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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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Related in: MedlinePlus

Effect of ionizing irradiation either alone or in combination with pUC and pU2 in human meningioma cell lines.(A, B) Conditioned medium of two meningioma cell lines, IOMM-Lee and CH-157MN, was collected after giving 5 and 10 gy doses of γ-rays irradiation. Human microvascular endothelial cells were seeded in 96-well plates and cultured with conditioned medium of meningioma cells. The cells were maintained for 48 hrs at 21% O2, washed, fixed, stained with Hema-3, and photographed. The percentages of angiogenic cells were assessed using Cell Quest software. Columns represent means ± SD of three independent experiments. *p<0.05, significant difference from pSV (C) 1×105 IOMM-Lee and CH-157MN cells were transfected with pSV, pUC and pU2 and cell lysates were subjected to SDS-PAGE, and immunoblot analysis was carried out to detect uPAR level. Similarly, tissue lysates isolated from nude mice were also subjected for western blotting to detect uPAR expression. (D) Human microvascular endothelial cells were seeded in 48-well plates and cultured with conditioned medium collected from pSV, pUC and pU2 transfected IOMM-Lee and CH-157MN meningioma cells. The cells were kept in a humidified atmosphere containing 5% CO2 at 37°C for 48 h, washed, fixed and stained with Hema-3 and photographed. (E) Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pSV and pSV+IR, respectively.
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pone-0017123-g001: Effect of ionizing irradiation either alone or in combination with pUC and pU2 in human meningioma cell lines.(A, B) Conditioned medium of two meningioma cell lines, IOMM-Lee and CH-157MN, was collected after giving 5 and 10 gy doses of γ-rays irradiation. Human microvascular endothelial cells were seeded in 96-well plates and cultured with conditioned medium of meningioma cells. The cells were maintained for 48 hrs at 21% O2, washed, fixed, stained with Hema-3, and photographed. The percentages of angiogenic cells were assessed using Cell Quest software. Columns represent means ± SD of three independent experiments. *p<0.05, significant difference from pSV (C) 1×105 IOMM-Lee and CH-157MN cells were transfected with pSV, pUC and pU2 and cell lysates were subjected to SDS-PAGE, and immunoblot analysis was carried out to detect uPAR level. Similarly, tissue lysates isolated from nude mice were also subjected for western blotting to detect uPAR expression. (D) Human microvascular endothelial cells were seeded in 48-well plates and cultured with conditioned medium collected from pSV, pUC and pU2 transfected IOMM-Lee and CH-157MN meningioma cells. The cells were kept in a humidified atmosphere containing 5% CO2 at 37°C for 48 h, washed, fixed and stained with Hema-3 and photographed. (E) Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pSV and pSV+IR, respectively.

Mentions: To assess the effect of ionizing radiation on angiogenesis, both meningioma cell lines were irradiated at a dose of 5 and 10gy, and in vitro capillary-like structure formation assay was performed with human microvascular endothelial cells to examine the importance of tumor-conditioned medium in the angiogenic process. We observed that 5gy of irradiation induced angiogenesis in IOMM-Lee when compared with control (1.5 fold; p<0.05) and 10gy irradiation (1.3 fold) made them radio resistant; however, reduced angiogenesis was shown by the CH-157MN cells conditioned medium at both the 5gy (30%) and 10gy (40%) dose in human microvascular endothelial cells (Fig. 1A, B). We wanted to observe the effect of treatment on radioresistant and radiosensitive cells and elected to conduct further experiments with a radiation dose of 5gy for IOMM-Lee and 10 gy for the CH-157MN cell line. To further test the role of uPA, uPAR and cathepsin B we silenced these genes by si-RNA-mediated pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) constructs. We found a significant decrease of uPAR in IOMM-Lee, CH-157MN and in vivo samples when transfected with pUC and pU2 either alone or in combination with irradiation (Fig. 1C). To determine the effect of pUC/pU2 bicistronic constructs on angiogenesis of conditioned medium from meningioma cells, we performed vessel formation assay in human microvascular endothelial cells (Fig. 1D). Results showed that si-RNA-mediated bicistronic constructs of uPAR and cathepsin B (pUC) efficiently reduced angiogenesis compared to pU2 (IOMM-Lee: 37.5%; CH-157MN: 50%) in both cells. We further studied the combined effect of these bicistronic constructs and irradiation on the angiogenesis of cells, and found that combined treatment of pUC+IR reduced vessel formation in both cell lines when compared with pSV+IR (IOMM Lee: 75%; CH-157MN: 85%; p<0.05), and pU2+IR (IOMM Lee: 33%; CH-157MN: 55%) (Fig. 1E). Results are expressed as mean ± SD of three independent experiments. These results also show that angiogenesis enhanced in endothelial cells by irradiation in conditioned medium of IOMM-Lee cells was again being suppressed by pUC+IR and pU2+IR; however pUC+IR was found to be relatively more pronounced.


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)

Effect of ionizing irradiation either alone or in combination with pUC and pU2 in human meningioma cell lines.(A, B) Conditioned medium of two meningioma cell lines, IOMM-Lee and CH-157MN, was collected after giving 5 and 10 gy doses of γ-rays irradiation. Human microvascular endothelial cells were seeded in 96-well plates and cultured with conditioned medium of meningioma cells. The cells were maintained for 48 hrs at 21% O2, washed, fixed, stained with Hema-3, and photographed. The percentages of angiogenic cells were assessed using Cell Quest software. Columns represent means ± SD of three independent experiments. *p<0.05, significant difference from pSV (C) 1×105 IOMM-Lee and CH-157MN cells were transfected with pSV, pUC and pU2 and cell lysates were subjected to SDS-PAGE, and immunoblot analysis was carried out to detect uPAR level. Similarly, tissue lysates isolated from nude mice were also subjected for western blotting to detect uPAR expression. (D) Human microvascular endothelial cells were seeded in 48-well plates and cultured with conditioned medium collected from pSV, pUC and pU2 transfected IOMM-Lee and CH-157MN meningioma cells. The cells were kept in a humidified atmosphere containing 5% CO2 at 37°C for 48 h, washed, fixed and stained with Hema-3 and photographed. (E) Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pSV and pSV+IR, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017123-g001: Effect of ionizing irradiation either alone or in combination with pUC and pU2 in human meningioma cell lines.(A, B) Conditioned medium of two meningioma cell lines, IOMM-Lee and CH-157MN, was collected after giving 5 and 10 gy doses of γ-rays irradiation. Human microvascular endothelial cells were seeded in 96-well plates and cultured with conditioned medium of meningioma cells. The cells were maintained for 48 hrs at 21% O2, washed, fixed, stained with Hema-3, and photographed. The percentages of angiogenic cells were assessed using Cell Quest software. Columns represent means ± SD of three independent experiments. *p<0.05, significant difference from pSV (C) 1×105 IOMM-Lee and CH-157MN cells were transfected with pSV, pUC and pU2 and cell lysates were subjected to SDS-PAGE, and immunoblot analysis was carried out to detect uPAR level. Similarly, tissue lysates isolated from nude mice were also subjected for western blotting to detect uPAR expression. (D) Human microvascular endothelial cells were seeded in 48-well plates and cultured with conditioned medium collected from pSV, pUC and pU2 transfected IOMM-Lee and CH-157MN meningioma cells. The cells were kept in a humidified atmosphere containing 5% CO2 at 37°C for 48 h, washed, fixed and stained with Hema-3 and photographed. (E) Columns represent means ± SD of three independent experiments. * and # denotes p<0.05, significant difference from pSV and pSV+IR, respectively.
Mentions: To assess the effect of ionizing radiation on angiogenesis, both meningioma cell lines were irradiated at a dose of 5 and 10gy, and in vitro capillary-like structure formation assay was performed with human microvascular endothelial cells to examine the importance of tumor-conditioned medium in the angiogenic process. We observed that 5gy of irradiation induced angiogenesis in IOMM-Lee when compared with control (1.5 fold; p<0.05) and 10gy irradiation (1.3 fold) made them radio resistant; however, reduced angiogenesis was shown by the CH-157MN cells conditioned medium at both the 5gy (30%) and 10gy (40%) dose in human microvascular endothelial cells (Fig. 1A, B). We wanted to observe the effect of treatment on radioresistant and radiosensitive cells and elected to conduct further experiments with a radiation dose of 5gy for IOMM-Lee and 10 gy for the CH-157MN cell line. To further test the role of uPA, uPAR and cathepsin B we silenced these genes by si-RNA-mediated pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) constructs. We found a significant decrease of uPAR in IOMM-Lee, CH-157MN and in vivo samples when transfected with pUC and pU2 either alone or in combination with irradiation (Fig. 1C). To determine the effect of pUC/pU2 bicistronic constructs on angiogenesis of conditioned medium from meningioma cells, we performed vessel formation assay in human microvascular endothelial cells (Fig. 1D). Results showed that si-RNA-mediated bicistronic constructs of uPAR and cathepsin B (pUC) efficiently reduced angiogenesis compared to pU2 (IOMM-Lee: 37.5%; CH-157MN: 50%) in both cells. We further studied the combined effect of these bicistronic constructs and irradiation on the angiogenesis of cells, and found that combined treatment of pUC+IR reduced vessel formation in both cell lines when compared with pSV+IR (IOMM Lee: 75%; CH-157MN: 85%; p<0.05), and pU2+IR (IOMM Lee: 33%; CH-157MN: 55%) (Fig. 1E). Results are expressed as mean ± SD of three independent experiments. These results also show that angiogenesis enhanced in endothelial cells by irradiation in conditioned medium of IOMM-Lee cells was again being suppressed by pUC+IR and pU2+IR; however pUC+IR was found to be relatively more pronounced.

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