特异性Aurora激酶抑制剂对人乳腺癌原代细胞增殖、周期、自噬、凋亡的影响及其机制  

Effects of Aurora kinase inhibitor on proliferation,cell cycle,autophagy,and apoptosis of human primary breast cancer cells

作　　者：张月[1] 孙光源 姜伟华[1] 孙健[1] 范敬静[1] 宋文丽[1] 信国峰[1] 张志生[1] ZHANG Yue;SUN Guangyuan;JIANG Weihua;SUN Jian;FAN Jingjing;SONG Wenli;XING Guofeng;ZHANG Zhisheng(The First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,chin)

机构地区：[1]河北北方学院附属第一医院,河北张家口075000

出　　处：《山东医药》2018年第15期12-16,共5页Shandong Medical Journal

基　　金：河北省医学科学研究重点课题资助项目(ZD20140243)

摘　　要：目的观察特异性丝/苏氨酸蛋白(Aurora)激酶抑制剂AT9283对乳腺癌原代细胞增殖、细胞周期、自噬、凋亡的影响,并探讨其可能作用机制。方法取对数生长期乳腺癌原代细胞,加入AT9283,培养48 h免疫荧光染色后镜下观察乳腺癌原代细胞细胞核和纺锤体变化。取对数生长期乳腺癌原代细胞,分为实验组(1、2、3、4、5组)和对照组,实验组分别加入2 m L的0.001、0.01、0.1、1、10μmol/L AT9283,对照组不做任何处理,MTT法测算实验组细胞增殖抑制率,流式细胞仪分析各组细胞周期,Western Blotting法检测各组Aurora A、组蛋白H3(Histone H3)、p-Aurora A、p-Histone H3、p21、p53、Bcl-2、Bax蛋白。另取适量乳腺癌原代细胞,分为A、B、C、D四组,A、B、C组分别加入0.1、1、10μmol/L的AT9283,D组不做任何处理,共培养24 h。采用吖啶橙染色法观察各组细胞自噬情况,采用流式细胞术观察各组细胞凋亡情况。结果未处理乳腺癌原代细胞的细胞核和纺锤体没有改变,加入AT9283处理的乳腺癌原代细胞镜下可见细胞核形态改变,出现核分叶现象,产生多核细胞,纺锤体由双极变为单极,出现纺锤体紊乱。与对照组比较,培养24、48 h时1、2、3、4、5组细胞增殖抑制率均升高,且呈浓度依赖性(P均<0.05)。与对照组比较,各组G2/M期细胞所占比例升高,G0/G1、S期细胞比例降低(P均<0.05),且呈剂量依赖性。与对照组比较,各组p-Aurora A、p-Histone H3、Bcl-2相对表达量降低,p21、p53、Bax相对表达量增加(P均<0.05)。A、B、C、D组自噬率分别为1.03%±0.01%、5.02%±0.31%、10.11%±0.31%、14.52%±0.21%,细胞凋亡率分别为3.03%±0.14%、8.25%±0.31%、12.15%±0.31%、44.52%±1.34%,组间两两比较,P均<0.05。结论 AT9283可抑制乳腺癌原代细胞的增殖,使G2/M期细胞所占比例升高,促进细胞自噬、凋亡,且作用呈剂量依赖性。其机制可能为AT9283抑制乳腺癌原代细胞p-Aurora A、p-Histone H3、Bcl-Objective To investigate the effects and mechanism of a new Aurora kinase inhibitor AT9283 on the proliferation,cell cycle,autophagy,and apoptosis of primary breast cancer cells in vitro. Methods The primary breast cancer cells in the logarithmic phase were cultured with AT9283 in vitro for 48 h. The changes of nucleus and spindle were tested by immunofluorescence. The breast cancer primary cells in the logarithmic phase were divided into the experimental groups 1,2,3,4,and 5,which were added with 2 m L AT9283 with the concentrations of 0. 001,0. 01,0. 1,1,and 10μmol/L）,and the control group without any treatment. The cell inhibition rate was examined by MTT assay. Cell cycle of breast cancer primary cells was determined by flow cytometry. The levels of Aurora A,Histone H3,p-Aurora A,p-Histone H3,p21,p53,Bcl-2,and Bax were detected by Western blotting. The primary breast cancer cells were divided into groups A,B,C,and D. Cells in the groups A,B,and C were added with 0. 1,1,10μmol/L AT9283 and then were cultured for 24 h,while cells in the group D were not treated. Autophagy was examined by acridine orange staining method.The apoptosis was tested by flow cytometry. Results AT9283 induced multinuclear and unipolar spindle tested by immunofluorescence. The untreated cells had no change. The experimental groups had cell nuclear morphological changes,and the spindle was monopolar and disordered. AT9283 inhibited the proliferation of primary breast cancer cells after 24 h or 48 h treatment in a dose-dependent and time-dependent manner. Compared with the control group,the percentage of cells in the G2/M phase increased,the percentage of cells in the S and G0/G1 phases decreased in the other groups in a dose-dependent manner（all P〈0. 05）. Compared with the control group,the relative expression levels of p-Aurora A,p-Histone H3,and Bcl-2 decreased in the other groups,and the relative expression levels of p21,p53,and Bax increased（all P〈0. 05）. Autophagy rates in the groups A,B,C,and D were 1. 03% ± 0. 03%

关 键 词：丝/苏氨酸蛋白激酶 乳腺癌 细胞增殖 细胞周期 细胞自噬 细胞凋亡 

分 类 号：R735.1[医药卫生—肿瘤]