岩大戟内酯B刺激MCF-7细胞条件培养基降低人脐静脉血管内皮细胞的增殖和迁移

目的:观察岩大戟内酯B(jolkinolide B,JB)刺激MCF-7条件培养基对人脐静脉血管内皮细胞的影响,并分析岩大戟内酯B的作用机制。方法:分别用25,55,85 mg·L~(-1)岩大戟内酯B刺激MCF-7人乳腺癌细胞为JB刺激组,正常培养的MCF-7细胞为MCF-7组。获得25,55,85 mg·L~(-1)JB刺激的MCF-7细胞上清液为相应浓度的条件培养基。利用各组条件培养基分别培养人脐静脉血管内皮细胞(HUVEC)为25,55,85 mg·L~(-1)JB组,正常培养的HUVEC为非条件培养基组。分别用噻唑蓝(MTT)比色法,Annexin V-FITC细胞凋亡实验,细胞划痕实验和transwell细胞小室迁移实验观察25,55,85 mg·L~(-1)JB组HUVEC增殖、凋亡、迁移的变化;并利用蛋白免疫印迹法(Western blot)检测JB对MCF-7细胞的作用机制,酶联免疫吸附试验(ELISA)分析各组条件培养基中血管内皮细胞生长因子(VEGF)的含量变化。结果:与正常组比较,25,55,85 mg·L~(-1)JB组HUVEC细胞凋亡数目逐渐升高(P0.01),HUVEC增殖显著降低(P0.01);25,55,85 mg·L~(-1)JB组HUVEC细胞划痕面积闭合率和细胞迁移率较非条件培养基组均显著降低(P0.01)。与MCF-7比较,25,55,85 mg·L~(-1)JB均能够明显抑制MSC-7细胞蛋白激酶B/信号传导及转录激活因子3/哺乳动物雷帕霉素靶蛋白(Akt/STAT3/m TOR)信号通路表达,下调MCF-7细胞表达的VEGF因子。结论:岩大戟内酯B通过抑制Akt/STAT3/m TOR信号通路下调MCF-7细胞旁分泌VEGF,抑制血管内皮细胞的增殖或迁移活性。

Effect of Conditioned Medium of MCF-7 Stimulated by Jolkinolide B in Reducing Proliferation and Migration of Human Umbilical Vein Endothelial Cells

Objective: To observe the effect of conditioned medium of MCF-7 cells stimulated by jolkinolide B (JB) on human umbilical vein endothelial cells (HUVECs), and analyze the mechanism of JB. Method: The 25, 55, 85 mg ·L^-1 of JB were used to stimulate MCF-7 human breast cancer cells, respectively. The cells stimulated by different concentrations of JB were taken as the JB groups, and the normal cultured MCF-7 cells were taken as control group. The supernatants of tumor cells stimulated by different concentrations of JB were collected as the conditioned medium (CM) of the corresponding concentrations. According to the corresponding concentrations of JB, the conditioned medium was used to culture HUVECs, and these cells were taken as 25-CM group, 55-CM group and 85-CM group, respectively. The normally cultured HUVECs were taken as the non-conditioned medium group (NCM). Methylthiazolyldiphenyl-tetrazoliumbromide (MTT) assay, Annexin V-FITC, cell scratch and transwell chamber experiment were separately used to detect the proliferation, apoptosis and migration of HUVEC in the above groups. The Western blot assay was used to detect the mechanism of JB on MCF-7 cells, and the enzyme linked immunosorbent assay (ELISA) was used to analyze the changes in the content of vascular endothelial growth factor (VEGF) in the conditioned medium of each group. Result: Compared with NCM group, the apoptosis rate of HUVEC in 25-CM group, 55-CM group and 85-CM group were gradually increased(P<0.01), and the A values of HUVEC proliferation in the above groups were significantly decreased (P<0.01); compared with NCM group, the closure rate and the migration rate of HUVEC in 25-CM group, 55-CM group and 85-CM group were significantly decreased (P<0.01). There were significant differences in apoptosis rate, the A value of cell proliferation, the scratch closure rate, and cell migration rate among 25-CM group, 55-CM group and 85-CM group (P<0.01). Compared with control group, both JB with the concentrations of 25, 55, 85 mg ·L^-1 can inhibit the expression of Akt/STAT3/mTOR signaling pathway in MCF-7 cells, and reduce the expression of VEGF in MCF-7 cells. Conclusion: JB can reduce the secretion of VEGF in MCF-7 cells by inhibiting the Akt/STAT3/mTOR signaling pathway, so as to inhibite the proliferation and migration of HUVEC.