Co-depletion of cathepsin B and uPAR induces G0/G1 arrest in glioma via FOXO3a mediated p27 upregulation.
BACKGROUND
Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.


RESULTS
Cathepsin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27(Kip1) and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by alphaVbeta3/PI3K/AKT/FOXO pathway as observed by the decreased alphaVbeta3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27(Kip1) and FOXO3a when treated with Ly294002 (10 microM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27(Kip1) was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.


CONCLUSIONS
Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27(Kip1) accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.
Gopinath
Sreelatha
S
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.
Malla
Rama Rao
RR
Gondi
Christopher S
CS
Alapati
Kiranmai
K
Fassett
Daniel
D
Klopfenstein
Jeffrey D
JD
Dinh
Dzung H
DH
Gujrati
Meena
M
Rao
Jasti S
JS
D000818:Animals; D015153:Blotting, Western; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D056742:Cyclin D2 / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008819:Mice, Nude; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002867:Chromones; D056742:Cyclin D2; D019927:Cyclin E; D051858:Forkhead Transcription Factors; D009025:Morpholines; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D002401:Cathepsin B