摘要
目的:检测热休克蛋白90(heat shock protein 90,HSP90)抑制剂格尔德霉素(geldanamycin,GA)对人肝癌耐多柔比星(adriamycin,ADR)HepG2/ADR细胞耐药性的影响,并进一步探讨其可能的作用机制。方法:用不同浓度的ADR(50、100、150、200和250μmol/L)处理HepG2/ADR细胞后,分别采用实时荧光定量PCR和蛋白质印迹法检测多药耐药1(multiple drug resistance 1,MDR1)基因mRNA及其编码的蛋白P-糖蛋白(P-glycoprotein,P-gp)的表达情况,筛选出能引起MDR1 mRNA和P-gp表达水平显著升高的ADR浓度。将HepG2/ADR细胞维持培养于筛选获得的ADR浓度中,随后分别加入不同浓度的GA(0、100、200、400、600和800 nmol/L)处理细胞24 h,分别采用实时荧光定量PCR法和蛋白质印迹法检测GA对HepG2/ADR细胞中HSP90、突变型p53(mutation p53,Mut-p53)、转录因子SP1(specifi city protein 1)、MDR1mRNA及其蛋白表达的影响;FCM法检测GA对HepG2/ADR细胞摄入ADR能力的影响;MTT法检测的GA联合ADR对HepG2/ADR细胞增殖的影响。结果:当ADR浓度为200μmol/L时,与对照组(未用ADR处理)相比,MDR1 mRNA和P-gp的表达水平分别上调了76.8%和89.3%(P值均<0.01)。将细胞培养于含200μmol/L ADR的细胞培养液中,梯度增加GA的浓度。结果发现与阴性对照(ADR单药作用)组相比,当GA浓度达到100 nmol/L时,HepG2/ADR细胞中MDR1 mRNA和P-gp的表达水平均显著降低(P<0.01和P<0.05)。当GA浓度为800 nmol/L时,HepG2/ADR细胞内MDR1 mRNA和P-gp的表达水平分别降至阴性对照的58.6%和38.3%(P值均<0.01)。FCM法检测结果显示,GA可以增加HepG2/ADR细胞对ADR的摄取量,与阴性对照组相比,当GA浓度为800 nmol/L时,ADR在细胞内的积聚量从13.6%上升至59.7%(P<0.01)。MTT法检测结果显示,联合用药组中GA对HepG2/ADR细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)值为(263.8±5.3)nmol/L,显著低于GA单独作用于HepG2/ADR细胞的(735.5±2.8)nmol/L(P<0.01)。结论:GA能逆转HepG2/ADR细胞对ADR的抗药性,其机制可能是通过HSP90/Mut-p53/SP1信号通路,阻止MDR1基因的转录和表达而实现的。
Objective：To investigate the effect of heat shock protein 90（HSP90） inhibitor geldanamycin（GA） on adriamycin（ADR） resistance of human hepatocellular carcinoma HepG2/ADR cells,and to explore its possible molecular mechanism.Methods：After treatment with different concentrations of ADR（50,100,150,200 and 250 μmol/L）,the expression levels of multiple drug resistance（MDR1） mRNA and P-glycoprotein（P-gp） in HepG2/ADR cells were detected by real-time fluorescent quantitative-PCR and Western blotting,respectively.The certain concentration of ADR which could significantly up-regulate the expression levels of MDR1 mRNA and P-gp was screened out for the next study.The HepG2/ADR cells were cultured with the selected concentration of ADR,then treated with a serial concentrations of GA（0,100,200,400,600 and 800 nmol/L） for 24 h,respectively.The expression levels of HSP90,Mut-p53,specificity protein 1（SP1）（a transcription factor） and MDR1 in HepG2/ADR cells were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.The effect of GA on ADR-intake ability of HepG2/ADR cells was detected by FCM method.The synergistic effect of GA combined with ADR on the proliferation of HepG2/ADR cells was detected by MTT assay.Results：Compared with the blank control group（without ADR treatment）,the levels of MDR1 mRNA and P-gp in HepG2/ADR cells treated with 200 μmol/L ADR could be up-regulated by 76.8% and 89.3%（both P 〈0.01）,respectively.When the HepG2/ADR cells were treated with 200 μmol/L ADR and 100 nmol/L GA,the expression levels of MDR1 mRNA and P-gp were significantly down-regulated as compared with the negative control group（treated with ADR alone）（P 〈0.05）.Furthermore,in HepG2/ADR cells treated with 200 μmol/L ADR and800 nmol/L GA,the levels of MDR1 mRNA and P-gp were decreased to 58.6%（P 〈0.01） and38.3 %（P 〈0.01） of the negative group,respectively.GA（800 nmol/L） could significantly up-regulate the intake level of ADR in HepG2/ADR cells compared with GA-untreated group（59.7% vs 13.6%）（P 〈0.01）.After treatment with 200 μmol/L ADR combined with GA,the half maximal inhibitory concentration（IC50） of GA on HepG2/ADR cells was significantly decreased as compared with GA alone group [（263.8±5.3） nmol/L vs（735.5±2.8） nmol/L]（P 〈0.01）.Conclusion：GA can reverse the ADR-resistance of HepG2/ADR cells.The mechanism may be related to inhibiting the transcription and expression of MDR 1 gene through HSP90/Mut-p53/SP1 signal pathway.
出处
《肿瘤》
CAS
CSCD
北大核心
2016年第4期414-423,共10页
Tumor
