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. 2014 May 27:12:148.
doi: 10.1186/1479-5876-12-148.

Induction of proline-rich tyrosine kinase 2 activation-mediated C6 glioma cell invasion after anti-vascular endothelial growth factor therapy

Cheng-Shi Xu, Ze-Fen Wang, Li-Ming Dai, Sheng-Hua Chu, Ling-Ling Gong, Ming-Huan Yang, Zhi-Qiang Li  1
Affiliations

Induction of proline-rich tyrosine kinase 2 activation-mediated C6 glioma cell invasion after anti-vascular endothelial growth factor therapy

Cheng-Shi Xu et al. J Transl Med. .

Abstract

Background: Anti-angiogenic therapy inhibits tumor growth and is considered as a potential clinical therapy for malignant glioma. However, inevitable recurrences and unexpected tumor resistance, particularly increased invasion ability of glioma cell, were observed after anti-angiogenic treatment. The underlying mechanism remains undetermined. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration; therefore, we investigated the possible role of these kinases in rat C6 glioma cell invasion induced by bevacizumab, a recombinant monoclonal antibody against vascular endothelial growth factor (VEGF).

Methods: The effects of bevacizumab on migration and invasion of C6 glioma cells were investigated in vitro and in vivo. The cells proliferation, migration, and invasion were determined by MTT assay, wound healing, and transwell assay, respectively. Invasive potential of glioma cells in vivo was assessed by counting vimentin-positive cells crossing the solid tumor rim by immunohistochemical staining. The total and phosphorylated protein levels of FAK and Pyk2 were detected by Western blotting.

Results: Bevacizumab exposure increased migration and invasion of cultured C6 cells in a concentration-dependent manner. In addition, the continuous bevacizumab treatment also promoted tumor invasion in rat C6 intracranial glioma models. Bevacizumab treatment enhanced Pyk2 phosphorylation at Tyr402, but no effect on FAK phosphorylation at Tyr397 both in vitro and in vivo. Knockdown of Pyk2 by siRNA or inhibition of Pyk2 phosphorylation by Src kinase specific inhibitor PP1 partially inhibited bevacizumab-induced cell invasion in cultured C6 glioma cells. Furthermore, the combined administration of bevacizumab and PP1 significantly suppressed glioma cell invasion into surrounding brain tissues compared to bevacizumab treatment alone in experimental rats.

Conclusions: These results suggest that anti-VEGF treatment promotes glioma cell invasion via activation of Pyk2. Inhibition of Pyk2 phosphorylation might be a potential target to ameliorate the therapeutic efficiency of anti-VEGF treatment.

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Figures

Figure 1
Figure 1
Effect of bevacizumab (Bev) on glioma cell proliferation. After treatment with the indicated concentrations of Bev for 72 hours, cell proliferation rates were analyzed by MTT assay. The growth rates of cells with the IgG control were defined as 1.0 (*p < 0.05 vs the group treated with IgG control).
Figure 2
Figure 2
Effect of bevacizumab (Bev) on glioma cell migration and invasion in vitro. (A) The migratory ability of C6 glioma cells treated with 5 mg/ml Bev was evaluated by wound healing assay. The representative images at 0 hour and 24 hours post-wounding are shown at 100 × magnification. (B) C6 glioma cell invasion was evaluated by transwell assay after 5 mg/ml Bev treatment for 24 hours. The stained invasive cells were photographed under an inverted light microscope at 100 × magnification. (C) Quantitative results of C6 glioma cell invasion in vitro. The experiments were performed in triplicate with three independent experiments.
Figure 3
Figure 3
Effect of bevacizumab (Bev) on glioma cell migration and invasion in vivo. (A) Rat C6 glioma xenografts treated with or without Bev were stained immunohistochemically for vimentin to show invasion of the tumor cells. Tumor cells were shown to have invaded the surrounding normal brain after Bev treatment, while the leading edge of the control tumor showed a clear rim. Scale bar: 100 μm. (B) Quantification of vimentin-positive cells outside the tumor rim with or without Bev treatment. The number of individual cells crossing the solid tumor rim was counted in five fields of view. The invasive tumor cell number of the group without Bev treatment was expressed as 100% (*p < 0.05, **p < 0.01, vs. the group treated with IgG control).
Figure 4
Figure 4
Effect of bevacizumab (Bev) on phosphorylation of FAK and Pyk2 in cultured glioma cells. (A) Protein expression and phosphorylation of FAK at Tyr397 and Pyk2 at Tyr402 in C6 glioma cells. (B, C) Quantification of total protein levels and phosphorylation of Pyk2 in (A). (D, E) Quantification of total protein levels and phosphorylation of FAK in (A). (**p < 0.01, vs. IgG control).
Figure 5
Figure 5
Effect of bevacizumab (Bev) on phosphorylation of FAK and Pyk2 in rat intracranial glioma model. (A) Protein expression and phosphorylated FAK at Tyr397 and Pyk2 at Tyr402 in intracranial glioma tissue of rats treated with Bev or IgG control. (B, C) Quantification of total protein levels and phosphorylation of Pyk2 in (A). (D, E) Quantification of total protein levels and its phosphorylation of FAK in (A). (**p < 0.01, vs. IgG control).
Figure 6
Figure 6
Effect of Pyk2 inhibition on glioma cell invasion in cultured glioma cells. (A) Total Pyk2 protein in C6 glioma cells treated with Pyk2 siRNA or control siRNA (**p < 0.01, vs. control siRNA group). (B) Quantification of the number of invasive glioma cells (*p < 0.05, vs. Bev group). (C) Phosphorylated Pyk2 in C6 glioma cells treated with PP1 (*p < 0.05, vs. control group). (D) Quantification of the number of invasive glioma cells after treatment with Bev, Bev plus DMSO, and Bev plus PP1 (*p < 0.05, vs. Bev group).
Figure 7
Figure 7
Effect of Pyk2 inhibition by PP1 on tumor cell invasiveness after bevacizumab (Bev) treatment in vivo. (A) Rat C6 glioma xenografts treated with Bev alone or Bev plus PP1 were stained immunohistochemically for vimentin to show the invasion of tumor cells. In the Bev alone treated group, tumor cells had clearly invaded the surrounding normal brain. However, the necrotic areas (N) in the Bev plus PP1 treated tumors increased and the tumor cells invading the surrounding normal brain decreased. The right upper corner shows a higher magnification image (boxed inset). (B) Quantification of vimentin-positive cells invading the surrounding normal brain with Bev alone or Bev plus PP1 treatment. The number of individual cells crossing the solid tumor rim was counted in five fields. The invasive tumor cell number of the group treated with Bev alone were expressed as 100% (**p < 0.01, vs. the group treated with Bev alone).
Figure 8
Figure 8
Effect of bevacizumab treatment alone or combination with PP1 on survival of rat C6 glioma xenografts. Kaplan-Meier survival curves for control IgG versus treated rats. Bev and Bev plus PP1 treatments resulted in a longer median survival duration compared with control, but no difference was found between Bev group and Bev plus PP1 group.
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