青葙苷A抑制缺氧乳腺癌MDA-MB-231细胞增殖、迁移和侵袭能力的研究

Study on Celosin A Inhibiting the Abilities of Proliferation,Migration and Invasion in MDA-MB-231 Breast Cancer Cells During Oxygen Deprivation

目的探索中药青葙子中的齐墩果烷型三萜化合物青葙苷A(celosin A,CA)对缺氧人乳腺癌MDAMB-231细胞的抗肿瘤作用及其相关机制。方法CoCl2造模MDA-MB-231细胞缺氧状态,建立细胞体外缺氧的CA给药培养体系。采用细胞计数试剂盒(MTT)检测细胞活力,免疫荧光法观察细胞形态学和核凋亡变化。流式细胞术(FCM)法检测细胞凋亡比例;细胞划痕实验检测细胞迁移能力;Transwell法检测CA对肿瘤细胞迁移和侵袭的抑制能力;Western blot实验检测缺氧相关蛋白表达水平和EMT标志蛋白表达水平。结果在缺氧状态下,高剂量的CA(20、40μmol/L)能抑制乳腺癌MDA-MB-231细胞增殖,诱导肿瘤细胞核凋亡的形态学改变,并且能有效促进早晚期细胞的凋亡率(PI-Annexin V-FITC促凋亡率:20μmol/L:+6.09%;40μmol/L:+18.50%),有效抑制乳腺癌MDA-MB-231细胞的缺氧诱导因子-1α(HIF-1α)、单核细胞趋化蛋白(MCP)、血管内皮生长因子(VEGF)表达;同时低剂量的CA(5、10μmol/L)能抑制乳腺癌MDA-MB-231细胞的Vimentin和N-cadherin因子的表达,并减少乳腺癌MDA-MB-231细胞迁移和侵袭细胞的数量(转移抑制率:5μmol/L,26.90%;10μmol/L,66.13%;侵袭抑制率:5μmol/L,23.71%;10μmol/L,75.00%)。结论CA能对缺氧乳腺癌MDA-MB-231细胞起到有效地诱导凋亡、抗肿瘤血管形成以及抑制侵袭与转移的作用。

Objective To explore the anti-tumor effect and mechanism of celosin A(CA),an oleanolic triterpene compound,on human breast cancer MDA-MB-231 cells line during oxygen deprivation.Methods The establishment of hypoxic conditions was induced by chemical hypoxia reagent CoCl2,MDAMB-231 cells were incubated under hypoxia treated with CA.Cell counting Kit(MTT)was used to detect the cell viability,the immunofluorescence assay was used to observe the cell morphological changes and nucleus apoptosis.The percentage of cell apoptosis was measured by FCM.The migration ability was detected using a wound healing assay.The effects of CA on cell migratory and invasive ability were assessed by Transwell assay.The expression level of hypoxia related factors and EMT marker proteins were detected by western blot.Results Under hypoxia,high-dose CA(20μmol/L,40μmol/L)can inhibit MDAMB-231 cells proliferation and induce the cell nuclear apoptosis in morphological changes.High-dose CA can also effectively promote tumor cell apoptosis in early and late phase(PI Annexin V-FITC apoptosis rates:20μmol/L:+6.09%;40μmol/L:+18.50%),and effectively inhibit the expression of HIF-1α,MCP,VEGF;meanwhile low-dose CA(5μmol/L,10μmol/L)can inhibit the expression of vimentin and N-cadherin factors in breast cancer MDA-MB-231 cells,thereby reducing the amount of migration and the invasive cells(migration inhibition rates:5μmol/L 26.90%and 10μmol/L 66.13%;invasion inhibition rates:5μmol/L 23.71%;10μmol/L 75.00%).Conclusion CA can effectively induce MDA-MB-231 cell apoptosis and attenuate the abilities of angiogenesis and metastasis in breast cancer MDA-MB-231 cell line during oxygen deprivation.