Mechanism of inhibition of tumor angiogenesis by Bletilla colloid: an experimental study

OBJECTIVE: To study the mechanism of inhibition of tumor angiogenesis by Bletilla colloid. METHODS: Human Hep-G2 hepatocellular carcinoma cells were cultured and treated with Bletilla colloid of different concentrations. Pure culture of Hep-G2 cells was used as control and pure culture medium without Hep-G2 cell was used as blank control. The concentration of vascular endothelial growth factor (VEGF) in the supernatant was detected by ELISA. The apoptosis and proliferation of the Hep-G2 cells were examined by flow cytometry. Cells of human endothelial cell line ECV-304 were cultured in the supernatant of culture media of Hep-G2 treated with Bletilla colloid of different concentrations. MTT method was used to observe the growth of the ECV-304 endothelial cells. Eighty rats were made animal model of transplanted Walker-256 hepatoma and then randomly divided into 4 groups of 20 rats 12 - 14 days after to undergo transarterial chemoembolization (TACE) treated with normal saline, 5-fluouracil (5-Fu), iodized poppy seed oil, and 5-Fu-Blettila microsphere respectively. Two weeks after, the rats were killed and the tumors were taken out. Immunohistological staining was conducted to detect the expression and localization of factor VIII, VEGF, and basic fibroblast growth factor (b-FGF). The microvcascular density (MVD) was calculated by counting the factor VIII positive endothelial cells. RESULTS: There was no statistically significant difference in the apoptosis rate, proliferation rate and supernatant VEGF level between the control group and the Bletilla groups. The inhibition rates of ECV-304 endothelial cell growth were 57.6%, 66.7%, 86.4%, 87.5%, and 94.8% in the groups of Bletilla of the concentrations of 0.5, 1.0, 2.0, 4.0, and 8.0 micro g/ml respectively in a dose-dependent manner. The MVD of tumor was 59 +/- 34 per field in the 5-Fu-Blatilla group,significantly lower than those in the other 3 groups (F = 5.177, P = 0.003). The expression of VEGF and the expression of b-FGF were not significantly different among the 4 TACE groups. CONCLUSION: Bletilla colloid inhibits angiogenesis after TACE, potentially, through inhibition of the binding of vascular endothelial growth factor to its receptor.