mTOR信号通路在乳腺癌拉帕替尼耐药细胞系中的作用

Effects of mammalian-target-of-rapamycin pathway on lapatinib resistance in breast cancer MDA-MB-231 cells

目的建立乳腺癌拉帕替尼耐药细胞系，探讨雷帕霉素靶蛋白（mTOR）信号通路在其中的作用。方法拉帕替尼逐步增加剂量诱导人乳腺癌MDA．MB-231细胞构建耐药细胞系rMDA．MB-231；四甲基偶氮唑盐（MW）细胞毒实验检测拉帕替尼对细胞的生长抑制作用；Western印迹检测蛋白的表达改变；Lipofectin2000转染小分子双链RNA干扰序列沉默rMDA-MB-231细胞mTOR基因的表达；异硫氰酸荧光素（FITC）．膜联蛋白V和碘化丙啶双染流式细胞仪检测细胞凋亡率的变化。结果Mrrr细胞毒实验结果显示拉帕替尼对MDA-MB．231和rMDA．MB．231细胞的半数抑制率（IC50）值分别为（6．1±0．6）和（34．9±2．7）Ixmol／L（P〈0．01），耐药5．68倍（P〈0．01）。Western印迹检测显示rMDA-MB-231细胞中p-mTOR蛋白的表达水平明显高于MDA-MB-231细胞。roTOR靶向小干扰RNA（siRNA）特异性的下调了rMDA-MB．231细胞中mTOR的表达。对照组、阴性RNA干扰对照组、roTOR特异性RNA干扰组分别在20Ixmol／L拉帕替尼作用48h后，细胞的凋亡率分别为：13．4％±2．5％、14．2％±2．8％、34．6％±5．8％，对照组和roTOR特异性RNA干扰组之间差异有统计学意义（P〈0．01），对照组和阴性对照之间差异无统计学意义（P〉0．05）。结论乳腺癌拉帕替尼耐药细胞rMDA-MB-231出现了roTOR信号通路的激活，siRNA干扰roTOR基因表达可显著提高拉帕替尼对rMDA．MB-231致凋亡作用，为拉帕替尼耐药的防治和临床用药的选择提供了理论依据。

[ Abstract] Objective To establish a lapatinib resistance cell line for elucidating the mechanisms of drug resistance of lapatinib in human breast cancer cells. Methods The human breast cancer MDA-MB- 231 cells were exposed in an incremental dose of lapatinib to establish a lapatinib resistance rMDA-MB-231 cell line. The assay of methyl thiazolyl tetrazolium （ MTT ） was used to detect the cytotoxic activity of lapatinib against MDA-MB-231 and rMDA-MB-231 cells. The protein expression was detected by Western blot. Small interfering RNA was used to specifically knock down mammalian-target-of-rapamycin （roTOR） in rMDA-MB-231 cells. Apoptosis was determined by fluorescein isothioeyanate （FITC）-annexin V/PI staining and flow cytometry. Results The human breast cancer lapatinib resistance cell line rMDA-MB-231 was induced by lapatinib. The half maximal inhibitory concentration （ ICs0 ） values of lapatinib against MDA-MB- 231 and rMDA-MB-231 cells were （ 6. 1 ± 0. 6 ） and （ 34. 9 ± 2. 7 ） tzmol/L respectively （ P 〈 0. 01 ）. Compared with MDA-MB-231 cells, the protein expresssion of mTOR in rMDA-MB-231 cells was significantly up-regulated. The protein expression of roTOR was significantly down-regulated by specific siRNA duplexes in rMDA-MB-231 cells. After siRNA interference, 20 p, mol/L lapatinib was added into control, negative siRNA control and mTOR-targeted siRNA groups respectively. The percents of cell apoptosis in control, negative control and targeted siRNA groups were 13.4% ±2. 5%, 14. 2% ±2. 8% and 34. 6% ±5.8% respectively, there was no significance between the first two groups（P 〉0. 05）, and there was significant difference between the control and targeted siRNA group （P 〈 0. 01 ）. Conclusions The up- regulation of mTOR plays an important role in the lapatinib-resistant phenotype of human breast cancer rMDA-MB-231 cells. And the down-regulation of mTOR increases the apoptotic death of lapatinib againstrMDA-MB-231 cells.