毛兰素通过调节HIF-1α/VEGF/VEGFR2信号通路抑制恶性黑素瘤细胞血管生成

ERIANIN INHIBITS ANGIOGENESIS OF MALIGNANT MELANOMA CELLS BY REGULATING HIF-1A/VEGF/VEGFR2 SIGNALING PATHWAY

目的 探究毛兰素通过调节缺氧诱导因子1α(HIF-1α)/血管内皮生长因子(VEGF)/血管内皮生长因子受体2(VEGFR2)信号通路对恶性黑素瘤细胞血管生成的影响。方法 不同浓度毛兰素处理B16F10细胞筛选合适的药物作用浓度。体外培养B16F10细胞、人脐静脉血管内皮细胞(HUVECs)并随机分为对照组、毛兰素低、高剂量组、空载组、毛兰素高剂量+HIF-1α过表达组,分组处理后,提取B16F10细胞条件培养基,酶联免疫吸附反应(ELISA)测定B16F10细胞培养基中VEGF水平;并用提取的B16F10细胞条件培养基按上述分组处理HUVECs,小管形成实验检验各组HUVECs细胞成管长度。建立B16F10移植瘤小鼠模型并按上述分组进行处理,免疫组化染色检测各组移植瘤微血管密度(CD31表达)及VEGF表达;免疫印迹检测各组B16F10细胞及其移植瘤小鼠肿瘤组织中HIF-1α/VEGF/VEGFR2通路蛋白表达。结果 与对照组相比,毛兰素低、高剂量组B16F10细胞培养基中VEGF水平、HUVECs细胞成管长度、移植瘤CD31及VEGF阳性细胞比例、B16F10细胞及其移植瘤小鼠肿瘤组织中HIF-1α、VEGF、VEGFR2蛋白表达均降低(P <0.05);与毛兰素高剂量组相比,毛兰素高剂量+HIF-1α过表达组B16F10细胞培养基中VEGF水平、HUVECs细胞成管长度、移植瘤CD31及VEGF阳性细胞比例、B16F10细胞及其移植瘤小鼠肿瘤组织中HIF-1α、VEGF、VEGFR2蛋白表达升高(P<0.05)。结论 毛兰素通过下调HIF-1α/VEGF/VEGFR2通路而在体内外抑制恶性黑素瘤细胞血管生成。

Objective To explore the impact of Erianin on the angiogenesis of malignant melanoma cells by regulating hypoxia-inducible factor 1α(HIF-1α)/vascular endothelial growth factor(VEGF)/vascular endothelial growth factor receptor 2(VEGFR2) signaling pathway.Methods B16F10 cells treated with different concentration of maolin were screened for appropriate drug action concentration.The B16F10 cells and human umbilical vein endothelial cells(HUVECs) cultured in vitro were randomly grouped into control group,low-dose and high-dose Erianin group,empty-load group,and high-dose Erianin+HIF-1α overexpression group.After group treatment,B16F10 cell conditioned medium was extracted,and the level of VEGF in B16F10 cell medium was determined by enzyme-related immunosorbent assay(ELISA).HUVECs were treated with extracted B16F10 cell conditioned medium according to the above groups.Tubule formation experiment was conducted to test the tubule length of HUVECs cells in each group.The B16F10 transplanted tumor mouse model was established and treated according to the above groups.Immunohistochemical staining was performed to detect the microvascular density(CD31 expression) and VEGF expression of transplanted tumors in each group.Western blotting was used to detect HIF-1α/VEGF/VEGFR2 pathway protein expression in B16F10 cells and tumor tissue of transplanted mice.Results Compared with the control group,the levels of VEGF in the culture medium of B16F10 cells,the tube-forming length of HUVECs cells,the proportions of CD31 and VEGF-positive cells in the transplanted tumor,and the protein expressions of HIF-1α,VEGF and VEGFR2 in the B16F10 cells and tumor tissue of the transplanted mice were all decreased in the low-dose and high-dose Erianin groups(P<0.05);compared with the high-dose Erianin group,the tube-forming length of HUVECs cells,the proportions of CD31 and VEGF-positive cells in the transplanted tumor,and the protein expressions of HIF-1α,VEGF and VEGFR2 in the B16F10 cells and tumor tissue of the transplanted mice in the high-dose Erianin + HIF-1α overexpression group increased(P<0.05).Conclusion Erianin inhibits the angiogenesis of malignant melanoma cells in vitro and in vivo by down-regulating the HIF-1α/VEGF/VEGFR2 pathway.