Abstract
BackgroundGlioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.Methodology/Principal FindingsIn the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.Conclusions/SignificanceIn conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.
Highlights
Apoptosis is a tightly regulated form of programmed cell death involving a series of biochemical events that leads to a variety of morphological changes including membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation [1,2]
To gain insight into the molecular roles of uPAR and cathepsin B, we knocked down the expression of these molecules using siRNA in U251 and 5310 glioma cells and analyzed the effect on apoptosis
Discussion uPAR and cathepsin B are overexpresssed in high-grade glioma, and this overexpression strongly correlates with invasive cancer phenotype and poor prognosis [26,27,28,29]
Summary
Apoptosis is a tightly regulated form of programmed cell death involving a series of biochemical events that leads to a variety of morphological changes including membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation [1,2]. The key events of mitochondrial apoptosis include the release of cytochrome C, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and anti-apoptotic Bcl-2 family proteins [6]. Bcl-2 and Bcl-xL are anti-apoptotic members predominantly localized in mitochondria that regulate mitochondrial membrane integrity and cytochrome C release. Proapoptotic members, such as Bax (Bcl-2–associated X protein) and BAD (Bcl-2-associated death promoter), mainly reside in the cytoplasm and redistribute into mitochondria in response to death stimuli [6,9,10,11]. Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes
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