Effect of the vascular endothelial growth factor expression level on angiopoietin-2-mediated nasopharyngeal carcinoma growth

Abstract

Background: The overexpression of angiopoietin-2 (Ang-2) has both pro-tumorigenic and anti-tumorigenic effects. However, the mechanisms of this protein's dual effects are poorly understood, and it remains unclear how Ang-2 cooperates with vascular endothelial growth factor (VEGF). In the current study, we investigated the effects of Ang-2 overexpression on nasopharyngeal carcinoma growth in the presence of different levels of VEGF.

Methods: Ang-2 was introduced into the CNE2 cell line by liposome transfection, and the expression of endogenous VEGF was inhibited by microRNA-mediated RNA interference. CNE2 cells expressing varying levels of Ang-2 and VEGF were injected subcutaneously into the flanks of nude mice. Tumor growth was measured, and vessels from the harvested tumors were analyzed.

Results: The overexpression of Ang-2 had no obvious effect on CNE2 tumor growth in the presence of endogenous VEGF but significantly inhibited CNE2 tumor growth when the expression of endogenous VEGF was silenced, and the Ang-2/VEGF ratio is negatively correlated with tumor growth. Ang-2 overexpression decreased the percentage of α-SMA-positive cells around the tumor vessels but reduced the microvessel density only in the absence of VEGF.

Conclusions: Our results indicate that the effects of Ang-2 on nasopharyngeal carcinoma are highly dependent on the level of VEGF expression, Ang-2/VEGF ratio may offer a novel therapeutic approach for treating human cancer.

Figure 1

Effects of VEGF silencing on CNE2 cells. CNE2 cells were treated with the transfection reagent only (mock), a scrambled miRNA (scrambled miRNA), or a VEGF-specific miRNA (VEGF miRNA). (a): Real-time RT-PCR demonstrated low VEGF mRNA expression in VEGF-interfered CNE2 cells. *P < 0.05, compared with the mock and scrambled miRNA groups. (b): Western blotting demonstrated lower VEGF protein expression in the VEGF-interfered CNE2 cells.

Figure 2

Expression levels of Ang-2 and VEGF in different engineered CNE2 cell groups. (a): mRNA expression was determined by qRT-PCR. All the PCR amplification was performed in triplicate and repeated in three independent experiments. The relative quantities of selected mRNAs in cell samples were normalized to that of GAPDH. Higher Ang-2 mRNA expression was observed in C, D, and E (**P < 0.01, vs. B, no significant difference among A, B and F). Lower VEGF mRNA expression was observed in group E and F (## P < 0.01, # P < 0.05, vs. A, B, C, D). (b): Western blots of the above samples. (c): Densitometric quantitation of the band intensities shown in (b). The densitometric intensity of the target band in each lane relative to the intensity of the GAPDH band from the same sample was calculated, and plotted. Data were expressed as means ± SD of three independent experiments. The level of total Ang-2 protein in the Ang-2-transfected group C,D,E was significantly higher than that in control group B (*P < 0.05 vs. B; no significant difference among A, B and F), and the level of endogenous VEGF protein in the VEGF miRNA-transfected group E,F was significantly decreased (## P < 0.01, vs. A, B, C, D). The ratio of Ang-2/VEGF was significantly higher in E and F than in other groups.

Figure 3

Effect of Ang-2 overexpression on CNE2 tumor growth under different VEGF level. Six groups of engineered CNE2 cells with different Ang-2 and VEGF levels were subcutaneously inoculated into six groups of nude mice (A-F) respectively. Tumor volumes were calculated dynamically (a), and tumors were weighed (b) when the mice were sacrificed on the 27^th day after inoculation. There was no significant difference on tumor development among A,B,C. (P > 0.05), while E and F exhibited greatly suppressed tumor growth compared with A,B,C,D (**P < 0.01, *P < 0.05 vs A,B,C,D, ## P < 0.01 vs F). E had higher inhibitory effects than F on tumor growth.

Figure 4

Images for CD34 staining in harvested tumor tissues with microvessel density measured. It showed that there were higher microvessel density (MVD) in A,B,C,D group, while lower MVD in E and F (VEGF silenced group). a: mock, b: control plasmid, c: Ang-2 plasmid, d: Ang-2 + scrambled miRNA, e: Ang-2 + VEGF miRNA, f: VEGF miRNA. Original magnifications: 200× for a,b,c,d,e,f.

Figure 5

Immunohistochemical staining for α-SMA in harvested tumor tissues. The results showed that there were fewer α-SMA-positive cells around endothelial cells in the Ang-2-transfected groups (c,d,e) than in non- Ang-2-transfected groups (a,b,f). In addition, this staining showed vessels in c,d,e displayed an aberrant structure characterized by distorted and dilated lumina (vessel indicated by arrow). a: mock, b: control plasmid, c: Ang-2 plasmid, d: Ang-2 + scrambled miRNA, e: Ang-2 + VEGF miRNA, f: VEGF miRNA, g: negative control. Original magnifications: 400× for a,b,c,d,e,f,g.