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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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Depletion of cathepsin B and uPAR increases p27Kip1 nuclear localization.A. Expression of p27Kip1 and p-p27 (Ser10 and Thr187) were studied using immunoblot analysis. GAPDH was used as loading control. B. Total RNA isolated from untreated and treated SNB19 and U251 cells was subjected to semi-quantitative RT-PCR analysis using p27Kip1 primers. Data represents average of triplicates normalized to GAPDH (**p<0.01). C. 36 hrs after transfection with SV, pU, pC and pCU, cells were fixed, immunostained with anti-p27 antibody followed by Texas Red-conjugated anti-mouse secondary antibody. DAPI was used for nuclear staining. Representative images of three independent experiments are shown. D. SNB19 and U251 cells were transfected with siRNA against p27 (p27si) individually and in combination with pU, pC and pCU. The cells were also transfected with control siRNA (C-si) and SV. Thirty six hours post-transfection, cells were lysed and the total lysates were immunoblotted for p27Kip1, p-p27 (Ser10), and p-p27 (Thr187). E. Effect of the above stated treatments on proliferation was assessed using BrdU incorporation assay. The graph represents the percent of proliferating cells and the data represented are the average of three separate experiments (*p<0.05, **p<0.01, in comparison with the control).
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pone-0011668-g002: Depletion of cathepsin B and uPAR increases p27Kip1 nuclear localization.A. Expression of p27Kip1 and p-p27 (Ser10 and Thr187) were studied using immunoblot analysis. GAPDH was used as loading control. B. Total RNA isolated from untreated and treated SNB19 and U251 cells was subjected to semi-quantitative RT-PCR analysis using p27Kip1 primers. Data represents average of triplicates normalized to GAPDH (**p<0.01). C. 36 hrs after transfection with SV, pU, pC and pCU, cells were fixed, immunostained with anti-p27 antibody followed by Texas Red-conjugated anti-mouse secondary antibody. DAPI was used for nuclear staining. Representative images of three independent experiments are shown. D. SNB19 and U251 cells were transfected with siRNA against p27 (p27si) individually and in combination with pU, pC and pCU. The cells were also transfected with control siRNA (C-si) and SV. Thirty six hours post-transfection, cells were lysed and the total lysates were immunoblotted for p27Kip1, p-p27 (Ser10), and p-p27 (Thr187). E. Effect of the above stated treatments on proliferation was assessed using BrdU incorporation assay. The graph represents the percent of proliferating cells and the data represented are the average of three separate experiments (*p<0.05, **p<0.01, in comparison with the control).

Mentions: It is well known that p27Kip1 plays an important role in G0/G1 arrest. Hence, we checked the expression of p27Kip1 using RT-PCR and western blot analysis. RT-PCR and immunoblot analysis of pC- and pU-treated cell lysates showed increased expression of p27Kip1. The pCU-treated cells showed a further increase in p27Kip1 expression in both the cell lines. Untreated control and SV-treated cells showed very low expression of p27Kip1 (Fig. 2A &B). The p27Kip1 protein is generally phosphorylated at Ser10 and Thr187 positions and its activity depends on its phosphorylation status. Therefore, we checked the phosphorylation status of p27Kip1 by immunoblot analysis and found that the treatments reduced the phosphorylation of p27Kip1 at Ser10 and Thr187 in both the cells lines compared to the controls. Immunoflourescence staining assay revealed that the treatments induced an increase in p27Kip1 localization in the nucleus when compared with control cells (Fig. 2C) and a higher number of cells expressing p27Kip1 in the nuclei was observed with the pCU treatment (Fig. S2A). These results were further confirmed by immunoblot analysis for p27Kip1 protein in cytosolic and nuclear fractions (Fig. S2B).


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)

Depletion of cathepsin B and uPAR increases p27Kip1 nuclear localization.A. Expression of p27Kip1 and p-p27 (Ser10 and Thr187) were studied using immunoblot analysis. GAPDH was used as loading control. B. Total RNA isolated from untreated and treated SNB19 and U251 cells was subjected to semi-quantitative RT-PCR analysis using p27Kip1 primers. Data represents average of triplicates normalized to GAPDH (**p<0.01). C. 36 hrs after transfection with SV, pU, pC and pCU, cells were fixed, immunostained with anti-p27 antibody followed by Texas Red-conjugated anti-mouse secondary antibody. DAPI was used for nuclear staining. Representative images of three independent experiments are shown. D. SNB19 and U251 cells were transfected with siRNA against p27 (p27si) individually and in combination with pU, pC and pCU. The cells were also transfected with control siRNA (C-si) and SV. Thirty six hours post-transfection, cells were lysed and the total lysates were immunoblotted for p27Kip1, p-p27 (Ser10), and p-p27 (Thr187). E. Effect of the above stated treatments on proliferation was assessed using BrdU incorporation assay. The graph represents the percent of proliferating cells and the data represented are the average of three separate experiments (*p<0.05, **p<0.01, in comparison with the control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908539&req=5

pone-0011668-g002: Depletion of cathepsin B and uPAR increases p27Kip1 nuclear localization.A. Expression of p27Kip1 and p-p27 (Ser10 and Thr187) were studied using immunoblot analysis. GAPDH was used as loading control. B. Total RNA isolated from untreated and treated SNB19 and U251 cells was subjected to semi-quantitative RT-PCR analysis using p27Kip1 primers. Data represents average of triplicates normalized to GAPDH (**p<0.01). C. 36 hrs after transfection with SV, pU, pC and pCU, cells were fixed, immunostained with anti-p27 antibody followed by Texas Red-conjugated anti-mouse secondary antibody. DAPI was used for nuclear staining. Representative images of three independent experiments are shown. D. SNB19 and U251 cells were transfected with siRNA against p27 (p27si) individually and in combination with pU, pC and pCU. The cells were also transfected with control siRNA (C-si) and SV. Thirty six hours post-transfection, cells were lysed and the total lysates were immunoblotted for p27Kip1, p-p27 (Ser10), and p-p27 (Thr187). E. Effect of the above stated treatments on proliferation was assessed using BrdU incorporation assay. The graph represents the percent of proliferating cells and the data represented are the average of three separate experiments (*p<0.05, **p<0.01, in comparison with the control).
Mentions: It is well known that p27Kip1 plays an important role in G0/G1 arrest. Hence, we checked the expression of p27Kip1 using RT-PCR and western blot analysis. RT-PCR and immunoblot analysis of pC- and pU-treated cell lysates showed increased expression of p27Kip1. The pCU-treated cells showed a further increase in p27Kip1 expression in both the cell lines. Untreated control and SV-treated cells showed very low expression of p27Kip1 (Fig. 2A &B). The p27Kip1 protein is generally phosphorylated at Ser10 and Thr187 positions and its activity depends on its phosphorylation status. Therefore, we checked the phosphorylation status of p27Kip1 by immunoblot analysis and found that the treatments reduced the phosphorylation of p27Kip1 at Ser10 and Thr187 in both the cells lines compared to the controls. Immunoflourescence staining assay revealed that the treatments induced an increase in p27Kip1 localization in the nucleus when compared with control cells (Fig. 2C) and a higher number of cells expressing p27Kip1 in the nuclei was observed with the pCU treatment (Fig. S2A). These results were further confirmed by immunoblot analysis for p27Kip1 protein in cytosolic and nuclear fractions (Fig. S2B).

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