ABSTRACT
Inactivation of the tumor suppressor gene PTEN and overexpression of VEGF are two of the most common events observed in high-grade malignant gliomas. The purpose of this study was to determine whether PTEN controls VEGF expression in gliomas under normoxic conditions. Transfer of PTEN to human glioma cells resulted in the transduction of a functional PTEN protein as evidenced by the upregulation of p27 and modification of the phosphorylation status of Akt. Under normoxic conditions, enzyme-linked immunosorbent assay and Northern blot analyses showed downregulation of VEGF in PTEN-treated cells. Moreover, conditioned media from PTEN-treated glioma cells significantly diminished the ability of endothelial cells to grow and migrate. Western blot assays demonstrated that, in a normoxic environment, PTEN downregulates HIF-1 alpha. Finally, promoter activity assays showed that the VEGF promoter region containing the HIF-1alpha binding site is necessary and sufficient for PTEN-mediated downregulation of VEGF. Experiments with PI3-K inhibitors and kinase assays suggested that PI3-K is mediating the effect of PTEN on VEGF, and not the p42/p48 or p38 MAP kinases. These results indicate that restoration of PTEN function in gliomas may induce therapeutic effect by downregulating VEGF. Furthermore, this close functional relationship between PTEN and VEGF suggests that a better understanding of the transduction signal regulated by PTEN might enhance the knowledge of the cause and physiology of vascular and inflammatory diseases.
Subject(s)
Neovascularization, Pathologic/physiopathology , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Protein Serine-Threonine Kinases , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Vascular Endothelial Growth Factor A/genetics , Cell Division , Cell Line, Tumor , Down-Regulation , Gene Expression Regulation, Neoplastic , Glioma , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Oxygen/pharmacology , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Promoter Regions, Genetic/physiology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock- or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.
Subject(s)
Adenoviridae/genetics , Brain Neoplasms/genetics , Cell Division/genetics , Glioma/genetics , Matrix Metalloproteinase 9/genetics , Neoplasm Invasiveness/genetics , Animals , Base Sequence , Brain Neoplasms/enzymology , Brain Neoplasms/pathology , DNA Primers , Glioma/enzymology , Glioma/pathology , Mice , Rats , Tumor Cells, CulturedABSTRACT
We previously showed that enhanced expression of MMP-9, an endopeptidase that digests basement-membrane type IV collagen, is related to tumor progression in vitro and in vivo; antisense-MMP-9 stably transfected clones were less invasive than untransfected parental cells and did not form tumors in nude mice. In this study, we examined the role of ERK-1 in the regulation of MMP-9 production and the invasive behavior of the human glioblastoma cell line SNB19, in which ERK1 is constitutively activated. SNB19 cells were stably transfected with mt-ERK, a vector encoding ERK-1 cDNA in which the conserved lysine at codon 71 was changed to arginine, thus impairing the catalytic efficiency of this enzyme. Gelatin zymography showed reduced levels of MMP-9 in the mt-ERK-transfected cell lines relative to those in vector-transfected and parental control cells. Reductions in MMP-9 protein mRNA levels were also detected in the mt-ERK-transfected cells by Western and Northern blotting. The mt-ERK-transfected cells were much less invasive than parental or vector control cells in a Matrigel invasion assay and in a spheroid coculture assay. Thus an ERK-dependent signaling pathway seems to regulate MMP-9 mediated glioma invasion in SNB19 cells; interfering with this pathway could be developed into a therapeutic approach, which aims at a reduction of cancer cell invasion.