CAJ | 학술논문

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(MTT)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；Lipofectin 2000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 MTT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)μmol/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.mTOR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、mTOR特异性RNA干扰组分别在20 μmol/L拉帕替尼作用48 h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mTOR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mTOR信号通路的激活,siRNA干扰mTOR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.
목적 건립유선암랍파체니내약세포계,탐토뢰파매소파단백(mTOR)신호통로재기중적작용.방법 랍파체니축보증가제량유도인유선암MDA-MB-231세포구건내약세포계rMDA-MB-231；사갑기우담서염(MTT)세포독실험검측랍파체니대세포적생장억제작용；Western인적검측단백적표체개변；Lipofectin 2000전염소분자쌍련RNA간우서렬침묵rMDA-MB-231세포mTOR기인적표체；이류청산형광소(FITC)-막련단백V화전화병정쌍염류식세포의검측세포조망솔적변화.결과 MTT세포독실험결과현시랍파체니대MDA-MB-231화rMDA-MB-231세포적반수억제솔(IC50)치분별위(6.1±0.6)화(34.9±2.7)μmol/L(P ＜0.01),내약5.68배(P＜0.01).Western인적검측현시rMDA-MB-231세포중p-mTOR단백적표체수평명현고우MDA-MB-231세포.mTOR파향소간우RNA (siRNA)특이성적하조료rMDA-MB-231세포중mTOR적표체.대조조、음성RNA간우대조조、mTOR특이성RNA간우조분별재20 μmol/L랍파체니작용48 h후,세포적조망솔분별위:13.4％±2.5％、14.2％±2.8％、34.6％±5.8％,대조조화mTOR특이성RNA간우조지간차이유통계학의의(P＜0.01),대조조화음성대조지간차이무통계학의의(P＞0.05).결론 유선암랍파체니내약세포rMDA-MB-231출현료mTOR신호통로적격활,siRNA간우mTOR기인표체가현저제고랍파체니대rMDA-MB-231치조망작용,위랍파체니내약적방치화림상용약적선택제공료이론의거.
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 (MTF) 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 (mTOR) in rMDA-MB-231 cells.Apoptosis was determined by fluorescein isothiocyanate (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 (IC50) values of lapatinib against MDA-MB-231 and rMDA-MB-231 cells were (6.1 ± 0.6) and (34.9 ± 2.7) μmol/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 mTOR was significantly down-regulated by specific siRNA duplexes in rMDA-MB-231 cells.After siRNA interference,20 μmol/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 upregulation 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 against rMDA-MB-231 cells.