Novel Insights into the Antagonistic Effects of Losartan against Angiotensin II/AGTR1 Signaling in Glioblastoma Cells

Figures

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  Figure 1. AGTR1 expression in SVG p12 normal glial cells and U-87 MG and T98G glioblastoma cells. (A) Real-time RT-PCR for AGTR1 in SVG p12, U-87 MG, and T98G cells; mRNA is shown relative to SVG p12 normal glial cells. (B) Immunoblotting showing AGTR1 protein expression. β-actin was used as a loading control. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over SVG12 for U-87 MG and T98G. (C) Immunofluorescence of AGTR1 in SVG p12, U-87 MG, and T98G cells. DAPI staining for nuclear detection. Scale bars = 5 µm. Original magnification, ×100. Data are expressed as means ± SD of three different experiments, each performed in triplicate. ** p < 0.01; *** p < 0.001.

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  Figure 2. AGTR1 and clinical outcomes in GBM patients. Kaplan–Meier survival analysis relating AGTR1 levels and overall survival (OS) (A), progression-free survival (PFS) (B), OS in chemotherapy-treated (C) GBM patients (TCGA dataset).

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  Figure 3. Effects of Ang II receptor antagonist Losartan on Ang II-induced AGTR1 expression in U-87 MG and T98G glioblastoma cells. (A,B) Real-time RT-PCR and immunoblotting assay for AGTR1 mRNA and protein expression in U-87 MG and T98G cells treated with vehicle (−) or the Ang II 0.1, 5, and 10 µM for 24 h. β-actin was used as a loading control. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over vehicle (−) for Ang II treatment. (C,D) AGTR1 mRNA and protein expression in U-87 MG and T98G cells treated with vehicle (−) and Ang II 5 µM alone or in combination with LOS 5 µM for 24 h. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over vehicle (−) for Ang II treatment or fold over Ang II for Ang II in combination with LOS. β-actin was used as a loading control. Data represent the mean ± SD of three different experiments, each performed in triplicate. ** p < 0.01; *** p < 0.001.

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  Figure 4. Effects of LOS on Ang II-induced U-87 MG and T98G glioblastoma cell proliferation, migration, and invasiveness. Cell proliferation was determined by the [3H]thymidine (A,C) and soft agar growth (B,D) assays in U-87 MG and T98G glioblastoma cells treated with vehicle (−) and Ang II 5 µM alone or in combination with LOS 5 µM for 24 h. (E,F) Wound-healing assays in U-87 MG and T98G glioblastoma cells treated for 12 h with vehicle (−) and Ang II 5 µM alone or in combination with LOS 5 µM. Images are representative of three independent experiments. The percentage of wound closure has been represented on histograms calculated using ImageJ software version 1.51q. Squares, time 0. Original magnification, ×10. Boyden chamber transmigration (G,I) and invasion (H,J) assays in U-87 MG and T98G glioblastoma cells treated with vehicle (−) and Ang II 5 µM alone or in combination with the LOS 5 µM for 12 h. Data represent the mean ± SD of three different experiments, each performed in triplicate. ** p < 0.01; *** p < 0.001.

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  Figure 5. Aromatase expression in SVG p12 normal glial cells and U-87 MG and T98G glioblastoma cells. (A) Real-time RT-PCR for CYP19A1 in SVG p12 normal glial cells, U-87 MG, and T98G glioblastoma cells; mRNA is shown relative to SVG p12 normal glial cells. (B) Immunoblotting showing Arom protein expression. β-actin was used as a control for equal loading and transfer. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over SVG p12 for U-87 MG and T98G. (C) Immunofluorescence of Arom in SVG p12 normal glial cells, U-87 MG, and T98G glioblastoma cells. DAPI staining for nuclear detection. Scale bars = 5 µm. Original magnification, ×100. Data are expressed as means ± SD of three different experiments, each performed in triplicate. *** p < 0.001.

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  Figure 6. Aromatase and clinical outcomes in GBM patients. Kaplan–Meier survival analysis relating CYP19A1 levels and overall survival (OS) in GBM patients (TCGA dataset).

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  Figure 7. Effects of Ang II and LOS on aromatase expression and activity in U-87 MG and T98G glioblastoma cells. (A,B) Real-time RT-PCR and immunoblotting assay for CYP19A1 mRNA and protein expression in U-87 MG and T98G glioblastoma cells, treated with vehicle (−) or the Ang II 0.1, 5, and 10 µM for 24 h. GAPDH was used as a loading control. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over vehicle (−) for Ang II treatment. (C,D) CYP19A1 mRNA and protein expression in U-87 MG and T98G glioblastoma cells treated with vehicle (−) and Ang II 5 µM alone or in combination with LOS 5 µM for 24 h. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over vehicle (−) for Ang II treatment or fold change over Ang II for Ang II in combination with LOS, 5 µM. GAPDH was used as a loading control. (E,G) Aromatase activity in U-87 MG and T98G glioblastoma cells treated with vehicle (−) and Ang II 5 µM alone or in combination…

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  Figure 8. Ang II potentiates the estradiol levels produced by an aromatizable steroid androst-4-ene-3,17-dione in U-87 MG glioblastoma cells. (A) ELISA for Estradiol secretion treated with vehicle (−), Ang II 5 µM, androst-4-ene-3,17-dione (AD 10 nM), Ang II 5 µM plus AD 10 nM, and Ang II 5 µM plus AD 10 nM plus LOS 5 µM for 24 h. (B) Cell proliferation was determined by the [3H]thymidine assay in U-87 MG cells treated with vehicle (−), estradiol (E2 10 nM), Ang II 5 µM, AD 10 nM, Ang II 5 µM plus AD 10 nM, and Ang II 5 µM plus AD 10 nM plus LOS 5 µM for 24 h. Data are expressed as means ± SD of three different experiments, each performed in triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 9. Aromatase promoters pI.f/I.4 in U-87 MG glioblastoma cells are stimulated upon Ang II exposure. (A,B) Upper panel, schematic map of the pI.f and pI.4 aromatase constructs. U-87 MG glioblastoma cells were transiently transfected with the reported constructs and treated with vehicle (−) or Ang II 5 µM, alone or in combination with LOS 5 µM for 24 h. (C,D) U-87 MG glioblastoma cells were treated in the presence of vehicle (−) or Ang II 5 µM for 3 h. Nuclear extracts were incubated with a biotinylated oligonucleotide containing the AP-1 or AP-1 mutant site in the aromatase promoters pI.f (left panel) or with a biotinylated oligonucleotide containing the STAT3 or STAT3 mutant site in the aromatase promoters pI.4 (right panel) and subjected to DNA affinity precipitation assay. Specifically bound proteins were subjected to Western blotting analysis. The specificity of the binding was tested by loading the unbound fraction (Negative Control). U-87 MG glioblastoma cells nuclear extracts were used as positive control. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed…

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  Figure 10. LOS Inhibits Ang II-induced tumor growth of U-87 MG xenografts. (A) U-87 MG glioblastoma cells were injected subcutaneously in female nude mice (five mice per group) and then treated with vehicle (−), Ang II alone, or in combination with LOS or ANA. Relative tumor volume (RTV) was calculated by the following formula: RTV = (Vx/V1) where Vx is the tumor volume on day x and V1 is the tumor volume at initiation of treatment (day 0). y-axis: means ± SD of the RTV. (B) Images of representative individual tumors and average tumor weight from vehicle (−), Ang II alone, or in combination with LOS or ANA. (C) Hematoxylin and eosin (H&E) staining of tumor sections from vehicle (−), LOS and Ang II alone, or in combination with LOS or ANA. (D) Immunohistochemical analysis in U-87 MG xenograft tumor of Ki-67, Arom, upon Ang II treatment w/o LOS or w/o ANA and both LOS and ANA. Scale bars = 12.5 μm. * p < 0.05; ** p < 0.01; *** p < 0.001.