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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 the drug resistance of HepG2 / ADR cells induced by adriamycin (ADR) And further explore its possible mechanism of action. Methods: HepG2 / ADR cells were treated with different concentrations of ADR (50, 100, 150, 200 and 250 μmol / L). Real time quantitative PCR and Western blotting were used to detect the expression of multiple drug resistance 1 ) Gene and P-glycoprotein (P-glycoprotein) expression were detected by RT-PCR. The concentrations of ADR which caused the significant increase of MDR1 mRNA and P-gp expression were screened out. HepG2 / ADR cells were maintained in the ADR concentration screening, and then were treated with different concentrations of GA (0,100,200,400,600 and 800 nmol / L) for 24 hours, respectively, using real-time fluorescence quantitative PCR And Western blotting were used to detect the effect of GA on the expression of HSP90, mutation p53 (Mut p53), SP1 (specifi c city protein 1) and MDR1 mRNA and protein in HepG2 / ADR cells; FCM was used to detect the effect of GA on HepG2 / ADR cells into ADR ability; MTT assay GA combined with ADR on HepG2 / ADR cell proliferation. Results: Compared with the control group (without ADR treatment), the expression levels of MDR1 mRNA and P-gp were up-regulated by 76.8% and 89.3% (P <0.01) when ADR concentration was 200μmol / L. The cells were cultured in cell culture medium containing 200 μmol / L ADR, and the concentration of GA was increased in a gradient. The results showed that the expression of MDR1 mRNA and P-gp in HepG2 / ADR cells were significantly decreased (P <0.01 and P <0.05, respectively) when the concentration of GA was 100 nmol / L compared with the negative control ). When the concentration of GA was 800 nmol / L, the expression levels of MDR1 mRNA and P-gp in HepG2 / ADR cells decreased to 58.6% and 38.3% of the negative control (P <0.01). The result of FCM showed that GA could increase ADR uptake in HepG2 / ADR cells. Compared with the negative control group, when GA concentration was 800 nmol / L, the accumulation of ADR in cells increased from 13.6% to 59.7% (P <0.01). The results of MTT assay showed that the half maximal inhibitory concentration (IC50) of GA on HepG2 / ADR cells was (263.8 ± 5.3) nmol / L in combination group, which was significantly lower than that of GA alone on HepG2 / ADR cells (735.5 ± 2.8) nmol / L (P <0.01). CONCLUSION: GA can reverse the resistance of HepG2 / ADR cells to ADR. The mechanism may be through the HSP90 / Mut-p53 / SP1 signaling pathway, preventing the transcription and expression of MDR1 gene.