吉西他滨、紫杉醇时序联合对膀胱癌细胞系细胞毒效应研究

Study on the efficacy of scheduled combination of gemcitabine with paclitaxel against human bladder cancer cell lines

目的评价吉西他滨(GEM)、紫杉醇(TAX)时序联合抗膀胱癌细胞系效应。方法MTT法检测GEM与TAX同时(A组)、先后间隔24h时序(B组为GEM先于TAX24h,C组为TAX先于GEM24h)联合用药对膀胱癌细胞系5637和EJ细胞的细胞毒活性,Chou和Talalay方法评估两药物作用关系;流式细胞仪碘化丙啶法检测药物作用细胞后的细胞周期分布变化;AnnexinV检测各组细胞凋亡率的变化;蛋白印迹法检测细胞周期CyclinD1和CyclinB1蛋白的变化。结果药物浓度为1.0μg/ml时,A组5637、EJ细胞生长抑制率为38%、40%,细胞凋亡率为29.3%、15.4%,细胞毒联合指数(CI)均<1;C组5637、EJ细胞生长抑制率为15%、13%,细胞凋亡率为11.5%、6.7%,CI均>1;B组结果与A组相仿。给药第48h细胞周期分布显示细胞B组主峰在G1期积聚(≥60%),A组G1、G2/M期略有增加,C组主峰在G2/M期积聚(≥65%)。A组CyclinD1蛋白表达较无药物对照组下降,CyclinB1表达上调。结论联合应用GEM和TAX治疗膀胱癌,存在时序效应,即同时用药产生协同作用,先用GEM,后用TAX也可产生较好的细胞毒效应,而先用TAX,后用GEM,则产生拮抗作用。

Objective To evaluate the most effective treatment regimen of gemcitabine (GEM) and paclitaxel (TAX) against human bladder cancer cell lines. Methods The bladder cancer cell lines 5637 and EJ were treated with 3 types of drug combinations,ie,GEM plus TAX simultaneously (group A),GEM 24 h ahead of TAX (group B) and TAX 24 h ahead of GEM (group C).Then the cytotoxic activity was evaluated by MTT assay,and the type of drug interaction was assessed using the methods of Chou and Talalay.Cell cycle perturbations and apoptosis were evaluated by flow cytometry.CyclinD_1 and CyclinB_1 expression was determined by Western blot. Results In group A,at the drug concentration of 1.0 μg/ml,the cell growth inhibition rates of 5637 and EJ were 38% and 40%,respectively; and the apoptosis rates were 29.3 % and 15.4%,respectively.The cytotoxic activity combination indices (CI) were less than 1.In group C,the cell growth inhibition rates of 5637 and EJ cell lines were 15% and 13%,respectively,and the apoptosis rates were 11.5% and 6.7%,respectively;and the CIs were more than 1.In group B,the parameters were similar to those in group A.At 48 h after treatment,the cell cycle distribution showed slightly increased G_1,G_2/M phase in group A;accumulated main peaks at G_1 phase (≥60%) in group B;accumulated main peaks at G_2/M phase(≥65%) in group C.In group A,an increase in CyclinB_1 expression and a decrease in CyclinD_1 expression were observed in cell lines. Conclusions The combination of GEM and TAX in vitro yields superior cytotoxic efficacy,if given simultaneously or with GEM first;while if given with TAX first,the antagonistic action of the 2 drugs is yielded.