12-去氧佛波醇-13-棕榈酸酯对人乳腺癌MCF-7细胞VEGF表达的影响及其相关机制

Effect and Related Machanism of 12-deoxyphorbol-13-palmitate on Expression of VEGF in Breast Cancer MCF-7 Cells

目的：观察狼毒大戟根部提取物12-去氧佛波醇-13-棕榈酸酯（12-deoxyphorbol 13-palmitate,DP）对MCF-7细胞血管内皮生长因子（vascular endothelial growth factor,VEGF）表达的影响,及其是否通过Von Hippel-Lindau蛋白（VHL）/低氧诱导因子（HIr-1α）信号通路进行调节.方法：实验分为常氧空白组、常氧实验组（10,20,40 μmol·L^-1DP）、乏氧空白组和乏氧实验组（10,20,40 μmol·L^-1DP）,其中乏氧空白组和乏氧实验组用150 μmol·L^-1CoCl2预处理细胞.当细胞密度达70％ ～80％时进行药物处理;MTT法检测DP对MCF-7细胞在6,12,24 h细胞存活率的影响;酶联免疫吸附测定（ELISA）检测DP对MCF-7细胞分泌VEGF的影响;实时荧光定量PCR法（RT-qPCR）检测DP对VEGF mRNA表达的影响;Western blot检测DP对MCF-7细胞中VEGF,HIF-1α和VHL蛋白表达;细胞免疫荧光法（IF）检测DP对HIF-1α表达及其在细胞中分布的影响.结果：MTT法结果显示,乏氧状态下细胞存活率在12,24 h且DP浓度为20,40 μmol·L^-1时与对照组相比有显著性差异（P＜0.05）;乏氧条件下,VEGF和HIF-1α的表达较常氧条件下升高,与乏氧组相比,DP能降低VEGF（P ＜0.05）和HIF-1α（P＜0.05）的表达;乏氧条件下VHL表达较常氧条件下降低,当药物处理后,相较于乏氧组,VHL浓度明显上升（P＜0.05）.结论：DP可能通过调节VHL/HIF-1α信号通路从而下调MCF-7细胞VEGF表达.

Objective：To observe the effect of 12-deoxyphorbol-13-palmitate （DP） which isolated from the roots of Euphorbia fischeriana on the vascular endothelial growth factor （VEGF） expression,and whether it shows anti-VEGF effects via Von Hippel-Lindau （VHL） /hypoxia induced 1 alphal （HIF-1α） signaling pathway.Method：The cells were divided into normoxia control group,normoxia experimental groups （10,20,40 μmol · L^-1 DP）,hypoxia control group,hypoxia experimental groups （10,20,40 μmol ·L^-1 DP）.The hypoxia control group and experimental groups were pretreated with CoC12 （150 μmol ·L^-1）.The viability of MCF-7 cells was measured by MTT method at a cell density of 70%-80% and at 6,12,24 h.The secretion of VEGF was investigated by enzyme-linked irnmuno sorbent assay （ELISA）.The mRNA level of VEGF was detected by RT-qPCR.The protein expressions of VEGF,HIF-1α and VHL were determined by Western blot.The localization and expression of HIF-lα were detected by immunofluorescence.Result：MTT assay showed that DP significantly decreased the cell viability at 12,24 h （P 〈0.05） and at DP concentration of 20 μmol ·L-1 and 40 μmol ·L^-1 Under hypoxia condition,the expression of VEGF and HIF-1α was increased compared with that under normoxia condition.But the up-regulation was reversed （P 〈 0.05） by the addition of DP.The VHL level was down regulated under hypoxia condition.However after treatment of DP,the expression of VHL was increasing significantly （P 〈 0.05）.Conclusion：DP could down-regulate VEGF by affecting VHL/HIF-1α signaling pathway.