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uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma.

Gupta R, Nalla AK, Gogineni VR, Chetty C, Bhoopathi P, Klopfenstein JD, Tsung AJ, Mohanam S, Rao JS - PLoS ONE (2011)

Bottom Line: This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF).We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment.In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation.

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: Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression.

Methodology/principal findings: In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model.

Conclusion/significance: These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.

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Over expression of uPAR and cathepsin B rescues vascular phenotype in meningiomas both in vitro and in vivo.(A) Nonirradiated and irradiated control and treated tissue lysates collected after co- transfection with FL-uPAR/FL-cathepsin B (FLpU/C) were subjected to SDS-PAGE. Immunoblot analysis was carried out to detect uPAR and cathepsin level. (B) Band intensities were quantified by densitometry. Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pUC and pUC+IR, respectively. (C) Vessel formation assay was performed in FL-uPAR/FL-cathepsin B (FLpU/C) transfected IOMM-Lee and CH-157MN cells and in nude mice.
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pone-0017123-g004: Over expression of uPAR and cathepsin B rescues vascular phenotype in meningiomas both in vitro and in vivo.(A) Nonirradiated and irradiated control and treated tissue lysates collected after co- transfection with FL-uPAR/FL-cathepsin B (FLpU/C) were subjected to SDS-PAGE. Immunoblot analysis was carried out to detect uPAR and cathepsin level. (B) Band intensities were quantified by densitometry. Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pUC and pUC+IR, respectively. (C) Vessel formation assay was performed in FL-uPAR/FL-cathepsin B (FLpU/C) transfected IOMM-Lee and CH-157MN cells and in nude mice.

Mentions: To prove that the inhibition of uPAR and cathepsin B leads to reduced vessel formation, uPAR and cathepsin B were over-expressed in pUC-transfected cells via co-transfection with FL-uPAR and FL-cathepsin B. We found rescued uPAR and cathepsin B (Fig. 4A) expression in nonirradiated pUC-transfected cells (IOMM-Lee: uPAR 1.8 fold, cathepsin B 1.78 fold; CH-157MN: uPAR 4.5 fold, cathepsin B 4.75 fold; nude mice: uPAR 2.25 fold, cathepsin B 1.78 fold), and irradiated pUC-transfected cells (IOMM-Lee: uPAR 3 fold, cathepsin B 3.2 fold; CH-157MN: uPAR 2 fold, cathepsin B 2.6 fold; nude mice: uPAR 4.8 fold, cathepsin B 1.65 fold) after co-transfection with FL-uPAR and FL-cathepsin B (Fig. 4B). Both in vivo and in vitro findings suggest that co-transfection with FL-uPAR and FL-cathepsin B rescues the vessel formation in pUC-transfected cells (Fig. 4C).


uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma.

Gupta R, Nalla AK, Gogineni VR, Chetty C, Bhoopathi P, Klopfenstein JD, Tsung AJ, Mohanam S, Rao JS - PLoS ONE (2011)

Over expression of uPAR and cathepsin B rescues vascular phenotype in meningiomas both in vitro and in vivo.(A) Nonirradiated and irradiated control and treated tissue lysates collected after co- transfection with FL-uPAR/FL-cathepsin B (FLpU/C) were subjected to SDS-PAGE. Immunoblot analysis was carried out to detect uPAR and cathepsin level. (B) Band intensities were quantified by densitometry. Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pUC and pUC+IR, respectively. (C) Vessel formation assay was performed in FL-uPAR/FL-cathepsin B (FLpU/C) transfected IOMM-Lee and CH-157MN cells and in nude mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037969&req=5

pone-0017123-g004: Over expression of uPAR and cathepsin B rescues vascular phenotype in meningiomas both in vitro and in vivo.(A) Nonirradiated and irradiated control and treated tissue lysates collected after co- transfection with FL-uPAR/FL-cathepsin B (FLpU/C) were subjected to SDS-PAGE. Immunoblot analysis was carried out to detect uPAR and cathepsin level. (B) Band intensities were quantified by densitometry. Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pUC and pUC+IR, respectively. (C) Vessel formation assay was performed in FL-uPAR/FL-cathepsin B (FLpU/C) transfected IOMM-Lee and CH-157MN cells and in nude mice.
Mentions: To prove that the inhibition of uPAR and cathepsin B leads to reduced vessel formation, uPAR and cathepsin B were over-expressed in pUC-transfected cells via co-transfection with FL-uPAR and FL-cathepsin B. We found rescued uPAR and cathepsin B (Fig. 4A) expression in nonirradiated pUC-transfected cells (IOMM-Lee: uPAR 1.8 fold, cathepsin B 1.78 fold; CH-157MN: uPAR 4.5 fold, cathepsin B 4.75 fold; nude mice: uPAR 2.25 fold, cathepsin B 1.78 fold), and irradiated pUC-transfected cells (IOMM-Lee: uPAR 3 fold, cathepsin B 3.2 fold; CH-157MN: uPAR 2 fold, cathepsin B 2.6 fold; nude mice: uPAR 4.8 fold, cathepsin B 1.65 fold) after co-transfection with FL-uPAR and FL-cathepsin B (Fig. 4B). Both in vivo and in vitro findings suggest that co-transfection with FL-uPAR and FL-cathepsin B rescues the vessel formation in pUC-transfected cells (Fig. 4C).

Bottom Line: This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF).We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment.In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation.

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: Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression.

Methodology/principal findings: In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model.

Conclusion/significance: These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.

Show MeSH
Related in: MedlinePlus