Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas.
BACKGROUND
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.


RESULTS
In 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
In 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.
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.
Ramarao
R
Malla
Sreelatha
S
Gopinath
Kiranmai
K
Alapati
Christopher S
CS
Gondi
Meena
M
Gujrati
Dzung H
DH
Dinh
Sanjeeva
S
Mohanam
Jasti S
JS
Rao
D017209:Apoptosis; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015536:Down-Regulation; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000502:physiology*
D019253:Proto-Oncogene Proteins c-bcl-2; D055293:Receptors, Urokinase Plasminogen Activator; D051028:bcl-2-Associated X Protein; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D002401:Cathepsin B