AT9283对基底样乳腺癌细胞侵袭及转移的影响

Effects of Aurora kinase inhibitor AT9283 on invasion and metastasis of basal-like breast cancer cells

目的研究Aurora激酶抑制剂AT9283对基底样乳腺癌细胞(BLBC)系MDA-MB-231细胞运动能力和凋亡的影响,探讨AT9283降低BLBC侵袭和转移的作用机制。方法取不同浓度(0、0.01、0.1、1、10μmol/L)AT9283处理MDA-MB-231细胞,MTT法检测细胞增殖抑制率;PI染色流式细胞术检测多倍体细胞形成,Annexin V/PI双染流式细胞术、荧光染料显色观察不同浓度下AT9283诱导MDA-MB-231细胞凋亡情况;Western blotting检测凋亡相关蛋白的表达变化和Aurora激酶、Cofilin-1及磷酸化水平的改变;划痕试验及趋化试验观察AT9283对细胞迁移、趋化能力的影响。结果AT9283(10μmol/L)处理MDA-MB-231细胞24 h后,细胞增殖抑制率为(89.17±0.03)%,荧光染色可见AT9283处理的胞核绿染、碎裂;流式细胞术检测1μmol/L AT9283作用于乳腺癌细胞48 h形成多倍体细胞,0.1、1、10μmol/L AT9283作用下MDA-MB-231细胞凋亡率分别为(13.4±2.5)%、(29.5±3.4)%、(52.8±6.7)%,与对照组的(0.7±0.1)%相比,均明显升高(P<0.05)。同时,抗凋亡蛋白Bcl-XL表达减少,凋亡相关蛋白Caspase-3和PARP蛋白剪切增加。AT9283可显著延长划痕愈合时间,减少趋化穿膜细胞个数(P<0.05),并有效抑制Aurora激酶及Cofilin-1的磷酸化水平。结论AT9283可通过抑制Aurora激酶的磷酸化及Cofilin-1磷酸化水平而诱导BLBC细胞系凋亡,并抑制其运动,从而抑制侵袭及转移。

Objective To investigate the effects of Aurora kinase inhibitor AT9283 on the apoptosis,motility of basal-like breast cancer(BLBC)cells MDA-MB-231,and to explore the mechanism of AT9283 to reduce the invasion and metastasis of BLBC.Methods The different concentrations of AT9283(0,0.01,0.1,1,10μmol/L)were designed to deal with MDA-MB-231.Inhibitory rate of cell proliferation was detected by MTT.The variety of polyploid formulation was tested by flow cytometry with cells stained by propidium iodide(PI).Morphological change of apoptotic cells was analyzed by Yo-pro-1 staining.The apoptotic rate of MDA-MB-231 cells were determined by Annexin V/PI double-staining.The expression of apoptosis related proteins(Bcl-XL,Caspase-3 and PARP)and phosphorylation levels of Aurora kinase and Cofilin-1 were analyzed by Western blotting.The capability of motility was measured by wound-healing assay and chemotaxis assay.Results After treatment of AT9283(10μmol/L)for 24 h,inhibitory rate of cell proliferation was(89.17±0.03)%.Fluorescence staining showed that the nuclei treated with AT9283 were stained green and the nuclei were fragmented.The formation of polyploid cells began at 48 hours after the addition of 1μmol/L AT9283 to MD-MB-231 cells.AT9283 induced obviously the apoptosis of MDA-MB-231 cells(P<0.05),and the apoptotic rates were(13.4±2.5)%,(29.5±3.4)%,(52.8±6.7)%.Compared with(0.7±0.1)%of the control group,they were significantly increased(P<0.05).AT9283 effectively downregulated the expressions of Bcl-XL and promoted the dissection of Caspase-3 and PARP.The effect displayed obvious dose-effect relationship.AT9283 could significantly extend the healing time of scratches,decrease the migration cell count(P<0.05),and inhibit the phosphorylation of Aurora kinase and Cofilin-1 in a dose-dependent manner.Conclusion Aurora kinase inhibitor AT9283 can induce apoptosis and the motility of breast cancer cell line MDA-MB-231 through inhibiting phosphorylation of Aurora kinase and Cofilin-1.