中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2015年
5期
983-985
,共3页
杨永刚%周敏%江学庆%江明
楊永剛%週敏%江學慶%江明
양영강%주민%강학경%강명
乳腺癌%溶酶体相关膜蛋白2A%阿霉素
乳腺癌%溶酶體相關膜蛋白2A%阿黴素
유선암%용매체상관막단백2A%아매소
Breast cancer%Lysosome-associated membrane protein type 2A%Doxorubicin
目的 探讨溶酶体相关膜蛋白2A (LAMP2A)对乳腺癌细胞分子伴侣介导自噬(CMA)的作用及机制,以及对阿霉素敏感性的影响.方法 使用小分子干扰RNA敲除以及质粒DNA过表达乳腺癌细胞中的LAMP2A.Western blot法检测蛋白水平的变化,CellTiter-Glo发光法检测细胞活性,2',7'-二氯氢化荧光素乙二脂(DCF-DA)染色法结合荧光酶标仪检测细胞内活性氧(ROS)水平,原位缺口末端标记法(TUNEL)法检测细胞凋亡.结果 过表达乳腺癌细胞LAMP2A后CMA活性、细胞生存能力升高,MCF-7与T47D细胞系中敲除或过表达LAMP2A后,与对照组相比,CMA活性、细胞生存能力在300nmol/L时差异最明显[(70.0±2.1)％比(52.0±1.8)％、(54.0±3.4)％比(76.0±1.5)％;(62.4±1.9)比(50.0±3.2)％、(60.0±1.1)％比(72.0±1.8)％,P＜0.05].敲除或过表达LAMP2A后,在T47D和MCF-7细胞中的ROS水平(0.89 ±0.32比1.11±0.41;1.00±0.00比1.37±0.24)、细胞凋亡(2.10±0.52比1.59 ±0.38;0.91 ±0.12比1.37±0.24)及对阿霉素的敏感性增加(84.33±3.20比54.73±2.90)或降低(82.70±3.70比53.90±2.30,P＜0.05).结论 LAMP2A能够诱导乳腺癌细胞的分子伴侣介导的自噬并影响对阿霉素的敏感性.
目的 探討溶酶體相關膜蛋白2A (LAMP2A)對乳腺癌細胞分子伴侶介導自噬(CMA)的作用及機製,以及對阿黴素敏感性的影響.方法 使用小分子榦擾RNA敲除以及質粒DNA過錶達乳腺癌細胞中的LAMP2A.Western blot法檢測蛋白水平的變化,CellTiter-Glo髮光法檢測細胞活性,2',7'-二氯氫化熒光素乙二脂(DCF-DA)染色法結閤熒光酶標儀檢測細胞內活性氧(ROS)水平,原位缺口末耑標記法(TUNEL)法檢測細胞凋亡.結果 過錶達乳腺癌細胞LAMP2A後CMA活性、細胞生存能力升高,MCF-7與T47D細胞繫中敲除或過錶達LAMP2A後,與對照組相比,CMA活性、細胞生存能力在300nmol/L時差異最明顯[(70.0±2.1)％比(52.0±1.8)％、(54.0±3.4)％比(76.0±1.5)％;(62.4±1.9)比(50.0±3.2)％、(60.0±1.1)％比(72.0±1.8)％,P＜0.05].敲除或過錶達LAMP2A後,在T47D和MCF-7細胞中的ROS水平(0.89 ±0.32比1.11±0.41;1.00±0.00比1.37±0.24)、細胞凋亡(2.10±0.52比1.59 ±0.38;0.91 ±0.12比1.37±0.24)及對阿黴素的敏感性增加(84.33±3.20比54.73±2.90)或降低(82.70±3.70比53.90±2.30,P＜0.05).結論 LAMP2A能夠誘導乳腺癌細胞的分子伴侶介導的自噬併影響對阿黴素的敏感性.
목적 탐토용매체상관막단백2A (LAMP2A)대유선암세포분자반려개도자서(CMA)적작용급궤제,이급대아매소민감성적영향.방법 사용소분자간우RNA고제이급질립DNA과표체유선암세포중적LAMP2A.Western blot법검측단백수평적변화,CellTiter-Glo발광법검측세포활성,2',7'-이록경화형광소을이지(DCF-DA)염색법결합형광매표의검측세포내활성양(ROS)수평,원위결구말단표기법(TUNEL)법검측세포조망.결과 과표체유선암세포LAMP2A후CMA활성、세포생존능력승고,MCF-7여T47D세포계중고제혹과표체LAMP2A후,여대조조상비,CMA활성、세포생존능력재300nmol/L시차이최명현[(70.0±2.1)％비(52.0±1.8)％、(54.0±3.4)％비(76.0±1.5)％;(62.4±1.9)비(50.0±3.2)％、(60.0±1.1)％비(72.0±1.8)％,P＜0.05].고제혹과표체LAMP2A후,재T47D화MCF-7세포중적ROS수평(0.89 ±0.32비1.11±0.41;1.00±0.00비1.37±0.24)、세포조망(2.10±0.52비1.59 ±0.38;0.91 ±0.12비1.37±0.24)급대아매소적민감성증가(84.33±3.20비54.73±2.90)혹강저(82.70±3.70비53.90±2.30,P＜0.05).결론 LAMP2A능구유도유선암세포적분자반려개도적자서병영향대아매소적민감성.
Objective To sutdy the effect of lysosome-associated membrane protein type 2A (LAMP2A)-induced chaperone-mediated autophagy on sensitivity to doxorobicin in breast cancer cells.Methods Small interfering RNA (siRNA) and plasmid cDNA (pcDNA) were used to knock down or overexpress LAMP2A in breast cancer cells respectively.Western blotting analysis was used to detect the expression of LAMP2A,and DCF-DA was performed to evaluate the level of reactive oxygen species (ROS).The terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) teschnique was used to measure cell apoptosis.Results In cell lines T47D 与 MCF-7,LAMP2A overexpression or knock-out changed the CMA activity and cell suvival ability in breast cancer cells when the concentration ofH2O2is300nmol/L[(70.0±2.1)％ vs.(52.0±1.8)％,(54.0±3.4)％ vs.(76.0±1.5)％;(62.4 ± 1.9) vs.(50.0 ± 3.2) ％,(60.0 ± 1.1) ％ vs.(72.0 ± 1.8) ％,P ＜ 0.05].Furthermore,inhibition of LAMP2A stimulates accumulation of ROS (0.89 ± 0.32 vs.1.11 ± 0.41;1.00 ± 0.00 vs.1.37 ±0.24),and induction of cellular apoptosis in breast cancer cells(2.10 ±0.52 vs.1.59 ±0.38;0.91 ± 0.12 vs.1.37 ± 0.24).Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to doxorubicin (84.33 ±3.20 vs.54.73 ±2.90),(82.70 ±3.70 vs.53.90 ±2.30,P＜0.05).Conclusion Inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.
