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Co-depletion of cathepsin B and uPAR induces G0/G1 arrest in glioma via FOXO3a mediated p27 upregulation.

Gopinath S, Malla RR, Gondi CS, Alapati K, Fassett D, Klopfenstein JD, Dinh DH, Gujrati M, Rao JS - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.

ABSTRACT

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.

Methodology/principal findings: 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.

Conclusion/significance: 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.

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Cathepsin B and uPAR knockdown decreases Cdk2 activity and the expression of αVβ3 integrin.A. Cell lysates were collected from SNB19 and U251 after transfection with SV, pU, pC or pCU. Western blot analysis of 50 µg of total cell lysates was performed to check the expression of cyclin D1, cyclin D2, Cdk2, cyclin E, Rb, p-Rb, p21, αV, β3, αVβ3 and Ki67. GAPDH was used as a loading control. B. Total lysates from the untreated control and SV, pU, pC or pCU-transfected cells were immunoprecipitated for Cdk2 and β3 individually and then immunoblotted for cyclin E and αV, respectively. The figure also shows the expression of αVβ3 integrin on native gel.
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pone-0011668-g003: Cathepsin B and uPAR knockdown decreases Cdk2 activity and the expression of αVβ3 integrin.A. Cell lysates were collected from SNB19 and U251 after transfection with SV, pU, pC or pCU. Western blot analysis of 50 µg of total cell lysates was performed to check the expression of cyclin D1, cyclin D2, Cdk2, cyclin E, Rb, p-Rb, p21, αV, β3, αVβ3 and Ki67. GAPDH was used as a loading control. B. Total lysates from the untreated control and SV, pU, pC or pCU-transfected cells were immunoprecipitated for Cdk2 and β3 individually and then immunoblotted for cyclin E and αV, respectively. The figure also shows the expression of αVβ3 integrin on native gel.

Mentions: Cell cycle regulators at the G0/G1 and G1/S phase transition were analyzed after the above mentioned treatments. As p27Kip1 is both an inhibitor and a substrate of cyclin E-Cdk2 complex [25], we analyzed the expression of these molecules using immunoblot analysis and found that the treatments decreased the expression of cyclin E whereas the expression of Cdk2 was unaffected (Fig. 3A). Similarly, expression of αV, β3, αVβ3 integrins decreased with the treatments. Further, Cdk2 was immunoprecipitated from the cell lysates of untreated and SV, pU, pC and pCU treated SNB19 and U251 cells and immunoblotted for cyclin E. The results revealed little or no expression of cyclin E in pU, pC and pCU treated cell lysates compared to control and SV transfected cells indicating that the treatments reduced the cyclinE-Cdk2 complex formation. pCU-treated cells showed significant downregulation of cyclin E as compared to pU and pC treatments. Dimerization of αVβ3 integrin was checked by immunoprecipitating the cell lysate with β3 integrin and immunoblotted for αV integrin and found that the treatments significantly reduced the dimer formation. It was further confirmed by native gel electrophoresis by using the αVβ3 antibody (Fig. 3B).


Co-depletion of cathepsin B and uPAR induces G0/G1 arrest in glioma via FOXO3a mediated p27 upregulation.

Gopinath S, Malla RR, Gondi CS, Alapati K, Fassett D, Klopfenstein JD, Dinh DH, Gujrati M, Rao JS - PLoS ONE (2010)

Cathepsin B and uPAR knockdown decreases Cdk2 activity and the expression of αVβ3 integrin.A. Cell lysates were collected from SNB19 and U251 after transfection with SV, pU, pC or pCU. Western blot analysis of 50 µg of total cell lysates was performed to check the expression of cyclin D1, cyclin D2, Cdk2, cyclin E, Rb, p-Rb, p21, αV, β3, αVβ3 and Ki67. GAPDH was used as a loading control. B. Total lysates from the untreated control and SV, pU, pC or pCU-transfected cells were immunoprecipitated for Cdk2 and β3 individually and then immunoblotted for cyclin E and αV, respectively. The figure also shows the expression of αVβ3 integrin on native gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908539&req=5

pone-0011668-g003: Cathepsin B and uPAR knockdown decreases Cdk2 activity and the expression of αVβ3 integrin.A. Cell lysates were collected from SNB19 and U251 after transfection with SV, pU, pC or pCU. Western blot analysis of 50 µg of total cell lysates was performed to check the expression of cyclin D1, cyclin D2, Cdk2, cyclin E, Rb, p-Rb, p21, αV, β3, αVβ3 and Ki67. GAPDH was used as a loading control. B. Total lysates from the untreated control and SV, pU, pC or pCU-transfected cells were immunoprecipitated for Cdk2 and β3 individually and then immunoblotted for cyclin E and αV, respectively. The figure also shows the expression of αVβ3 integrin on native gel.
Mentions: Cell cycle regulators at the G0/G1 and G1/S phase transition were analyzed after the above mentioned treatments. As p27Kip1 is both an inhibitor and a substrate of cyclin E-Cdk2 complex [25], we analyzed the expression of these molecules using immunoblot analysis and found that the treatments decreased the expression of cyclin E whereas the expression of Cdk2 was unaffected (Fig. 3A). Similarly, expression of αV, β3, αVβ3 integrins decreased with the treatments. Further, Cdk2 was immunoprecipitated from the cell lysates of untreated and SV, pU, pC and pCU treated SNB19 and U251 cells and immunoblotted for cyclin E. The results revealed little or no expression of cyclin E in pU, pC and pCU treated cell lysates compared to control and SV transfected cells indicating that the treatments reduced the cyclinE-Cdk2 complex formation. pCU-treated cells showed significant downregulation of cyclin E as compared to pU and pC treatments. Dimerization of αVβ3 integrin was checked by immunoprecipitating the cell lysate with β3 integrin and immunoblotted for αV integrin and found that the treatments significantly reduced the dimer formation. It was further confirmed by native gel electrophoresis by using the αVβ3 antibody (Fig. 3B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.

ABSTRACT

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.

Methodology/principal findings: 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.

Conclusion/significance: 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.

Show MeSH
Related in: MedlinePlus