冬凌草甲素对人黑色素瘤A375细胞侵袭和转移的影响及机制研究

Effects and mechanism of oridonin on invasion and migration abilities of human melanoma A375 cells

目的研究冬凌草甲素对人黑色素瘤A375细胞侵袭和转移的抑制作用及其作用机制。方法采用细胞培养技术体外培养A375细胞,用不同浓度冬凌草甲素处理细胞,采用CCK-8法检测细胞增殖率;划痕实验分析细胞的迁移能力;Transwell小室实验研究细胞的侵袭能力;黏附实验评价细胞的黏附能力;Western blotting法检测转录因子Snail、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)、基质金属蛋白酶-2(MMP-2)和MMP-9蛋白的表达。结果冬凌草甲素对A375细胞48 h半数抑制浓度(IC50)为47.94μmol/L;与对照组比较,5、10、20μmol/L冬凌草甲素可明显降低A375细胞的体外迁移、侵袭及黏附能力(P<0.05),且具有量效关系。冬凌草甲素作用于细胞后,E-cadherin蛋白表达水平明显上调(P<0.05),Snail、N-cadherin、vimentin、MMP-2和MMP-9蛋白表达水平明显下调(P<0.05)。结论冬凌草甲素具有体外抑制A375细胞侵袭和转移的作用,其机制可能与调控上皮-间质转化(EMT)及MMPs有关。

Objective To study the effects and the mechanism of oridonin on inhibiting the invasion and migration abilities of human melanoma A375 cells. Methods Human melanoma A375 cells were cultured and treated respectively with indicated concentrations of oridonin by cell culture technique. The proliferation rate was detected by CCK-8 method. The migration ability was measured by wound healing assay. The invasion ability was examined by Transwell assay. The adhesion capabilities were evaluated by adhesion assay. The epithelial-mesenchymal transition（EMT） and matrix metalloproteinases（MMPs） related protein expression levels were determined by Western blotting. Results CCK-8 assay showed the median inhibition concentration（IC50） of 48 h was 47.94 μmol/L. Oridonin（5, 10, and 20 μmol/L） inhibited the migration, invasion and adhesion abilities of human melanoma A375 cells in a dose-dependent manner（P 0.05）. After oridonin treatment, the protein expression levels of E-cadherin increased significantly（P 0.05） and the protein levels of Snail, N-cadherin, vimentin, MMP-2, and MMP-9 decreased significantly（P 0.05）. Conclusion Oridonin inhibits the migration, invasion and adhesion abilities of human melanoma A375 cells. The mechanism may be related with the regulating effects of oridonin on EMT and MMPs.