维拉帕米逆转人食管鳞状细胞癌细胞株对顺铂耐药性的研究

Reversal of cisplatin resistance in human esophageal squamous cell carcinoma cell lines by verapamil

目的观察维拉帕米逆转人食管癌细胞株对顺铂耐药程度,探讨维拉帕米逆转耐药与多药耐药蛋白P-gp的关系。方法CCK-8法测5-氟尿嘧啶、阿霉素、顺铂对2种食管癌细胞株(KYSE-150、KYSE-510)的IC50值(IC501);联合维拉帕米的IC50值(IC502),以IC501/IC502结果判定维拉帕米逆转耐药能力。实时荧光定量PCR测各组多药耐药基因hMDR-1表达。Western-blotting测各组P-gp表达。结果阿霉素、顺铂在KYSE-150的IC50值为7.33±0.67、3.23±0.06均小于KYSE-510的IC50值11.57±0.98、17.25±0.08,5-氟尿嘧啶在KYSE-150的IC50值24.33±1.67大于KYSE-510的IC50值14.67±1.02。联用维拉帕米后,各组IC50值均下降,提示维拉帕米提高药物敏感性。其中,顺铂组加入维拉帕米后,KYSE-510的IC50值变化较大(4.7倍)提示逆转敏感,KYSE-150的IC50值变化较小(1.84倍)提示逆转耐受。应用维拉帕米前后,各组hMDR1/P-gp表达没有明显变化。结论KYSE-510对维拉帕米联合顺铂逆转敏感,而KYSE-150逆转耐受。维拉帕米逆转人食管癌细胞株耐顺铂不是通过改变hMDR1/P-gp的数量,而可能通过阻碍hMDR1/P-gp作用或其他未知机制来实现。

Objective To the effect of verapamil on chemosensitivity of human esophageal squamous cell carcinoma cell lines and to investigate the relationship between the resistance of verapamil to reverse and P-gp.Methods The IC50 values(IC501)of three kinds of chemotherapy drugs:Adriamycin,cisplatin,and 5-fluorouracil were detected by CCK-8 method in 2 kinds of esophageal cancer cell lines(KYSE-150 and KYSE-510).Each cell was processed by verapamil(4.91μg/mL)combined with the chemotherapy treatment above,IC50(IC502)value was detected by CCK-8 assay combined with chemotherapy for the verapamil of the 2 esophageal carcinoma cell lines(KYSE-150 and KYSE-510).Real-time PCR was used to measure the expression of multidrug resistance genes hMDR1 in various treatment groups of esophageal cancer cells.Expression of P-glycoprotein in esophageal cancer cells in each treatment group by Western bolt method.Results The IC50 values of adriamycin and cisplatin in KYSE-150 cells 7.33±0.67,3.23±0.06 were both less than that in KYSE-510 cells 11.57±0.98,17.25±0.08,while the IC50 values of 5-fluorouracil in KYSE-150 cells 24.33±1.67 was significantly higher than that in KYES-510 cells 14.67±1.02.After adding verapamil,the IC50 values of Adriamycin,cisplatin and 5-fluorouracil in two esophageal cancer cell lines(KYSE-150 and KYSE-510)all decreased to different degrees,and the above three kinds of chemotherapeutic drugs had increased their sensitivity to varying degrees.Among them,the cisplatin group before adding verapamil had different degrees of resistance compared with KYSE-150 cells and KYSE-510 cells.After joining verapamil,IC50 value change for the KYSE-510 cells multiplied for 4.7 times,suggesting that the reversal was sensitive;IC50 value change for KYSE-150 cells was 1.84 times smaller,suggesting that the reversal was tolerant.The expression of hMDR1/P-gp in each cell group did not change significantly before and after the application of verapamil.Conclusion Tip the KYSE-510 cells(DDP)sensitivity to the reversal of verapamil,and KYSE-150 cells(DDP)reverse resistance of verapamil.Verapamil reverses drug.