Effects of a monoclonal antibody against (pro)renin receptor on gliomagenesis

Figures

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  Figure 1. (P)RR and SOX2 expression in human gliomas. (A) Expression of SOX2 and (P)RR in representative tumor samples of each WHO grade by immunohistochemistry (scale bar = 50 µm). (B) PS of (P)RR and (C) IS of SOX2 expression increased according to the WHO grade. (D) SOX2 expression weakly correlated with (P)RR expression (r = 0.40, P < 0.01). *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 2. Effect of the (P)RR Ab on Wnt signaling and stemness markers. Using Cells were treated with the (P)RR Ab at 400 µg/mL. (A) Representative western blot images showing that treatment with the (P)RR Ab reduced the activity of the Wnt/β-catenin signaling pathway [active β-catenin and c-myc] and expression of stemness markers (SOX2 and Oct3/4) in U251MG, U87MG and MGG23 cells. Original blots are presented in Supplementary Fig. S5. (B) Relative protein levels measured using western blotting, quantified and normalized to β-actin levels. C control, A antibody.

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  Figure 3. Antiproliferative effects of the (P)RR Ab on human glioblastoma cell lines in vitro and in vivo. The (P)RR Ab dose-dependently reduced cell proliferation in WST-1 (n = 3) and cell numbers (n = 4) in (A) U251MG cells and (B) U87MG cells (scale bar = 200 µm). (C) In vivo antitumor effect of systemic administration of the (P)RR Ab on engrafted tumors of U87MG cells in athymic nude mice 20 days after injection (left), and time dependent changes in tumor volume (right). The mean tumor volume after 20 days was 1730 ± 399 mL vs. 2739 ± 707 mL (P < 0.01, n = 7). *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 4. Antiproliferative effects of the (P)RR Ab on the human glioma stem cell line. (A) Dose-dependent changes in sphere formation of MGG23 after treatment with the (P)RR Ab (scale bar = 75 µm). The sphere size decreased dose-dependently, while the quantity of the spheres increased (n = 4). (B) The (P)RR Ab reduced cell proliferation in the MTT assay, both dose-dependently and time-dependently (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 5. The (P)RR Ab induces apoptosis of glioblastoma cell lines and the glioma stem cell line. (A) Detection of early and late apoptosis in U251MG, U87MG and MGG23 treated with control (left) or the (P)RR Ab (right). Cells were stained with FITC-labeled annexin V and PI, and the intensity of fluorescence was assessed at 525 nm (FITC) and 675 nm (PI). C1: dead cells; C2: late-staged apoptosis; C3: viable cells; C4: early-staged apoptosis. A significant increase in the proportion of late-staged apoptotic cells was observed. (B) Caspase-3 activity in U251MG, U87MG and MGG23 cells treated with the (P)RR Ab is shown. U251MG and MGG23 cells treated with the (P)RR Ab increased caspase-3 activity significantly (n = 3). *P < 0.05.

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  Figure 6. Effects of (P)RR Ab silencing on the migration of glioblastoma cell lines. Migration were evaluated in (A) U251MG and (B) U87MG by the wound healing assay. Cells were treated with 400 µg/mL of (P)RR Ab. Representative images were captured at 0, 24 and 48 h after scratching (scale bar = 75 µm). The white line shows the borders created by the original scratch. A graphical percentage of the wound closure in (A) U251MG and (B) U87MG is shown on the right side. The (P)RR Ab decreased cell invasion and migration significantly in U251MG and U87MG (n = 5). *P < 0.05; **P < 0.01.

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  Figure 7. Proposed model for the effect of the (P)RR Ab in glioblastoma. Treatment with the (P)RR Ab decreases cell proliferation and migration and induces apoptosis via inhibition of the Wnt signaling pathway. The (P)RR Ab suppresses SOX2 and Oct3/4 expression and cell proliferation of the glioma stem cell line, which indicates that the (P)RR Ab inhibits stemness of glioma. Fz frizzled.