丹皮酚对肝癌 HepG2／ADM 细胞株多药耐药性的逆转作用及其机制

摘要

目的：探讨丹皮酚体外给药对肝癌耐药细胞株HepG2／阿霉素（ADM）多药耐药性的逆转作用及其可能分子机制。方法采用人的肝癌细胞株HepG2，体外ADM浓度递增法诱导建立耐药肝癌细胞HepG2／ADM 耐药模型。分别用0、5、10、25、50、100μmol／L的丹皮酚培养液培养HepG2／ADM、HepG2细胞48 h，选定25、50μmol／L丹皮酚进行下一步实验。分别采用ADM（0．01、0．2、0．5、0．75、1．5、3．0μmol／L）和氟尿嘧啶（0、1、2、4、10、20、40μmol／L）单独刺激，及25、50μmol／L丹皮酚联合上述各浓度的氟尿嘧啶及ADM联合刺激2种细胞株。采用MTT法检测HepG2／ADM、HepG2细胞的增殖水平，流式细胞仪检测HepG2／ADM、HepG2细胞内细胞毒药物含量，蛋白免疫印迹法检测肿瘤细胞P-糖蛋白表达水平。结果 ADM抑制HepG2／ADM、HepG2细胞株的IC50依次为1．48、124．14μmol／L，耐药指数为83．9；丹皮酚（25、50μmol／L）能够明显增强ADM、氟尿嘧啶对HepG2／ADM细胞株的细胞毒作用，并降低IC50值（P均＜0．01），但对HepG2细胞的细胞毒作用无明显影响；丹皮酚（25、50μmol／L）能够明显增加HepG2／ADM细胞株对ADM的摄取能力（P均＜0．01），但对HepG2细胞株无明显影响；丹皮酚能显著降低HepG2／ADM细胞株P-糖蛋白表达水平（P均＜0．01），但对HepG2细胞株P-糖蛋白水平无明显影响。结论丹皮酚对HepG2／ADM细胞株多药耐药性具有明显的逆转作用，可能与其抑制HepG2／ADM细胞株P-糖蛋白表达、增加细胞毒药物细胞内摄取有关。%Objective To investigate the reversal activity of paeonol on multidrug resistance of liver cancer cell line HepG2/adriamycin ( ADM) and its potential molecular mechanism.Methods The human HepG2 cell line was used to make the HepG2/ADM model which was induced by the increased concentrations of adriamycin (ADM).MTT assay was performed to detect the proliferation of HepG2/ADM and HepG2.The flow cytometer was used to measure the intracellular accumulation of cytotoxic drugs in HepG2/ADM and HepG2.Western blotting was carried out to determine the levels of P-glycoprotein.Results Paeonol with the concentrations from 0 to 50 μmol/L had no cytotoxicity to the HepG2/ADM and HepG2.The IC50 values for inhibiting proliferation of HepG2/ADM and HepG2 were 1.48 μM and 124.14 μM, and the resistance index was 83.9.Paeonol (25 μmol/L, 50 μmol/L) could significantly enhance the cytotoxic effects of ADM and 5-fluorouracil on HepG2/ADM, and therefore reduced their IC50 values (P<0.01), while It had no effect on those of HepG2.Moreover, Paeonol increased the accumulation of ADM in HepG2/ADM ( all P<0.01) , but not in HepG2.The further study found that Paeonol obviously reduced the expression level of P-glycoprotein in HepG2/ADM (all P<0.01), while had no effect on that of HepG2.Conclusion Paeonol can reverse the multidrug resistance of HepG2/ADM signifi-cantly, which may be related with the inhibition of P-glycoprotein expression and the increased accumulation of cytotoxic drugs.