基于TGF-β1信号通路研究小檗碱联合芒柄花黄素抑制鼻咽癌细胞迁移的作用机制

Mechanism of berberine combined with formononetin on inhibiting migration of nasopharyngeal carcinoma cells based on TGF-β1 signaling pathway

目的研究小檗碱联合芒柄花黄素对鼻咽癌细胞迁移的影响,探讨转化生长因子-β1(transforming growth factor-β1,TGF-β1)信号通路在其效应中的作用。方法采用实时无标记细胞功能分析仪监测小檗碱和芒柄花黄素单用及联合使用对鼻咽癌5-8F、6-10B细胞增殖的影响;采用划痕实验和Transwell实验检测TGF-β1信号通路激活后,小檗碱联合芒柄花黄素抑制细胞迁移效应的变化;采用Western blotting检测TGF-β1信号通路关键蛋白和迁移相关蛋白的表达。结果与对照组比较,小檗碱和芒柄花黄素单用可抑制5-8F和6-10B细胞增殖,小檗碱(5、10μmol/L)和芒柄花黄素(5、10μmol/L)联合作用于5-8F和6-10B细胞后,显示小檗碱联合芒柄花黄素抑制鼻咽癌细胞增殖以协同效应为主;小檗碱联合芒柄花黄素显著降低鼻咽癌细胞TGF-β1和Smad3蛋白表达水平(P<0.05、0.01)。采用TGF-β1激活TGF-β1信号通路后,小檗碱联合芒柄花黄素抑制鼻咽癌细胞迁移的效应显著降低(P<0.01);与小檗碱10μmol/L+芒柄花黄素10μmol/L组相比,TGF-β1+小檗碱+芒柄花黄素组细胞TGF-β1、Smad3、磷酸化细胞外调节激酶1/2(phosphorylated extracellular signal regulated kinase 1/2,p-ERK1/2)蛋白表达水平显著上调(P<0.05、0.01),N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)表达水平升高,E-钙黏蛋白(E-cadherin)表达水平降低(P<0.01)。结论小檗碱联合芒柄花黄素能够协同抑制鼻咽癌细胞迁移,其作用机制可能与抑制TGF-β1信号通路有关。

Objective To study the effect of berberine combined with formononetin on migration of nasopharyngeal carcinoma cells and explore the role of transforming growth factor-β1(TGF-β1)signaling pathway in its effect.Methods Effects of berberine and formononetin on proliferation of nasopharyngeal carcinoma 5-8F and 6-10B cells were detected by real time cellular analysis technology;Inhibition of cell migration by berberine combined with formononetin after activation of TGF-β1 signaling pathway was detected by scratch and Transwell migration assay;Expressions of key proteins in TGF-β1 signaling pathway and migration related proteins were detected by Western blotting.Results Compared with control group,berberine and formononetin alone inhibited the proliferation of 5-8F and 6-10B cells.When 5-8F and 6-10B cells were treated with berberine(5,10μmol/L)and formononetin(5,10μmol/L),inhibitory effect of berberine and formononetin on proliferation were mainly synergistic.Berberine combined with formononetin could reduce the expressions of TGF-β1 and Smad3 in nasopharyngeal carcinoma cells(P<0.05,0.01).After TGF-β1 signaling pathway was activated by TGF-β1,inhibitory effect of berberine combined with formononetin on nasopharyngeal carcinoma cells migration was decreased(P<0.01).And expressions of TGF-β1,Smad3,phosphorylated extracellular signal regulated kinase(p-ERK1/2)in TGF-β1+berberine+formononetin group was up-regulated(P<0.05,0.01),N-cadherin and vimentin protein expressions were up-regulated,while E-cadherin protein expression was decreased(P<0.01).Conclusion Berberine combined with formononetin can synergistically inhibit the migration of nasopharyngeal carcinoma cells,its mechanism may be related to the inhibition of TGF-β1 signaling pathway.