Down-regulation of integrin alpha(v)beta(3) expression and integrin-mediated signaling in glioma cells by adenovirus-mediated transfer of antisense urokinase-type plasminogen activator receptor (uPAR) and sense p16 genes.

Interaction between the extracellular matrix and integrin receptors on cell surfaces leads not only to cell adhesion but also to intracellular signaling events that affect cell migration, proliferation, and survival. The vitronectin receptor alpha(v)beta(3) integrin is of key importance in glioma cell biology. The expression of urokinase-type plasminogen activator receptor (uPAR) was recently shown to co-regulate with the expression of alpha(v)beta(3) integrin. Moreover, restoration of the p16 protein in glioma cells inhibits the alpha(v)beta(3) integrin-mediated spreading of those cells on vitronectin. Thus we hypothesized that adenovirus-mediated down-regulation of uPAR and overexpression of p16 might down-regulate the expression of alpha(v)beta(3) integrin and the integrin-mediated signaling in glioma cells, thereby defeating the malignant phenotype. In this study, we used replication-deficient adenovirus vectors that contain either a uPAR antisense expression cassette (Ad-uPAR) or wild-type p16 cDNA (Ad-p16) and a bicistronic adenovirus construct in which both the uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16) are inserted in the E1-deleted region of the vector. Infecting the malignant glioma cell line SNB19 with Ad-uPAR, Ad-p16, or Ad-uPAR/p16 in the presence of vitronectin resulted in decreased alpha(v)beta(3) integrin expression and integrin-mediated biological effects, including adhesion, migration, proliferation, and survival Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

Elevated levels of cathepsin B in human glioblastoma cell lines.

Degradation of the extracellular matrix is a prerequisite for the invasive phenotype in glioma cells. Several proteases released by invading tumor cells seem to participate in the focal degradation of extracellular matrix proteins. Using enzymatic assays, Western blotting, and Northern blotting techniques, we investigated whether cathepsin B level was associated with malignant grade in seven human glioma cell lines. Cathepsin B activity and protein content levels were higher in glioblastoma cell lines than in anaplastic astrocytoma or low-grade glioma cell lines. Cathepsin B transcripts were overexpressed in glioblastoma cell lines relative to their expression in anaplastic astrocytoma and low-grade glioma cell lines. Cathepsin B promoter activity and amount of SP-1 complexes were much higher in glioblastoma cell lines than in anaplastic astrocytoma or low-grade glioma cell lines. Finally, E-64, an inhibitor of cathepsin B, inhibited both cathepsin B enzymatic activity and the invasiveness of glioblastoma cell lines. These results strongly support a role for cathepsin B in glioblastoma cell lines and suggest that inhibition of cathepsin B activity may be proven useful in cancer therapy.

A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas.

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa serine protease inhibitor found at high levels in extracellular matrix. Recombinant human TFPI-2 has recently been shown to be a strong inhibitor of trypsin, plasmin, plasma kallikrein, and factor XIa amidolytic activity. Earlier studies in our laboratory showed that the expression of TFPI-2 is lost during tumor progression in human gliomas. We stably transfected this protease inhibitor in multiform glioblastoma cell line (SNB-19) and in low-grade glioma cell line (Hs683) in sense and antisense orientation respectively. This confirmed that the upregulation/down-regulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas both in vitro and in vivo models. Collectively, these results suggested an idea to determine whether TFPI-2 is necessary for cell survival and inhibition of tumor formation in nude mice, due to apoptosis of intracerebrally injected SNB-19 cells. In the present study we determined p-ERK levels and found that they are decreased in TFPI-2 over-expressed clones (SNB-19) and increased in TFPI-2 down-regulated clones (Hs683). We also checked the levels of BAX/BCl-2, caspases (for e.g., 9, 7, 3, 8), PARP, cytochrome-c and Apaf-1. Moreover, the increase of apoptosis in vitro is associated with increased and decreased expression of apoptotic protein BAX in sense clones (SNB-19) and antisense clones (Hs683) respectively, when compared to controls and vice versa with Bcl-2 the anti-apoptotic protein. Caspases (9, 7 and 3), cytochrome-c, Apaf-1 and PARP levels are increased in SNB-19 and decreased in Hs683. Caspase 8 was not expressed in either cell line. Caspases 9 and 3 activity assay revealed higher activity in sense clones (SNB-19) but lesser in antisense clones (Hs683) compared to controls. This is the first report of TFPI-2 playing a novel role in cell survival in human gliomas.

Downregulation of urokinase-type plasminogen activator receptor (uPAR) induces caspase-mediated cell death in human glioblastoma cells.

Urokinase-type plasminogen activator receptors (uPARs) play an important role in tumor invasion by localizing degradative enzymes at the invasive zone. Our previous studies with human glioblastoma cell line SNB19 expressing AS-uPAR stable tranfectant lose their invasive properties when injected in vivo. The aim of the present study is to investigate whether the inhibition of tumor formation is due to apoptosis. Apoptosis is a highly conserved cell suicide program essential for development and tissue homeostasis of all metazoan organisms. Key to the apoptotic program is a family of cystein proteases termed caspases, which are important for execution of apoptosis by cleavage of essential cellular proteins. We found loss of mitochondrial transmembrane potential, release of cytochrome C from mitochondria and subsequent activation of Caspase-9 in SNB 19 AS-uPAR cells compared to parental and vector controls. Our results indicate that suppression of uPAR results in apoptosis and suggest that Caspase-9 dependent apoptosis plays an important role in SNB19 AS-uPAR-induced apoptosis.