Xiao Lan, Gao Rui, Lu Shi, Lu Mei-Song, Liang Ming-Lin, Ren Li-Rong, Wang Ze-Hua
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan 430022, China.
Zhonghua Fu Chan Ke Za Zhi. 2007 Jun;42(6):412-6.
To investigate the reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel.
shRNA plasmid vector specifically targeting MDR1 and MDR3 genes was transfected into A2780/taxol cells. The early stage cell apoptosis and the effect of intracellular rhodamine 123 (Rh123) accumulation were detected by flow cytometry (FCM). The late stage cell apoptosis rate was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/taxol cells was determined by methyl thiazolyl tetrazolium (MTT) assay. MDR1 and MDR3 mRNA were assessed by RT-PCR, and caspase-3 protein was detected by western blot.
After treatment with MDR1 and MDR3 shRNA plasmid vector, early apoptosis rate of A2780/taxol cells was (20.21 +/- 0.56)% and (10.87 +/- 1.24)%, respectively. MDR1 and MDR3 shRNA could increase cellular Rh123 accumulation (116.6 +/- 8.1 and 98.4 +/- 3.8, respectively). The late stage apoptosis rates detected by TUNEL displayed the same tendency as FCM results did. The IC(50) for paclitaxel of A2780/taxol cells was decreased significantly. The mRNA levels of MDR1 and MDR3 in A2780/taxol cells were decreased by (73.3 +/- 0.8)% and (51.6 +/- 0.4)% of control, and the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. The expression of caspase-3 protein of MDR1 and MDR3 shRNA vector transfected group in A2780/taxol cells was significantly increased [(80.8 +/- 2.6)% and (72.0 +/- 4.7)%, respectively].
MDR1 and MDR3 gene silencing could recover sensitivity of A2780/taxol cells to paclitaxel and induce cell apoptosis, thus reversing cell resistance to paclitaxel.
探讨MDR1和MDR3基因沉默对A2780/紫杉醇细胞紫杉醇耐药性的逆转作用。
将特异性靶向MDR1和MDR3基因的shRNA质粒载体转染至A2780/紫杉醇细胞。采用流式细胞术（FCM）检测早期细胞凋亡及细胞内罗丹明123（Rh123）蓄积情况。采用末端脱氧核苷酸转移酶（TdT）介导的脱氧尿苷三磷酸（dUTP）缺口末端标记法（TUNEL）检测晚期细胞凋亡率。采用甲基噻唑基四氮唑（MTT）法测定紫杉醇对A2780/紫杉醇细胞的半数抑制浓度（IC50）。采用逆转录聚合酶链反应（RT-PCR）检测MDR1和MDR3 mRNA，采用蛋白质免疫印迹法检测半胱天冬酶-3蛋白。
用MDR1和MDR3 shRNA质粒载体处理后，A2780/紫杉醇细胞早期凋亡率分别为（20.21±0.56）%和（10.87±1.24）%。MDR1和MDR3 shRNA可增加细胞内Rh123蓄积（分别为116.6±8.1和98.4±3.8）。TUNEL检测的晚期凋亡率与FCM结果趋势一致。A2780/紫杉醇细胞对紫杉醇的IC50显著降低。A2780/紫杉醇细胞中MDR1和MDR3的mRNA水平分别降低至对照的（73.3±0.8）%和（51.6±0.4）%，且MDR1和MDR3 mRNA的降低呈时间依赖性。转染MDR1和MDR3 shRNA载体组的A2780/紫杉醇细胞中半胱天冬酶-3蛋白表达显著增加[分别为（80.8±2.6）%和（72.0±4.7）%]。
MDR1和MDR3基因沉默可恢复A2780/紫杉醇细胞对紫杉醇的敏感性并诱导细胞凋亡，从而逆转细胞对紫杉醇的耐药性。
