High-grade
gliomas comprise the most malignant type of
primary brain tumor and are relatively frequent in adults. Recent studies have indicated that the loss of p16, an inhibitor of CDK4, promotes the acquisition of malignant characteristics in
gliomas. A correlation between overexpression of
urokinase-type plasminogen activator receptor (uPAR) and
glioblastoma invasion has also been established. Moreover, uPAR/
integrin binding has been shown to initiate or potentiate
integrin signaling through
focal adhesion kinase and/or
src kinases. Our previous studies demonstrated that downregulation of uPAR expression and restoration of p16 regress
glioma growth in nude mice and downregulate
alphavbeta3 integrin receptor expression. Here, we show the effect of a bicistronic construct on
alphavbeta5 integrin receptor expression, angiogenesis and the biochemical pathway that causes
glioma cell death. The U251
glioblastoma and a
glioblastoma xenograft cell line transduced with a recombinant replication-defective adenovirus vector containing the
cDNA of wild-type p16 and
antisense RNA of uPAR significantly inhibited human mammary epithelial cell capillary formation and
vascular endothelial growth factor (
VEGF) expression. Inactivation of anti-apoptotic molecules such as Akt, PARP, activation of
caspases and accumulation of heteroduplex chromosomal
DNA in pre-G1 phase of the cell cycle was demonstrated by Western blotting,
caspase activity assay and FACS analysis. Nuclear DNA fragmentation upon induction of apoptosis was scored using the TUNEL assay. Significant downregulation of
alphavbeta5 integrin receptor expression was also confirmed by FACS analysis, immunoprecipitation and RT-PCR. Taken together, the results demonstrate that the sense p16 and anti-sense uPAR bicistronic construct significantly inhibits angiogenesis, induces apoptosis by deregulation of the PI3K-Akt pathway and downregulates
alphavbeta5 integrin receptor expression.