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Activation of hypoxia signaling induces phenotypic transformation of glioma cells: implications for bevacizumab antiangiogenic therapy.

Xu H, Rahimpour S, Nesvick CL, Zhang X, Ma J, Zhang M, Zhang G, Wang L, Yang C, Hong CS, Germanwala AV, Elder JB, Ray-Chaudhury A, Yao Y, Gilbert MR, Lonser RR, Heiss JD, Brady RO, Mao Y, Qin J, Zhuang Z - Oncotarget (2015)

Bottom Line: Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro.These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells.These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.

ABSTRACT
Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

No MeSH data available.


Related in: MedlinePlus

Morphologic changes and expression of EMT inducers and markers in glioma cell lines in response to hypoxia(a) Phase-contrast microscopy of U87, C6 and U251 glioma cell lines in culture under varying oxygen concentrations and treatment with a pharmacological inhibitor of HIF1α or HIF2α as indicated. (b) Immunofluorescent staining for the mesenchymal marker vimentin in each of the cultures from (a). Vimentin expression is upregulated in a time- and oxygen concentration-dependent manner and is significantly reduced by cell treatment with an inhibitor of HIF1α or HIF2α. (c) Quantitative real-time PCR of multiple EMT inducers, MMP2 and MMP9 extracted from U87 glioblastoma cells under various oxygen concentrations for 24 or 48 hours. Hypoxia consistently increased expression of EMT-associated genes, and this effect was blocked by treatment with HIFα inhibitors. The y-axis reflects relative mRNA expression (standard deviation [S.D.]). (d) Immunoblot of Twist, MMP2 and MMP9 extracted from U87 cells under the same conditions as (c). Hypoxia increased expression of each marker at 48 but not 24 hours, and the increase at 48 hours was significantly reduced by treatment with a HIF inhibitor. β-actin was used as a loading control.
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Figure 4: Morphologic changes and expression of EMT inducers and markers in glioma cell lines in response to hypoxia(a) Phase-contrast microscopy of U87, C6 and U251 glioma cell lines in culture under varying oxygen concentrations and treatment with a pharmacological inhibitor of HIF1α or HIF2α as indicated. (b) Immunofluorescent staining for the mesenchymal marker vimentin in each of the cultures from (a). Vimentin expression is upregulated in a time- and oxygen concentration-dependent manner and is significantly reduced by cell treatment with an inhibitor of HIF1α or HIF2α. (c) Quantitative real-time PCR of multiple EMT inducers, MMP2 and MMP9 extracted from U87 glioblastoma cells under various oxygen concentrations for 24 or 48 hours. Hypoxia consistently increased expression of EMT-associated genes, and this effect was blocked by treatment with HIFα inhibitors. The y-axis reflects relative mRNA expression (standard deviation [S.D.]). (d) Immunoblot of Twist, MMP2 and MMP9 extracted from U87 cells under the same conditions as (c). Hypoxia increased expression of each marker at 48 but not 24 hours, and the increase at 48 hours was significantly reduced by treatment with a HIF inhibitor. β-actin was used as a loading control.

Mentions: To test the hypothesis that hypoxia induces mesenchymal change in glioblastoma, we evaluated the expression of the mesenchymal marker vimentin using immunofluorescence under various oxygen concentrations at 24 or 48 hours (Fig. 4a and 4b). Low levels of vimentin were detected under normoxic conditions in each of the cell lines evaluated, indicating some degree of mesenchymal change at baseline. At 24 hours, there was no appreciable change in vimentin expression under 1% oxygen concentration, and a slight increase was detected under 0.2% oxygen in U251 cells only. However, hypoxia consistently upregulated vimentin at 48 hours, indicating an oxygen concentration- and time-dependent acceleration of mesenchymal change under hypoxic conditions. This effect was reduced by treatment with a HIF inhibitor. This effect did not appear to differ between HIF1α and HIF2α.


Activation of hypoxia signaling induces phenotypic transformation of glioma cells: implications for bevacizumab antiangiogenic therapy.

Xu H, Rahimpour S, Nesvick CL, Zhang X, Ma J, Zhang M, Zhang G, Wang L, Yang C, Hong CS, Germanwala AV, Elder JB, Ray-Chaudhury A, Yao Y, Gilbert MR, Lonser RR, Heiss JD, Brady RO, Mao Y, Qin J, Zhuang Z - Oncotarget (2015)

Morphologic changes and expression of EMT inducers and markers in glioma cell lines in response to hypoxia(a) Phase-contrast microscopy of U87, C6 and U251 glioma cell lines in culture under varying oxygen concentrations and treatment with a pharmacological inhibitor of HIF1α or HIF2α as indicated. (b) Immunofluorescent staining for the mesenchymal marker vimentin in each of the cultures from (a). Vimentin expression is upregulated in a time- and oxygen concentration-dependent manner and is significantly reduced by cell treatment with an inhibitor of HIF1α or HIF2α. (c) Quantitative real-time PCR of multiple EMT inducers, MMP2 and MMP9 extracted from U87 glioblastoma cells under various oxygen concentrations for 24 or 48 hours. Hypoxia consistently increased expression of EMT-associated genes, and this effect was blocked by treatment with HIFα inhibitors. The y-axis reflects relative mRNA expression (standard deviation [S.D.]). (d) Immunoblot of Twist, MMP2 and MMP9 extracted from U87 cells under the same conditions as (c). Hypoxia increased expression of each marker at 48 but not 24 hours, and the increase at 48 hours was significantly reduced by treatment with a HIF inhibitor. β-actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494911&req=5

Figure 4: Morphologic changes and expression of EMT inducers and markers in glioma cell lines in response to hypoxia(a) Phase-contrast microscopy of U87, C6 and U251 glioma cell lines in culture under varying oxygen concentrations and treatment with a pharmacological inhibitor of HIF1α or HIF2α as indicated. (b) Immunofluorescent staining for the mesenchymal marker vimentin in each of the cultures from (a). Vimentin expression is upregulated in a time- and oxygen concentration-dependent manner and is significantly reduced by cell treatment with an inhibitor of HIF1α or HIF2α. (c) Quantitative real-time PCR of multiple EMT inducers, MMP2 and MMP9 extracted from U87 glioblastoma cells under various oxygen concentrations for 24 or 48 hours. Hypoxia consistently increased expression of EMT-associated genes, and this effect was blocked by treatment with HIFα inhibitors. The y-axis reflects relative mRNA expression (standard deviation [S.D.]). (d) Immunoblot of Twist, MMP2 and MMP9 extracted from U87 cells under the same conditions as (c). Hypoxia increased expression of each marker at 48 but not 24 hours, and the increase at 48 hours was significantly reduced by treatment with a HIF inhibitor. β-actin was used as a loading control.
Mentions: To test the hypothesis that hypoxia induces mesenchymal change in glioblastoma, we evaluated the expression of the mesenchymal marker vimentin using immunofluorescence under various oxygen concentrations at 24 or 48 hours (Fig. 4a and 4b). Low levels of vimentin were detected under normoxic conditions in each of the cell lines evaluated, indicating some degree of mesenchymal change at baseline. At 24 hours, there was no appreciable change in vimentin expression under 1% oxygen concentration, and a slight increase was detected under 0.2% oxygen in U251 cells only. However, hypoxia consistently upregulated vimentin at 48 hours, indicating an oxygen concentration- and time-dependent acceleration of mesenchymal change under hypoxic conditions. This effect was reduced by treatment with a HIF inhibitor. This effect did not appear to differ between HIF1α and HIF2α.

Bottom Line: Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro.These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells.These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.

ABSTRACT
Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

No MeSH data available.


Related in: MedlinePlus