The effect of alphastatin peptide suppressing the hypoxiainduced vasculogenic mimicry formation of glioma

Objective： To investigate the vasculogenic mimicry formation induced by hypoxia in Ⅱ-Ⅲ human glioma cell and the effect of alphastatin peptide suppressing the hypoxia-induced vasculogenic mimicry formation and the mechanism. Methods： MTT, Transwell and three- dimentional culture were used to detect the proliferation, migration and tubule formation of SHG44. The expression of vascular endothelial growth factor-α（VEGF-α, erythropoietin-producing hepatocellular carcinoma-A2 （EphA2） and matrix metalloproteinases 2 （MMP2） was detected by RT-PCR and Western blotting analysis. Results： The OD490 in hypoxia group was 0.60±0.06 and in control group was 0.46±0.05. The number of cell migration was 178.71±18.81 in hypoxia group and 85.86±17.92 in control group. The tubule formation was 56.80±12.21 in hypoxia group and 4.20±2.62 in control group. The proliferation, migration and tubule formation in hypoxia group were significantly higher than that in control group. The expression of VEGF-α, EphA2 and MMP2 was upregulated in hypoxia. When various concentrations of alphastatin （100, 1 000, 10 000 nmol/L） were added to hypoxia group, the numbers of cell migration were 142.57±12.12, 92.71±17.68, 30.00±7.72 and the tubule formation were 47.71±10.58, 18.86±8.40, 8.43±5.62. The cell migration and tubule formation were significantly suppressed by alphastatin in a dose-dependent manner. In alphastatin group, the phosphorylation of EphA2 protein （P=0.037, F=4,629） and activation of MMP2 protein （P=0.005, F=9.331） were significantly suppressed but there was no change in VEGF-α protein. Conclusion： Ⅱ-Ⅲ human glioma cell is able to form vasculogenic mimicry induced by hypoxia and alphastatin peptide can suppress the hypoxia-induced vasculogenic mimicry. VEGF-α induced EphA2 phospharilation and MMP2 activation maybe the key pathway to form vasculogenic mimicry.