Abstract
BackgroundCathepsin 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.Methodology/Principal FindingsCathespin 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 p27Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27Kip1 and FOXO3a when treated with Ly294002 (10 µM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27Kip1 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.Conclusion/SignificanceOur study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27Kip1 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.
Highlights
Malignant glioma, a common tumor among the intracranial tumors, remains formidable despite aggressive surgery, radiotherapy and chemotherapy [1]
Conclusion/Significance: Our study indicates that cathepsin B and urokinase plasminogen activator receptor (uPAR) knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27Kip1 accompanied by the binding of FOXO3a to its promoter
Knockdown of cathepsin B and uPAR decreases cell proliferation and induces G0/G1 arrest To gain insight into the molecular roles of cathepsin B and uPAR, we knocked down the expression of these molecules using shRNA in SNB19 and U251 glioma cells and analyzed the effects on cell proliferation and cell cycle
Summary
A common tumor among the intracranial tumors, remains formidable despite aggressive surgery, radiotherapy and chemotherapy [1]. Increased susceptibility to cancer and multi-organ hyperplasia have been reported in p27Kip1-null mice [9] It plays a crucial role in the control of cell proliferation by inhibiting the activities of complexes of G1 cyclins and Cdks and, as such, is an important candidate for therapeutic tumor suppression [10]. 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
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