原花青素对宫颈癌HeLa细胞上皮间质转化的抑制作用

Inhibitory effect of proanthocyanidins on epithelial mesenchymal transition of cervical cancer HeLa cells

目的观察原花青素抑制宫颈癌HeLa细胞的上皮间质转化的作用,为临床新的治疗宫颈癌方案奠定基础。方法采用MTT法检测原花青素对HeLa细胞的IC50,划痕和Transwell实验观察药物抑制宫颈癌细胞侵袭和迁移的作用,免疫荧光观察宫颈癌细胞EMT(上皮间质转化)生成相关蛋白在药物作用后改变情况,Western blot检测TGF⁃β1(转化生长因子⁃β1)、E⁃cadherin(上皮钙黏蛋白)等EMT相关蛋白在药物干预下的改变及在药物作用下Smad2、Smad3及其磷酸化的作用和Smad7蛋白表达,qPCR检测药物联合Smad通路抑制剂SB431542后E⁃cadherin mRNA表达变化。结果原花青素可抑制HeLa细胞的增殖。与对照组比较,原花青素组可抑制细胞侵袭和迁移。免疫荧光发现,原花青素作用后与EMT相关的TGF⁃β1、E⁃cadherin蛋白表达发生显著改变。Western blot结果显示,HeLa细胞EMT相关蛋白在原花青素干预下TGF⁃β1、E⁃cadherin、pSmad2、pSmad3、Smad7均发生显著变化,但对Smad2和Smad3蛋白的作用无统计学意义(P>0.05),在Smad通路抑制剂SB431542作用下原花青素对E⁃cadherin作用效果下降,提示原花青素通过Smad通路抑制细胞EMT的形成。结论原花青素对HeLa细胞EMT的生成有抑制作用,可抑制细胞侵袭和迁移,其作用机制与TGF⁃β1介导的Smad依赖性通路有关。

AIM To observe the inhibitory effect of procyanidins on epithelial mesenchymal transition(EMT)of cervical cancer HeLa cells,and to explore a new approach in treating cervical cancer.METHODS To HeLa cells,proanthocyanidins had their IC50 detected by MTT assay;their effects on the cellular invasion and observed by scratch test and Transwell test;and their impact on the changes of EMT production related proteins identified by immunofluorescence.Western blot was performed to determine the the changes of EMT related proteins,such as TGF⁃β1(transforming growth factor⁃β1),E⁃cadherin,etc.;the expression of Smad2,Smad3 and and their phosphorylation,and Smad7 as well.qPCR was applied in the detection of E⁃cadherin mRNA expression after concurrent use of Smad pathway inhibitor SB431542 and procyanidins.RESULTS Procyanidins exhibited their inhibitory effects to the proliferation of HeLa cells.Compared with the control group,the group intervened with proanthocyanidins shared inhibited invasion and migration of cells.Proanthocyanidins treatment brought forth significant change in the protein content of EMT⁃related TGF⁃β1 and E⁃cadherin as immunofluorescence revelation;and significant change in TGF⁃β1,E⁃cadherin,Psmad2,psmad3 and Smad7,but not in Smad2 and Smad3 as Western blot demonstration(P>0.05).With their effects on E⁃cadherin compromised by the inhibitor of Smad pathway SB431542,proanthocyanidins suggest their positive role in the formation of EMT via Smad pathway.CONCLUSION Being an inhibitor to EMT production in HeLa cells,procyanidins inhibit cell invasion and migration via Smad dependent TGF⁃β1⁃mediated pathway.