冬凌草甲素对乳腺癌MCF-7细胞氟维司群耐药的逆转作用及机制  

Effect and mechanism of oridonin on drug resistance reversal in breast cancer MCF-7 fulvestrant resistant cells

作　　者：胡高波[1] 邱惠萍[1] 金湛 姜莉苑[1] 姚水洪[1] HU Gao-bo;QIU Hui-ping;JIN Zhan;JIANG Li-yuan;YAO Shui-hong(Medical School,Quzhou College of Technology,Quzhou 324000,China)

机构地区：[1]衢州职业技术学院医学院,浙江衢州324000

出　　处：《中国药理学与毒理学杂志》2021年第11期809-815,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：衢州市2019年度科技攻关项目(2019K41);衢职院病毒致瘤研究科技创新团队(衢州市重点创新团队)。

摘　　要：目的探究冬凌草甲素(Ori)对乳腺癌MCF-7细胞氟维司群(Ful)耐药的逆转作用及机制。方法MCF-7细胞在含Ful(终浓度为1μmol·L^(-1))的DMEM培养液中培养12个月,压力筛选MCF-7耐药细胞,制备耐药细胞株MCF-7/Ful。MCF-7和MCF-7/Ful细胞分别加Ful 0,2,4,8和16μmol·L^(-1),孵育48 h,计算MCF-7/Ful细胞耐药指数(RI)。MCF-7/Ful细胞用Ori 5μmol·L^(-1)+Ful 0,2,4,8和16μmol·L^(-1)联合孵育48 h,计算IC50,根据Ful单独用药和与Ori联合用药的IC_(50)值计算逆转倍数。MCF-7/Ful细胞分为细胞对照组、Ful 8μmol·L^(-1)组、Ori 5μmol·L^(-1)组和Ful+Ori组,作用24 h,彗星实验检测细胞拖尾长度;流式细胞术检测细胞周期和凋亡率;吖啶橙染色法检测细胞自噬;Western印迹法检测γ-H2AX、Bcl-2、Bax、胱天蛋白酶3、胱天蛋白酶9、周期蛋白D1表达、细胞分裂周期蛋白2(cdc2)磷酸化水平和微管相关蛋白轻链3(LC3)Ⅱ/Ⅰ比值。结果MCF-7/Ful细胞RI为8.67,Ori对MCF-7/Ful细胞耐药逆转倍数为4.2;彗星实验结果显示,Ful联合Ori能够使MCF-7/Ful细胞拖尾现象更加明显;流式细胞术结果显示,Ful联合Ori能有效地阻滞MCF-7/Ful细胞于G0/G1期(P<0.05),MCF-7/Ful细胞凋亡率显著增加(P<0.05);吖啶橙染色结果显示,Ful联合Ori能促使MCF-7/Ful细胞呈现橙红色,表现出明显的自噬现象;Western印迹结果显示,Ful联合Ori显著上调γ-H2AX蛋白表达(P<0.01);显著降低Bcl-2表达(P<0.01),升高Bax、胱天蛋白酶3和胱天蛋白酶9蛋白表达(P<0.01);显著降低周期蛋白D1表达(P<0.05),cdc2磷酸化升高水平(P<0.01);LC3Ⅱ/Ⅰ比值显著增加(P<0.05)。结论Ori能够逆转乳腺癌MCF-7/Ful细胞获得性耐药,其机制可能是通过诱导MCF-7/Ful细胞产生DNA损伤,进而引起细胞凋亡、细胞周期阻滞及细胞自噬。OBJECTIVE To investigate the reversal effect and mechanism of oridonin(Ori)on fulvestrant(Ful)resistance in breast cancer MCF-7 cells.METHODS Ful was added into the culture medium,and the final concentration was 1μmol·L^(-1).After 12 months of culture,MCF-7 resistant cells were screened under pressure to prepare MCF-7/Ful cells.MCF-7 and MCF-7/Ful cells were added with Ful 0,2,4,8 and 16μmol·L^(-1),respectively,and incubated for 48 h to calculate the drug resistance index(RI)of MCF-7/Ful cells.MCF-7/Ful cells were incubated with Ori 5μmol·L^(-1)+Ful 0,2,4,8 and 16μmol·L^(-1)for 48 h,IC50 was calculated,and the reverse multiple was calculated according to the IC50 value of Ful alone or with Ori.MCF-7/Ful cells were divided into cell control group,Ful 8μmol·L^(-1)group,Ori 5μmol·L^(-1)group and Ful+Ori group.After 24 h,the tail length of MCF-7/Ful cells was detected by comet assay.The apoptosis rate and cycle were detected by flow cytometry.Acridine orange staining was used to detect autophagy.The expression levels ofγ-H2AX,Bcl-2,Bax,caspase 3,caspase 9,cyclin D1,ratio of phorphyated cell cycle-related protein 2(p-cdc2)/cdc2 and microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰwere detected by Western blotting.RESULTS The RI of MCF-7/Ful cells was 8.67,and the reversal ratio of Ori to MCF-7/Ful cells was 4.2.The results of comet assay showed that Ful+Ori could highlight the trailing of MCF-7/Ful cells.The results of flow cytometry showed that Ful+Ori could effectively arrest the MCF-7/Ful cells in G0/G1 phase(P<0.05),and signifi⁃cantly increase the apoptosis rate of MCF-7/Ful cells(P<0.05).The results of acridine orange staining showed that Ful+Ori could promote MCF-7/Ful cells into orange-red autophagy.The results of Western blotting showed that Ful+Ori could significantly up-regulate the expression of DNA damage markerγ-H2AX(P<0.01),reduce the protein expression of Bcl-2(P<0.01),increase the protein expressions of Bax,caspase 3 and caspase 9(P<0.01),reduce the expression of cyclin D1 protei

关 键 词：冬凌草甲素 DNA损伤 乳腺癌 氟维司群 耐药 

分 类 号：R285[医药卫生—中药学]