摘要
目的:探讨野菊花总黄酮对人乳腺癌MCF-7和MDA-MB-231细胞增殖、凋亡、转移、侵袭、细胞周期的影响及可能的分子机制。方法:体外培养MCF-7和MDA-MB-231细胞,分为对照组和不同浓度野菊花总黄酮组。显微镜观察细胞形态;CCK-8法评价细胞增殖;细胞划痕实验检测细胞迁移;Transwell实验检测细胞侵袭能力;AnnexinV-FITC/PI双染法及Hoechst染色检测细胞凋亡;流式细胞术检测细胞周期;实时荧光定量聚合酶链反应(Real-time PCR)检测Bax、P53 mRNA表达;Western Blot法检测β-catenin、E-cadherin蛋白表达。结果:野菊花总黄酮处理MCF-7、MDA-MB-231细胞后,细胞发生缩小、变形、解体;CCK-8结果显示,野菊花总黄酮对MCF-7、MDA-MB-231细胞的增殖抑制作用呈一定的浓度和时间依赖性,24 h的半数抑制浓度(IC_(50))分别为(42.17±0.59)、(35.01±0.83)μg/mL;划痕实验显示,野菊花总黄酮可显著降低MCF-7、MDA-MB-231细胞的迁移率(P<0.01);Transwell实验显示,野菊花总黄酮使MCF-7、MDA-MB-231细胞侵袭率显著降低(P<0.01);AnnexinV/PI双染结果显示,野菊花总黄酮使MCF-7、MDA-MB-231细胞的早期凋亡率升高,且Hoechst染色可观察到明显的细胞凋亡的核浓缩、核裂解;流式结果显示,野菊花总黄酮使MCF-7、MDA-MB-231细胞阻滞在S期和G_(2)期;Real-time PCR结果显示,经野菊花总黄酮处理后的MCF-7和MDA-MB-231细胞的Bax、P53 mRNA表达显著升高(P<0.01);Western Blot实验显示,野菊花总黄酮可下调MCF-7、MDA-MB-231细胞β-catenin蛋白表达(P<0.01),而对E-cadherin蛋白表达无明显影响(P>0.05)。结论:野菊花总黄酮在体外能抑制MCF-7、MDA-MB-231细胞的增殖、迁移和侵袭,诱导细胞凋亡,影响细胞周期分布,其机制可能与抑制Wnt/β-catenin信号通路及促进凋亡相关基因Bax、P53的表达有关,与EMT进程无明显关系。
Objective:To investigate the effects of total flavonoids of Chrysanthemum indicum on proliferation,apoptosis,migration,invasion,cell cycle of human breast cancer cells MCF-7 and MDA-MB-231 in vitro,to explore the potential mechanisms.Methods:MCF-7,MDA-MB-231 cells were cultured in vitro and divided into control group and different concentrations of total flavonoids of Chrysanthemum indicum groups.Cell morphology was observed under microscope,cell proliferation was evaluated by CCK-8 method,cell migration was detected by cell scratch assay,Transwell assay was used to detect the invasiveness of cells,AnnexinV-FITC/PI double staining and Hoechst staining were used to detect cell apoptosis,cell cycle was detected by flow cytometry.The expressions of Bax and P53 mRNA were detected by Real-time PCR.The proteins expressions ofβ-catenin and E-cadherin were detected by Western Blot.Results:MCF-7 and MDA-MB-231 cells were shrunk,deformed and disintegrated after treated with total flavonoids of Chrysanthemum indicum.The results of CCK-8 showed that the inhibitory effects of total flavonoids of Chrysanthemum indicum on MCF-7 and MDA-MB-231 cells were concentration and time-dependent,with IC_(50)of(42.17±0.59)μg/mL,(35.01±0.83)μg/mL at 24 hours,respectively.The scratch test results showed that total flavonoids of Chrysanthemum indicum could significantly reduce the mobility of MCF-7 and MDA-MB-231 cells(P<0.01).The Transwell experiment results showed that total flavonoids of Chrysanthemum indicum significantly reduced the invasion rates of MCF-7 and MDA-MB-231 cells(P<0.01),AnnexinV/PI double staining results showed that total flavonoids of Chrysanthemum indicum increased the early apoptosis rate of MCF-7 and MDA-MB-231 cells,and Hoechst staining results showed obvious nuclear concentration and cleavage of apoptosis cells,flow cytometry results showed that total flavonoids of Chrysanthemum indicum inhibited MCF-7 and MDA-MB-231 cells in S and G_(2)phases.Real-time PCR results showed that the expressions of Bax and P53 mRNA in MCF-7 and MDA-MB-231 cells were significantly increased after treatment with total flavonoids(P<0.01).Western Blot showed that total flavonoids of Chrysanthemum indicum could down-regulate the expression ofβ-catenin protein in MCF-7 and MDA-MB-231 cells(P<0.01),but had no significant effect on the expression of E-cadherin protein(P>0.05).Conclusion:The total flavonoids of Chrysanthemum indicum can inhibit the proliferation,migration and invasion of MCF-7 and MDA-MB-231 cells in vitro,induce apoptosis and affect the distribution of cell cycle.The mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway and the promotion of the expressions of apoptosis-related genes Bax and P53,but has no obvious relationship with EMT process.
作者
张惠娟
黄大元
谭敦勇
ZHANG Hui-juan;HANG Da-yuan;TAN Dun-yong(Medical college of Jishou University,Jishou 416099,China)
出处
《中药材》
CAS
北大核心
2021年第12期2908-2914,共7页
Journal of Chinese Medicinal Materials
基金
国家自然科学基金项目(81172497,81260396)
湖南省教育厅科学研究项目(18C0561)
湖南省中医药管理局青年项目(2020113)
吉首大学科研项目(2019012)
关键词
野菊花总黄酮
乳腺癌
增殖
凋亡
细胞周期
BAX
P53
Β-连环蛋白
Total flavonoids of Chrysanthemum indicum L.
Breast cancer
Proliferation
Apoptosis
Cell cycle
Bax
P53
β-Catenin
