RESUMEN
Although intrinsic tumours of the brain seldom metastasize to distant sites, their diffuse, infiltrative-invasive growth within the brain generally precludes successful surgical and adjuvant therapy. Hence, attention has now focused on novel therapeutic approaches to combat brain tumours that include the use of anti-invasive and anti-proliferative agents. The effect of four anti-invasive agents, swainsonine (a locoweed alkaloid), captopril (an anti-hypertensive drug), tangeretin and nobiletin (both citrus flavonoids), were investigated on various parameters of brain tumour invasion such as matrix metalloproteinase (MMP) secretion, migration, invasion and adhesion. A standard cytotoxicity assay was used to optimize working concentrations of the drugs on seven human brain tumour-derived cell lines of various histological type and grade of malignancy. A qualitative assessment by gelatin zymography revealed that the effect of these agents varied between the seven cell lines such that the low grade pilocytic astrocytoma was unaffected by three of the agents. In contrast, downregulation of the two gelatinases, MMP-2 and MMP-9 was seen in the grade 3 astrocytoma irrespective of which agent was used. Generally, swainsonine was the least effective whereas the citrus flavonoids, particularly nobiletin, showed the greatest downregulation of secretion of the MMPs. Furthermore, captopril and nobiletin were most efficient at inhibiting invasion, migration and adhesion in four representative cell lines (an ependymoma, a grade II oligoastrocytoma, an anaplastic astrocytoma and a glioblastoma multiforme). Yet again, the effects of the four agents varied between the four cell lines. Nobiletin was, nevertheless, the most effective agent used in these assays. In conclusion, the differential effects seen on the various parameters studied by these putative anti-invasive agents may be the result of interference with MMPs and other mechanisms underlying the invasive phenotype. From these pilot studies, it is possible that these agents, especially the citrus flavonoids, could be of future therapeutic value. However, further work is needed to validate this in a larger study.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Captopril/farmacología , Flavonas , Flavonoides/farmacología , Swainsonina/farmacología , Astrocitoma/tratamiento farmacológico , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno/metabolismo , Evaluación Preclínica de Medicamentos , Ependimoma/tratamiento farmacológico , Ependimoma/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Concentración 50 Inhibidora , Metaloproteinasas de la Matriz/metabolismo , Invasividad Neoplásica , Células Tumorales CultivadasRESUMEN
Matrix metalloproteinases (MMPs) are an homologous family of proteolytic enzymes capable of degrading components of the extracellular matrix (ECM) and thereby facilitating the invasion of tumour cells into normal tissues. The neural cell adhesion molecules (NCAMs) of neuronal and glial cells provide a Ca2+-independent mechanism for cell-cell and cell-ECM adhesion. NCAMs are downregulated to promote cell disaggregation during cell migration in the developing nervous system whereas MMPs facilitate migration. Recent studies have shown downregulation of MMP secretion in rat glioma cells transfected with an NCAM cDNA, implying an inverse correlation between NCAM and MMP expression. The purpose of this study was to establish whether such a correlation could be demonstrated in a panel of nine human glioma cell-lines, one metastatic carcinoma and one foetal astrocyte derived cell line. The secretion of two MMPs, 72 kDa gelatinase (MMP-2 or gelatinase-A) and 92 kDa gelatinase (MMP-9 or gelatinase-B), was investigated using SDS-PAGE zymography; NCAM-A was assayed by an immunochemiluminescent assay following SDS-PAGE of whole-cell extracts. An inverse correlation was found between the expression of NCAM-A and that of both MMPs studied although the patterns of expression showed no obvious correlation with histological type or grade of the parent tumours. Our results suggest that downregulation of NCAM-A may contribute to tumour invasiveness by promoting both cell disaggregation and protease secretion.
Asunto(s)
Neoplasias Encefálicas/enzimología , Colagenasas/metabolismo , Gelatinasas/metabolismo , Glioma/enzimología , Metaloendopeptidasas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Células Tumorales CultivadasRESUMEN
Although the mechanics of brain tumour cell motility are poorly documented, studies in other cell types-notably fibroblasts--have revealed cell motility to be dependent on dynamic remodelling of the actin cytoskeleton. Initially, protrusion of membrane microspikes or lamellipodia is associated with actin polymerisation and probably involves membrane-anchored myosin I. Subsequent attachment of the cell's leading edge to the substratum is via actin-anchored adhesion complexes and finally generation of tractile forces is believed to involve the formation of contractile actin structures. Co-ordination of these events is even less well understood but recent evidence points to the involvement of the Ras family of GTP-binding proteins, particularly the cdc42-Rac-Rho cascade which appears to choreograph membrane extension and attachment. Furthermore, the growth associated protein GAP-43 (neuromodulin) has recently been demonstrated in brain tumour cells. This protein stabilises membrane protrusions during neuritogenesis in response to external trophic factors and is likely to have a similar role in brain tumour cell invasion.
Asunto(s)
Actinas/fisiología , Neoplasias Encefálicas/fisiopatología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Citoesqueleto/fisiología , Animales , Neoplasias Encefálicas/patología , Dictyostelium , Glioma/fisiopatología , HumanosRESUMEN
Matrix metalloproteinases (MMPs) are zinc-dependent peptidases and are amongst those enzymes responsible for extracellular matrix (ECM) degradation during tumour-cell migration. Gangliosides are a family of acidic membrane glycolipids thought to play a role during cell development, differentiation and oncogenic transformation. In this descriptive study, we investigated the effects of six exogenous gangliosides (GM1, GM3, GD1a, GD1b, GD3 and GT1b) on the secretion of MMP-2 (72 kDa gelatinase or gelatinase-A) and MMP-9 (92 kDa gelatinase or gelatinase-B). Cell-conditioned media from eight human glioma-derived cell-lines served as the source of MMPs and were investigated using SDS-PAGE zymography. Six of the cell lines showed upregulation of secretion of both enzymes by all six gangliosides. Of the remaining two cell lines, one showed inhibition of MMP secretion by all gangliosides and the other had a small but differential response to the range of gangliosides investigated. These results suggest that gangliosides may stimulate glioma cell invasiveness by promoting MMP expression.
Asunto(s)
Colagenasas/metabolismo , Gangliósidos/farmacología , Gelatinasas/metabolismo , Glioma/enzimología , Metaloendopeptidasas/metabolismo , Medios de Cultivo Condicionados , Electroforesis en Gel de Poliacrilamida , Humanos , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Matrix metalloproteinases (MMP) are a family of zinc-dependent enzymes which degrade various components of the extracellular matrix (ECM) and play an important role in facilitating tumour cell invasion of the normal brain. The family includes the gelatinases, stromelysins and collagenases. Preliminary studies have shown that there is a differential expression four metalloproteinases in human brain tumour cell lines derived from neoplasms of various histological types and grades of malignancy. Morphological and antigenic changes in human glioma-derived cell lines over many serial in vitro passages have been reported in earlier studies. When established cell lines are maintained in culture over a long period, it is possible that the secretion of enzymes such as metalloproteinases may differ according to the passage level examined. This report presents a study on the secretion of four matrix metalloproteinases - interstitial collagenase (MMP-), 72-kDa and 92-kDa gelatinases (MMP-2 and MMP-9 respectively), and stromelysin (MMP-3) - in three human brain tumour-derived cell lines at sequentially increasing passage numbers, ranging from passage 2 to passage 50; foetal astrocytes were used as a positive control. Reverse zymography and substrate degradation analysis were employed to demonstrate the presence of these enzymes in cell- conditioned culture medium. Aminophenyl mercuric acetate (APMA) was used to activate latent zymogen. Results demonstrate that there is no definite pattern of change in the levels of enzyme secretion common to all cell lines studied. Instead, the fluctuations in APMA- activated metalloproteinase activity in serial passage seems to vary considerably depending on the cell line and the type of enzyme studied. The variation in metalloproteinase expression observed on serial passage may be due to in vitro selection processes or karyotype evolution where the transcription of either the enzyme and/or its inhibitor may be affected. Thus an imbalance of the two products could be occurring in serial passage. Ideally, experiments requiring the measurement of relative enzyme activities should use cultures as near to the biopsy stage as possible, i.e. very low passages, to avoid artifacts that may arise on prolonged culturing.