三阴性乳腺癌细胞CAL-51对多西他赛的耐药机制

Mechanisms of docetaxel resistance in triple negative breast cancer cell line CAL-51

目的通过比较分析多西他赛(多西紫杉醇,docetaxel)对三阴性乳腺癌细胞系CAL-51和非三阴乳腺癌细胞系T47D的杀伤敏感性差异,探讨CAL-51对多西他赛耐药的可能机制。方法 MTT法测定不同剂量多西他赛对CAL-51和T47D细胞生长的影响并计算IC_(50)值;瑞氏-吉姆萨染色分析多西他赛作用后对两种细胞形态的变化;流式细胞仪(FCM)分析多西他赛对细胞周期的分布及凋亡情况的影响;荧光定量PCR检测分析多个基因在两种细胞中的差异表达;Western印迹法检测抗凋亡蛋白Bcl-2和胱天蛋白酶(caspase)家族蛋白在两种细胞中表达水平的差异。结果 T47D细胞经多西他赛处理后,形态出现显著变化;流式细胞检测显示,多西他赛能明显诱导非三阴乳腺癌细胞系T47D凋亡,与三阴性乳腺癌细胞系CAL-51相比具有显著性差异(P<0.01);定量PCR结果显示,CAL-51中抗凋亡基因Bcl-2高表达,与T47D相比具有显著性差异(P<0.05);蛋白质印迹法显示加药后两种细胞皆能活化内源性凋亡途径,但下游效应caspase的活化途径有所不同。结论多西他赛诱导2种细胞产生凋亡的内源性途径有所不同,Bcl-2的高表达可能是CAL-51细胞对多西他赛耐药的机制之一。

Objective To compare sensitive difference of docetaxel between the triple negative breast cancer（TNBC）cell line CAL-51 and non TNBC line T47D and analyze mechanisms underlying docetaxel resistance in former cells. Methods Cell activi-ty was determined by MTT method and IC50 value was calculated;Wright-Giemsa stain was used to analyze the effect of docetaxel in the morphology of CAL-51 and T47D cell lines. Flow cytometry（FCM）was performed to determine cell cycle distribution and apoptosis. Realtime fluorescence quantitative PCR was used to compare the relative gene expression levels.The anti-apoptosis protein Bcl-2 and caspase family protein expression levels were determined by Western blot. Results Wright-Giemsa stain showed significant morpholo-gy change in T47D cells by docetaxel treatment. Further flow cytometry results confirmed that docetaxel could significantly induce apoptosis in T47D cells compared to CAL-51 cells（P〈0.01）. The result of realtime fluorescence quantitative PCR revealed that anti-apoptosis protein Bcl-2 was significantly higher expressed in CAL-51 cells（P〈0.05）. Immunoblot analysis revealed docetaxel treat-ment induced the instrinsic pathways in both CAL-51and T47D cells,but the activated pathway of executioner caspase was different. Conclusion Our present study shows that docetaxel induces different intrinsic apoptosis pathway in CAL-51 and T47D cell lines. An-ti-apoprosis protein Bcl-2 is highly expressed,which might be the underlying mechanism of docetaxel resistance in TNBC cell line-CAL-51.