Inhibitory Effect Of HB On Tumor Neoangiogenesis

Objective:In order to study the effects of HB (berberinea-hydroxy5-decanoylethyl sulfonate) on tumor neoangiogenesis and the expressions of angiogenesis factors in tumor cells, and evaluate the anti-angiogenic and anti-tumor effects of HB on hematogenous metastasis of Lewis lung carcinoma.Methods:1. Study the effects of HB on the proliferation, migration and tube-like structure formation of vascular endothelial cell:MTT assay was employed to determine the effects of HB on the proliferation of ECV304cells; The wound scratch assays were applied to determine the effect of HB on the migration capacity of ECV304cells; Tube formation was evaluated by two-dimensional collagen gel assay.2. Study the effects of HB on the expression of vascular endothelial growth factor of Lewis lung carcinoma (LLC) cells:MTT assay was employed to determine the effects of HB on the proliferation of LLC cells; Tranwell co-culture was used to determine the effect of HB on LLC-induced ECV304cells migration; The VEGF and MMP-9expression of LLC cells were determined by western blotting.3. The influence of HB on Lewis lung carcinoma in C57BL/6mice:The C57BL/6mice bearing Lewis lung carcinomas were intragastric adiministrated with HB (60,90,120mg/kg-d) for20d, the weight of mice and volume of the tumor measured. After22days, the mice were sacrificed and the tissue of xenografted tumor was taken and weighed. The samples were fixed by10%formaldehyde, embedded by general paraffin and sliced up continuously by4μm. The lung metastasis of LLC cells was determined by HE staining. Tumor angiogenesis was marked by CD34and the expression of microvessel density (MVD) were detected by imunohistochemical image analysis.Results:1. HB can effectively inhibited the proliferation, migration and tube-like structure formation of vascular endothelial cell:The inhibitory effect of proliferation of ECV304cells in a time-and dose-dependent manner, with the50%inhibitory concentration (IC50) of HB corresponding to treatment durations of24,48and72h being40.61,7.15and2.40mg/L respectively; The migration of endothelial cells reduced to38.56%,53.11%and78.81%respectively when incubated withl.25,2.5and5mg/L HB; HB also can reduce the tube-like structure formation, after treated with HB (20,30and40mg/L,18h), the rates of inhibition were26.99%,39.26%and65.64%respectively.2. HB can inhibit the expression of growth promoting protein VEGF in LLC cells:The assay revealed the inhibit rates of proliferation of LLC cells in a time-and dose-dependent manner, with the50%inhibitory concentration (IC50) of HB corresponding to treatment durations of24,48and72h being8.87>2.26and0.98mg/L respectively; After treated with HB (0,2.5,5and10mg/L,24h) the numbers of migration were283.3+14.2,178.7+26.6,130.5+14.6and104.0+12.5respectively in a dose-response relationship; In LLC cells following the increasing of the concentration of HB, the expressions of MMP-9was decreased gradually, and when the concentration of HB was20mg/L, the expressions of VEGF showed significant decreased.3. HB inhibit tumor growth and tumor angiogenesis in mice Lewis lung carcinoma:Compared with the model group, in the middle dose and high dose group, the MVD expression, tumor weight and number of the metastatic foci were lower, the tumor grew comparatively slow too, besides there was no significant decrease in body weigh.Conclusion:Evidence suggests that HB can direct inhibit of angiogenesis in vitro by inhibit the proliferation migration and tube-like structure formation of vascular endothelial cell. Besides HB can also inhibit Lewis lung carcinoma cells reduced angiogenesis by inhibit the expression of VEGF and MMP-9in vitro. In vivo, HB can remarkably inhibit the growth and metastasis of Lewis lung carcinoma with less toxic reaction in mice. The inhibitory effect partially due to the effect of HB inhibiting neovascularization induced by malignant tumor.