摘要
目的探讨汉防己甲素(Tet)联合阿霉素(DOX)体外逆转耐药型前列腺癌(DU145/DOX)细胞耐药的可行性及潜在机制。方法采用经典的DOX浓度梯度诱导法制备DU145/DOX细胞,然后采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法测定Tet干预对DU145/DOX细胞生长抑制的作用,凋亡试剂盒测定联合用药对耐药细胞凋亡诱导的影响,流式细胞仪和荧光分光光度计分别测定Tet对DOX和荧光探针罗丹明123(R123)被DU145/DOX细胞摄取行为,Pgp-Glo TM分析系统测定Tet对P-糖蛋白(P-gp)ATP酶活性的影响,Western blot法测定Tet对耐药细胞膜上P-gp蛋白表达的影响,RT-PCR法测定Tet对DU145/DOX细胞MDR1 m RNA表达的影响。结果 Tet组、DOX组、DOX+Tet(1/5)组对DU145/DOX细胞的半数抑制浓度(IC50,以COX浓度计算)分别为(7.16±0.54)、(1.62±0.09)、(0.37±0.03)μM,Tet能明显增强DOX的抗肿瘤作用。DOX+Tet(1/5)组诱导DU145/DOX细胞凋亡率是DOX组的2.1倍。Tet的参与能明显提高DU145/DOX细胞内DOX和R123的累积量,其机制可能与Tet刺激细胞内P-gp ATP酶活性、降低细胞膜上P-gp蛋白表达有关。Tet可能通过降低耐药基因表达的机制,联合DOX提高对DU145/DOX细胞杀伤作用。结论 Tet与DOX体外联合使用具有逆转DU145/DOX细胞耐药的潜力,作用机制可能涉及刺激耐药泵酶活性、降低耐药蛋白表达量、下调MDR1 m RNA表达等。
Objective To investigate the effect of combination of tetrandrine(Tet)with doxorubicin(DOX)on drug resistance of prostate carcinoma DU145/DOX cells and its molecular mechanisms.Methods Drug resistant prostate cancer DU145/DOX cells were induced,and treated with Tet,DOX or Tet+DOX,respectively.The cell growth was assayed by MTT method,cells apoptosis was determined with AnnexinV-PE/7-AAD kit,the intracellular accumulation of DOX and R123 was tested by flow cytometry and fluorospectrophotometer,the P-gp ATPase activity and P-gp expression were detected by Pgp-GloTM Assay Systems and Western blot method,respectively,the expression of MDR1 mRNA was detected by RT-PCR.Results The IC50 of Tet,DOX and Tet+DOX against DU145/DOX cells were(7.16±0.54),(1.62±0.09)and(0.37±0.03)滋M,respectively.The apoptosis induction ratio of DU145/DOX cells treated with Tet+DOX was 2.1-folded of DOX group.The intracellular accumulation of DOX and R123 increased significantly in the presence of Tet.The P-gp ATPase activity was elevated,the expression P-gp and MDR1 mRNA was down-regulated in DU145/DOX cells after Tet treatment.Conclusion The combination of Tet with DOX can reverse drug resistance of DU145/DOX cells in vitro,which is associated with the effects of Tet in interfering P-gp function and suppressing MDR1 mRNA expression.
作者
李忠孝
葛启斌
王俊龙
郑璐娜
袁菱
程红涛
CHENG Zhongxiao;GE Qibin;WANG Junlong(Department of Urology,Shulan(Hangzhou)Hospital,Hangzhou 310004,China)
出处
《浙江医学》
CAS
2018年第13期1432-1437,共6页
Zhejiang Medical Journal
