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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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In vivo inhibition of tumor growth.Stereotactic implantation of SNB19 and U251 (1×105) tumor cells was performed and, after one week, PBS (mock), SV or pCU was injected into the brain using an Alzet mini osmotic pump. Five animals per group were used. 30 days after implantation, the animals were sacrificed, the brains were removed and fixed, and paraffin sections were prepared. A. Hematoxylin and eosin staining of tissue sections to visualize tumor cells and to examine tumor volumes. Bar: 20 µM (*p<0.01) B. Immunohistochemical analysis of cathepsin B, uPAR, Ki67 and p27Kip1 in paraffin embedded tissue sections. Bar: 200 µM.
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pone-0011668-g006: In vivo inhibition of tumor growth.Stereotactic implantation of SNB19 and U251 (1×105) tumor cells was performed and, after one week, PBS (mock), SV or pCU was injected into the brain using an Alzet mini osmotic pump. Five animals per group were used. 30 days after implantation, the animals were sacrificed, the brains were removed and fixed, and paraffin sections were prepared. A. Hematoxylin and eosin staining of tissue sections to visualize tumor cells and to examine tumor volumes. Bar: 20 µM (*p<0.01) B. Immunohistochemical analysis of cathepsin B, uPAR, Ki67 and p27Kip1 in paraffin embedded tissue sections. Bar: 200 µM.

Mentions: The effect of RNAi-mediated inhibition of cathepsin B and uPAR on pre-established tumors was studied. H&E staining revealed a large spread of tumor growth in mock and SV-treated brain sections. Whereas, pre-established intracranial tumor growth was inhibited by 95% when treated with pCU (Fig. 6A). Immunohistochemical analysis of the tumor sections from control mice for cathepsin B and uPAR showed increased expression levels localized to the tumor region while the pCU-treated tumor sections revealed very little or no expression of the cathepsin B and uPAR. When probed for the expression of p27Kip1 and Ki67 proteins, mock and SV-treated brain sections showed very little expression of p27Kip1 and increased expression of Ki67. In contrast, pCU treated brain sections showed high expression of p27Kip1. However, pCU-treated brain sections showed very little or no expression of Ki67 as compared to the controls (Fig. 6B), indicating that cell proliferation is inhibited by these treatments through upregulation of p27Kip1. The effect of the pCU treatment on tumors induced by SNB19 and U251 cells was the same.


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)

In vivo inhibition of tumor growth.Stereotactic implantation of SNB19 and U251 (1×105) tumor cells was performed and, after one week, PBS (mock), SV or pCU was injected into the brain using an Alzet mini osmotic pump. Five animals per group were used. 30 days after implantation, the animals were sacrificed, the brains were removed and fixed, and paraffin sections were prepared. A. Hematoxylin and eosin staining of tissue sections to visualize tumor cells and to examine tumor volumes. Bar: 20 µM (*p<0.01) B. Immunohistochemical analysis of cathepsin B, uPAR, Ki67 and p27Kip1 in paraffin embedded tissue sections. Bar: 200 µM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011668-g006: In vivo inhibition of tumor growth.Stereotactic implantation of SNB19 and U251 (1×105) tumor cells was performed and, after one week, PBS (mock), SV or pCU was injected into the brain using an Alzet mini osmotic pump. Five animals per group were used. 30 days after implantation, the animals were sacrificed, the brains were removed and fixed, and paraffin sections were prepared. A. Hematoxylin and eosin staining of tissue sections to visualize tumor cells and to examine tumor volumes. Bar: 20 µM (*p<0.01) B. Immunohistochemical analysis of cathepsin B, uPAR, Ki67 and p27Kip1 in paraffin embedded tissue sections. Bar: 200 µM.
Mentions: The effect of RNAi-mediated inhibition of cathepsin B and uPAR on pre-established tumors was studied. H&E staining revealed a large spread of tumor growth in mock and SV-treated brain sections. Whereas, pre-established intracranial tumor growth was inhibited by 95% when treated with pCU (Fig. 6A). Immunohistochemical analysis of the tumor sections from control mice for cathepsin B and uPAR showed increased expression levels localized to the tumor region while the pCU-treated tumor sections revealed very little or no expression of the cathepsin B and uPAR. When probed for the expression of p27Kip1 and Ki67 proteins, mock and SV-treated brain sections showed very little expression of p27Kip1 and increased expression of Ki67. In contrast, pCU treated brain sections showed high expression of p27Kip1. However, pCU-treated brain sections showed very little or no expression of Ki67 as compared to the controls (Fig. 6B), indicating that cell proliferation is inhibited by these treatments through upregulation of p27Kip1. The effect of the pCU treatment on tumors induced by SNB19 and U251 cells was the same.

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