells did not produce tumors in nude mice. However, injection of the Ad-uPAR construct into previously established so U87-MG tumors in nude mice caused regression of those tumors. Our results support the therapeutic potential of targeting the uPA-uPAR system for the treatment of gliomas and other cancers.
Assuntos
Adenoviridae/genética , Neoplasias Encefálicas/terapia , DNA Antissenso/genética , Terapia Genética , Glioblastoma/terapia , Proteínas de Neoplasias/genética , Receptores de Superfície Celular/genética , Animais , Neoplasias Encefálicas/patologia , Progressão da Doença , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Transplante de Neoplasias , Organoides , Ratos , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Células Tumorais CultivadasRESUMO
The cell-surface urokinase plasminogen activator receptor (uPAR) plays a key role in regulating plasminogen cleavage during extracellular proteolysis. Our recent results demonstrated that uPAR expression is critical for the invasiveness of human gliomas and down regulation of uPAR caused by antisense cDNA transfection inhibits the invasion of these stable antisense uPAR-transfectant clones. To study the role of uPARs in glioma cell invasion, a human neuroglioma cell line (H4) that normally produces low numbers of uPARs was transfected with the expression vector containing full-length human uPAR cDNA. Stable transfectants were analyzed for uPAR mRNA expression, receptor number, in vitro invasion and secretion of uPA and MMP-2. The uPAR-overproducing clones showed a 4-fold increase in uPAR mRNA transcription and approximately 40% increase in receptor numbers. uPAR-overproducing clones also invaded through matrigel to a significantly greater extent than did parent cell line and vector clones. However, the uPAR-overexpressing clones and parent cell lines showed similar uPA and MMP-2 activities. These results suggest that the over-production of uPAR on the surface of neuroglioma cells enhances the invasiveness.
Assuntos
Glioma/patologia , Invasividade Neoplásica , Receptores de Superfície Celular/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sítios de Ligação , Northern Blotting , DNA Complementar/genética , Fibrina/metabolismo , Gelatinases/metabolismo , Glioma/metabolismo , Glioma/secundário , Humanos , Metaloproteinase 2 da Matriz , Metaloendopeptidases/metabolismo , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transfecção , Células Tumorais CultivadasRESUMO
Alterations in cytoskeleton and subsequent cell shape changes exert specific effects on the expression of various genes. Our previous results suggested that malignant human gliomas express elevated levels of matrix metalloproteinases compared with normal brain tissue and low grade gliomas. To understand the role of cell shape changes on matrix metalloproteinase expression in human glioma cells, we treated SNB19 cells with cytochalasin-D, an inhibitor of actin polymerization, and colchicine-B, a tubulin inhibitor, in the presence of phorbol 12-myristate 13-acetate. Cytochalasin-D treatment of SNB19 cells resulted in the loss of phorbol 12-myristate 13-acetate-induced matrix metalloproteinase-9 (also known as gelatinase-B) expression and coincided with inhibition of actin polymerization, resulting in cell rounding. Moreover, compared with monolayers, cells grown as spheroids or cell aggregates failed to express matrix metalloproteinase-9 in the presence of phorbol 12-myristate 13-acetate. Matrix metalloproteinase-9 expression was also inhibited by calphostin-C, a protein kinase inhibitor, suggesting the involvement of protein kinase C in matrix metalloproteinase-9 expression. Phorbol 12-myristate 13-acetate-induced invasion of SNB19 cells through Matrigel was inhibited by cytochalasin-D and calphostin-C. These results suggest that the actin polymerization transduces signals that modulate the expression of matrix metalloproteinase-9 expression and the subsequent invasion of human glioma cells.
Assuntos
Actinas/metabolismo , Neoplasias Encefálicas/enzimologia , Colagenases/biossíntese , Citoesqueleto/metabolismo , Glioma/enzimologia , Biopolímeros , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/ultraestrutura , Moléculas de Adesão Celular/metabolismo , Citocalasina D/farmacologia , Ativação Enzimática , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glioma/patologia , Glioma/ultraestrutura , Humanos , Metaloproteinase 9 da Matriz , NF-kappa B/metabolismo , Invasividade Neoplásica , Proteína Quinase C/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
OBJECTIVE: This study was conducted to obtain evidence that restoration of the retinoblastoma protein function may have therapeutic application for gliomas. BACKGROUND: The development of glioblastoma multiforme involves progressive inactivation of several tumor suppressor genes. Abnormalities of the retinoblastoma tumor suppressor gene are found in the majority of cancers, including at least 30% of malignant gliomas. No final evidence has been produced about the role of Rb in suppressing glioma growth. METHODS: To address this question, the Ad5CMV-Rb adenovirus carrying a 3.2-kb cDNA of the Rb gene was constructed. Expression of the exogenous protein was assessed by immunoblot and immunohistochemistry analyses. Growth curve assays were used to evaluate the effect of the Rb protein on glioma cell growth. Flow-cytometry analyses were used to analyze the phenotype of the cell cycle after the transfer of Rb. Human glioma xenografts implanted subcutaneously in nude mice were used for the tumorigenicity assay. RESULTS: After the transfer of Rb, 80% of the treated cells expressed high levels of the retinoblastoma protein for at least 7 days. Within 5 days of treatment, the cells lost the neoplastic morphology and showed marked growth suppression. The majority of the Rb-expressing cells were arrested in the G1 phase of the cell cycle. In addition, the restoration of the retinoblastoma activity rendered the human glioma cells unable to form tumors in nude mice. CONCLUSIONS: These findings provide direct evidence that inactivation of the retinoblastoma protein is a critical event in gliomas, and suggest that the restoration of wild-type retinoblastoma activity in these tumors may have therapeutic utility.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Genes do Retinoblastoma , Glioblastoma/terapia , Animais , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Vetores Genéticos , Humanos , Camundongos , Camundongos Nus , Fosforilação , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
We have sought to determine the production and activity of serine proteases in primary and metastatic spinal tumors and the association of these enzymes with the invasive and metastatic properties of spinal column tumors. Using immunohistochemical techniques, the cellular localization and expression of urokinase-type plasminogen activator (uPA) was assessed, whereas its activity was determined by fibrin zymography, and the amounts of enzyme were measured by an enzyme-linked immunosorbent assay (ELISA) in primary spinal column tumors (chordoma, chondrosarcoma, and giant cell tumor) and metastatic tumors of the spine arising from various malignancies (breast, lung, thyroid, and renal cell carcinomas, and melanomas). Metastatic tumors displayed higher levels of uPA activity than did primary spinal tumors (P<0.001). Immunohistochemical analysis revealed that uPA expression was highest in metastases from lung and breast carcinomas and melanomas, followed by metastatic tumors from thyroid and renal cell carcinomas. Similar results were obtained for uPA activity and enzyme level as determined by fibrin zymography and ELISA, respectively. We conclude that metastatic spinal tumors possess higher levels of uPA expression and activity than the primary spinal tumors, which tend to be less aggressive and only locally invasive malignancies. The results suggest that the plasminogen system may participate in the metastasis of tumors to the spinal column.
Assuntos
Neoplasias da Coluna Vertebral/enzimologia , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/metabolismo , Condrossarcoma/enzimologia , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Cordoma/enzimologia , Cordoma/patologia , Cordoma/secundário , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fibrina , Tumores de Células Gigantes/enzimologia , Tumores de Células Gigantes/metabolismo , Tumores de Células Gigantes/patologia , Humanos , Imuno-Histoquímica , Neoplasias da Coluna Vertebral/patologia , Neoplasias da Coluna Vertebral/secundárioRESUMO
Matrix metalloproteinases (MMPs) have been implicated in the process of tumor invasion and metastasis formation. Thus, we determined the expression of MMPs in various primary and metastatic spinal tumors in order to assess the role of these enzymes in spinal invasion. MMP expression was examined by immunohistochemical localization, and quantitative evaluation of MMP protein content was determined by enzyme-linked immunosorbant assay (ELISA) and Western blotting. MMP enzyme activity was determined by gelatin zymography. Lung carcinomas and melanomas metastatic to the spine were shown to have higher levels of MMP-9 activity than those of breast, thyroid, renal metastases and primary spinal tumors. Immunohistochemical analysis revealed similar difference in expression of MMP-9 in tissue samples. When the tissue samples were subjected to gelatin zymography for examination of MMP-2 and MMP-9 activity and to ELISA and Western blotting for quantitative estimation of protein content, the most striking results were obtained for lung carcinomas and melanomas relative to the other tumors. Lung carcinomas and melanomas metastatic to the spine had considerably higher levels of MMP-9 activity than those of primary spinal tumor or breast, thyroid, and renal carcinoma metastases. Within the metastatic tumor category, neoplasms that are known to be associated with the shortest overall survival rates and most aggressive behavior, such as lung carcinomas and melanomas, had the highest levels of MMP-2 and MMP-9 activity compared to those less aggressive metastatic tumors such as breast, renal cell, and thyroid carcinomas. Our results suggest that MMPs may contribute to the metastases to the spinal column, and overexpression of these enzymes may correlate with enhanced invasive properties of both primary and metastatic spinal tumors.
Assuntos
Colagenases/biossíntese , Colagenases/fisiologia , Gelatinases/biossíntese , Gelatinases/fisiologia , Metaloendopeptidases/biossíntese , Metaloendopeptidases/fisiologia , Neoplasias da Coluna Vertebral/enzimologia , Condrossarcoma/enzimologia , Condrossarcoma/patologia , Cordoma/enzimologia , Cordoma/patologia , Cordoma/secundário , Ensaio de Imunoadsorção Enzimática , Tumores de Células Gigantes/enzimologia , Tumores de Células Gigantes/metabolismo , Tumores de Células Gigantes/patologia , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias da Coluna Vertebral/patologia , Neoplasias da Coluna Vertebral/secundárioRESUMO
Malignant gliomas extensively infiltrate the surrounding normal brain, and their diffuse invasion is one of the most important barriers to successful therapy. Recent studies indicate that the progression of gliomas from low-grade to high-grade may depend on the acquisition of a new phenotype and the subsequent addition of genetic defects. One of the most frequent abnormalities in the progression of gliomas is the inactivation of tumor-suppressor gene p16, suggesting that loss of p16 is associated with acquisition of malignant characteristics. Consistent with this hypothesis, our previous studies showed that restoring wild-type p16 activity into p16-null malignant glioma cells modified their phenotype. In order to understand whether the biological consequences of p16 inactivation in high-grade gliomas included facilitating invasiveness, we used a recombinant replication-deficient adenovirus carrying the cDNA of the p16/CDKN2 gene to infect and express high levels of p16 protein in p16-null SNB19 glioma cells. Invasion of SNB19 glioma cells was tested into two models: invasion of glioma cells through Matrigel-coated transwell inserts and invasion of tumor-cell spheroids into fetal rat-brain aggregates in a co-culture system. Matrigel invasion assays showed that the SNB19 cells expressing exogenous p16 exhibited significantly reduced invasion. Similarly, invasion of p16-treated SNB19 cells into fetal rat-brain aggregates was reduced during a 72 h time period compared to invasion of the adenovirus-control and mock-infected cells. Expression of matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor-cell invasion, in SNB19 cells expressing p16 was significantly reduced compared to that of parental SNB19 and vector-infected cells. Our results show that restoring wild-type p16 activity into p16-null SNB19 glioma cells significantly inhibits tumor-cell invasion, thus suggesting a novel function of the p16 gene.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Gelatinases/metabolismo , Técnicas de Transferência de Genes , Genes Supressores de Tumor/genética , Glioma/genética , Glioma/secundário , Metaloendopeptidases/metabolismo , Proteínas de Neoplasias/metabolismo , Adenoviridae/genética , Animais , Neoplasias Encefálicas/secundário , Colágeno , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Combinação de Medicamentos , Vetores Genéticos/genética , Glioma/metabolismo , Humanos , Laminina , Metaloproteinase 2 da Matriz , Invasividade Neoplásica , Proteoglicanas , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/metabolismo , Esferoides Celulares/patologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Matrix metalloproteinases (MMPs) play an important role in various physiological and pathological conditions such as tissue remodeling, and cancer cell invasion and metastasis. The aim of this study was to determine the effect of the antitumor compounds cis-dichlorodiammine platinum (ii) (cisplatin) and 1, 3 bis (2-chloroethyl)-1-nitrosourea (BCNU) on 72-kDa type IV collagenase activity (MMP-2) in human gliomas. Human glioblastoma cell lines were treated with cisplatin (25 microM), and BCNU (50 microM), and the levels of MMP-2 were estimated in serum-free conditioned medium and in cell extracts at different time intervals. Gelatin zymography revealed increased levels of MMP-2 in serum-free conditioned medium and in cell extracts of untreated glioblastoma cell cultures during a 72-h period. In contrast, MMP-2 levels were significantly decreased in cisplatin-treated cells both in conditioned medium and cell extracts. However, no significant changes of MMP-2 levels were noted in BCNU-treated cells. Quantitative analysis of MMP-2 enzyme activity by densitometry and amount of MMP-2 protein by ELISA showed significantly decreased levels of MMP-2 in cisplatin-treated cells compared to BCNU and untreated glioblastoma cells. The results indicate that decreased levels of MMP-2 might represent an additional mechanism by which cisplatin provides its antineoplastic effects.
Assuntos
Carmustina/farmacologia , Cisplatino/farmacologia , Gelatinases/metabolismo , Glioblastoma/enzimologia , Metaloendopeptidases/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Meios de Cultura Livres de Soro , Densitometria/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática , Gelatina/química , Gelatinases/efeitos dos fármacos , Gelatinases/imunologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Metaloproteinase 2 da Matriz , Metaloendopeptidases/efeitos dos fármacos , Metaloendopeptidases/imunologia , Células Tumorais CultivadasRESUMO
Our previous studies showed that glioblastomas express increased urokinase-type plasminogen activator receptors (uPARs) in comparison to low-grade gliomas (Yamamoto et al., Cancer Res., 54, 5016-5020, 1994). To explore whether downregulation of uPAR inhibits tumor formation and invasiveness, a human glioblastoma cell line was transfected with a cDNA construct corresponding to 300 bp of the human uPAR's 5' end in an antisense orientation, resulting in a reduced number of uPA receptors. Co-culture studies with tumor spheroids and fetal rat brain aggregates showed that antisense SNB19-AS1 cells expressing reduced uPAR failed to invade fetal rat brain aggregates. Intracerebral injection of SNB19-AS1 stable transfectants failed to form tumors and were negative for uPAR expression in nude mice. Thus uPAR appears in this model to be essential for tumorigenicity and invasion of glioblastomas in vivo.
Assuntos
Elementos Antissenso (Genética)/farmacologia , Glioblastoma/genética , Glioblastoma/patologia , Receptores de Superfície Celular/genética , Animais , Elementos Antissenso (Genética)/genética , Encéfalo/embriologia , Encéfalo/patologia , Testes de Carcinogenicidade , Técnicas de Cocultura , Feminino , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neoplasias Experimentais/genética , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Glioblastomas extensively invade the surrounding normal brain tissue, with a concomitant expression of various proteolytic enzymes, in particular urokinase-type plasminogen activator (uPA). In this study we used cis-diamminedichloroplatinum (cisplatin) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), commonly used anti-cancer drugs for the treatment of glioblastomas, to study the expression of uPA in three human glioblastoma cell lines in vitro. Cells were treated with 25 microM cisplatin and 50 microM BCNU, and uPA levels were estimated by fibrin zymography during a 72-h time course. Treatment of glioblastoma cells with cisplatin resulted in significantly decreased levels of uPA in serum-free conditioned medium and cell extracts, compared to BCNU-treated and untreated cell lines. Quantitative levels of uPA enzyme activity assessed by scanning laser densitometry and uPA protein by ELISA using antibody against uPA showed decreased levels of uPA in cisplatin-treated glioma cell lines relative to BCNU and untreated cell lines. Our results suggest that anti-tumor compound, cisplatin, may exert its anti-neoplastic effects by inhibiting uPA in malignant glioblastomas.
Assuntos
Carmustina/farmacologia , Cisplatino/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Meios de Cultura Livres de Soro , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática , Fibrina/análise , Fibrina/metabolismo , Glioblastoma/metabolismo , Humanos , Invasividade Neoplásica , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/análise , Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacosRESUMO
The cell surface urokinase-type plasminogen activator receptor (uPAR) has been shown to be a key molecule in regulating plasminogen-mediated extracellular proteolysis. To investigate the role of uPAR in invasion of brain tumors, human glioblastoma cell line SNB19 was stably transfected with a vector capable of expressing an antisense transcript complementary to the 300 base pair of the 5' end of the uPAR mRNA. Parental and stably transfected (vector, sense, and antisense) cell lines were analysed for uPAR mRNA transcript by Northern blot analysis, and receptor protein levels were measured by radioreceptor assays and Western blotting. Significant reduction of uPAR sites was observed in the antisense transfected cell lines. The levels of uPAR mRNA were significantly decreased in antisense clones compared to control, vector and sense clones. The invasive potential of the cell lines in vitro was measured by Matrigel invasion assay and migration of cells from spheroids to monolayers. The antisense transfected cells showed a markedly lower level of invasion and migration than the controls. The antisense clones were more adhesive to the ECM components compared to parental, vector and sense clones. All transfected (vector, sense and antisense) clones and parental cells produced similar levels of uPA activity without any significant difference however, MMP-2 activity was decreased in antisense clones compared to controls. These results demonstrate that uPAR expression is critical for the invasiveness of human gliomas and down regulation of uPAR expression may be a feasible approach to decrease invasiveness.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Invasividade Neoplásica/prevenção & controle , Oligonucleotídeos Antissenso/farmacologia , Receptores de Superfície Celular/genética , Northern Blotting , Neoplasias Encefálicas/enzimologia , Adesão Celular/genética , Células Clonais , Gelatinases/metabolismo , Glioblastoma/enzimologia , Humanos , Metaloproteinase 2 da Matriz , Metaloendopeptidases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transfecção , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The interaction of urokinase-type plasminogen activator (uPA) with its cell-surface receptor (uPAR) is implicated in diverse biological processes such as cell migration, tissue remodeling, and tumor cell invasion. Recent studies indicated that uPAR can act as an extracellular matrix receptor during cell adhesion. Recently, we showed that transfection of the human glioma cell line SNB19 with antisense uPAR resulted in downregulation of uPAR at both the mRNA and protein levels. In this study, we used SNB19 to determine how the presence or absence of uPAR promotes cell spreading and associated changes in cell morphology. Microscopic analysis of cell spreading revealed that antisense uPAR-transfected cells were larger, remained round, and did not spread efficiently over extracellular matrix substrate type IV collagen and fibronectin, unlike parental SNB19 cells, which were smaller and spindle shaped. Biochemical studies showed that antisense uPAR-transfected cells, in addition to not spreading, exhibited increased expression of alpha 3 beta 1 integrin but not alpha 5 beta 1 integrin. However, we could not find a change in the expression of extracellular matrix components or altered growth rate in these cells. Furthermore, despite the increased alpha 3 beta 1 integrin expression, antisense uPAR-transfected cells failed to form an organized actin cytoskeleton when plated on type IV collagen or fibronectin, unlike parental SNB19 cells, which displayed an organized cytoskeleton. These findings show that the absence of uPAR in human glioma cells leads to morphological changes associated with decreased spreading and a disorganized cytoskeleton resulting in altered cell morphology, suggesting that coordinated expression of uPAR and integrin may be involved in spreading of antisense uPAR-transfected glioma cells.
Assuntos
Citoesqueleto/ultraestrutura , Glioblastoma/fisiopatologia , Receptores de Superfície Celular/biossíntese , Adesão Celular , Movimento Celular , DNA Antissenso , Regulação para Baixo , Proteínas da Matriz Extracelular , Glioblastoma/ultraestrutura , Humanos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transfecção , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Primary, malignant brain tumors show an extensive infiltrative invasion into surrounding normal brain. At present, little information is available regarding the local invasive behavior of human brain tumors and until now no animal model suitable to mimic human gliomas has been reported. To identify the infiltrative behavior of an established glioblastoma cell line (SNB19), we achieved a stable transfection of the SNB19 cell line with beta-galactosidase (lac-Z) plasmid. The stable beta-galactosidase-expressing cells were then injected intracerebrally into nude mice in an attempt to follow its pattern of spread. The mice were sacrificed at 3, 4, and 6 weeks postinjection. We could detect tumor formation in all of the animals, and the tumor size increased gradually over the 6 week time period. Three weeks after injection, tumor cells showed characteristic infiltrative invasion along the corpus callosum. We also observed tumor-cell invasion into the anterior commissure in some animals, and each tumor cell could be identified by lac-Z expression as visualized by its blue color. Further invasion was identified at 4 and 6 weeks postinjection. Our results suggest that this model could be used to study the molecular mechanisms involved in the invasion of gliomas so that appropriate therapeutic intervention strategies could be designed.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/secundário , Invasividade Neoplásica/patologia , beta-Galactosidase/metabolismo , Animais , Neoplasias Encefálicas/enzimologia , Feminino , Genes Reporter , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/genéticaRESUMO
The invasion of glioma into normal brain tissue is a major challenge to clinical intervention because these tumors often highly infiltrate the surrounding brain tissue. Total surgical resection of gliomas is impossible, and recurrence of tumor growth is a common phenomenon; patients have a mean survival time of 8-12 months. Although in recent years substantial progress has been made toward understanding the invasive behavior of gliomas in vitro and in vivo, the factors responsible for the extensive infiltration are still poorly documented. This review focuses on recent research concerning the invasion of gliomas, as well as the extracellular matrix components, and the proteolytic enzymes involved. A better understanding of cell-matrix interactions will help in developing therapeutic strategies to decrease the invasion of gliomas.