天然产物EM-E-11-4逆转人口腔上皮癌细胞KBV_(200)多药耐药作用及其机制

Reversal of multidrug resistance in human oral epithelial cancer cells KBV_(200) by a natural product

目的考察无毒浓度千金烷二帖化合物EM-E-11-4对P-糖蛋白(P-glycoprotein,P-gp)介导人口腔上皮癌多药耐药(multi-drug resistance,MDR)的逆转作用并检测其对P-gp功能的影响。方法MTT比色法测定EM-E-11-4对人口腔上皮癌KB及其具有多药耐药株表型的KBV_(200)细胞的细胞毒活性,采用维拉帕米为参比药,非细胞毒剂量EM-E-11-4(2.5、5、10μmol·L^(-1))与3种常用抗癌药合用考察其逆转肿瘤多药耐药的作用,并计算逆转倍数。EME-11-4对P-gp的转运功能采用阿霉素蓄积实验检测。EM-E-11-4对基础与维拉帕米诱导的P-gp ATPase活性采用P-gp-GloTM试剂盒检测。结果EM-E-11-4能够有效逆转P-gp介导的KBV_(200)的多药耐药,并具有良好的剂量依赖关系。10μmol·L^(-1)EM-E-11-4能够逆转KBV_(200)细胞对紫杉醇、长春新碱和阿霉素3种抗癌药物的耐药作用,逆转倍数分别为33.8、36.4、20.1。无细胞毒作用的EM-E-11-4对P-gp的转运功能有显著抑制作用,并且能够增加阿霉素在肿瘤细胞内的蓄积,具有良好的浓度依赖性。同时,EM-E-11-4能明显抑制维拉帕米刺激的重组P-gp的ATPase活性。结论EM-E-11-4能逆转P-gp介导的肿瘤多药耐药,其作用机制与抑制P-gp的转运功能有关,为其临床治疗口腔上皮癌肿瘤多药耐药提供了理论依据。

Objective To investigate the ability of a natural product EM-E-11-4 at non-cytotoxic concentrations to reverse P-gp-mediated multidrug resistance(MDR)in human oral epithelial cancer cells KBV_(200)and to reveal the mechanism of action.Methods The effect of EM-E-11-4 on the growth of KB cells and the corresponding resistant cells KBv200 was determined by MTT assay.Using VRP as positive control,the ability of non-cytotoxic concentrations of EM-E-11-4(2.5,5,10μmol·L^(-1))with/without such chemotherapeutic agents as paclitaxel vincristine and adriamycin to reverse MDR was evaluated,while the reversal folds(RF)of EM-E-11-4 and VRP were calculated and compared.The effect of EME-11-4 and VRP on the accumulation and efflux function of P-gp was studied.Furthermore,the effect of EM-E-11-4 on the ATPase activity of P-gp was measured by Pgp-GloTM kit.Results 10μmol·L^(-1)of EM-E-11-4 effectively reversed the MDR-phenotype of KBv200 cells to paclitaxel,vincristine and adriamycin.The RF was 33.8、36.4 and 20.1,respectively.In addition,the P-gp functional study suggested that EM-E-11-4 elevated the intracellular accumulation of adriamycin in a dose-dependent manner.EM-E-11-4 suppressed VRP-stimulated ATPase activity of P-gp in a dose-dependent manner.Conclusion EM-E-11-4 can be a potential agent that can overcome P-gp-mediated MDR by suppressing the transport function of P-gp.