醉茄素A通过抑制VEGF-VEGFR2轴的激活抑制肿瘤血管生成

Withaferin A inhibits tumor angiogenesis by targeting VEGF-VEGFR2 axis

目的探讨醉茄素A(Withaferin A,WFA)对血管生成的抑制作用及其机制。方法 CCK8法测定WFA对原代脐静脉内皮细胞(Primary Human Umbilical Vein Endothelial Cells,pHUVECs)的增殖抑制作用;Transwell法检测WFA对原代脐静脉内皮细胞迁移的抑制作用;管腔形成实验检测WFA对原代脐静脉内皮细胞小管作用;Western blotting法检测WFA对VEGF诱导原代脐静脉内皮细胞磷酸化VEGFR2表达的影响;QPCR法检测WFA作用于原代脐静脉内皮细胞后VEGF、bFGF、Ang-1、VEGFR2的mRNA表达。结果 WFA对原代脐静脉内皮细胞生长24h、48h、72h的IC50分别为1.12±0.09μmol/L、0.83±0.05μmol/L和0.71±0.04μmol/L,对原代脐静脉内皮细胞的增殖有较强的抑制作用;与对照组相比,WFA抑制VEGF诱导的原代脐静脉内皮细胞迁移和管腔形成,差异有显著性(P<0.05);WFA能够下调VEGF诱导的原代脐静脉内皮细胞中磷酸化VEGFR2的表达;与对照组相比,WFA抑制原代内皮细胞中的VEGF、bFGF、Ang-1、VEGFR2 mRNA的表达,差异有显著性(P<0.05)。结论 WFA可抑制VEGF-VEGFR2通路的激活,抑制原代脐静脉内皮细胞的增殖、迁移和管腔形成,这可能是WFA抑制肿瘤血管生成的重要机制。

Objective To investigate the inhibitory effect of Withaferin A on the angiogenensis and the mechanism of its antitumoral effect. Methods In vitro,the CCK8 assay was used to detect the effect of Withaferin A on pHUVECs proliferation. The transwell assay was used to detect the effect of Withaferin A on migration of pHUVECs. The anti - angiogenic effect of Withaferin A on pHUVEC was detected by tubule formation experiment. Western Bloting assay was used to detect the phospho - VEGFR2 protein expression. VEGF, bFGF, Ang - 1, VEGFR2 mRNA levels were detected by QCPR assay. Results The growth of pHUVECs was significantly inhibited by different concentrations of Withaferin A,and the 50% concentration of inhibition （ IC50 ） of Withaferin A were 1.12 ±0.09μmol/L, 0.83 ± 0.05μmol/L and 0.71 ± 0. 04μmol/L respectively at 24 h, 48h and 72h. Withaferin A （ 1,0.5 μmol/ L） could inhibit the migration abilities and the tubule structure formation of pHUVECs activated by VEGF165 （P 〈0.05）. Withaferin A had significantly downregulated expression of phospho -VEGFR2 protein activated by VEGF165 in HUVECs. WFA inhibits VEGF, bFGF, Ang - 1 mRNA levels of pHUVECs stimulated by the supernatant of hepatocellular carcinoma ceils. Conclusion Withaferin A significantly reduces the phosphorylation of VEGFR2 and formation of tumor vessels by inhibiting proliferation, migration and tubule structure formation of HUVECs , which may be one mechanism of its anti - cancer effect.