17-AAG联合紫杉醇对人未分化甲状腺癌FRO细胞增殖和凋亡影响的研究

Effect of 17-AAG Combining with Paclitaxel on Proliferation and Apoptosis of Human Anaplastic Thyroid Cancer FRO Cell Line

目的探讨热休克蛋白90(HSP90)抑制剂17-丙烯胺基-17-去甲氧格尔德霉素(17-AAG)联合紫杉醇对人未分化甲状腺癌FRO细胞增殖和凋亡的影响。方法 1采用四甲基偶氮唑盐微量酶反应比色法(MTT比色法)测定不同浓度(17-AAG:0.312 5、0.625 0、1.250 0、2.500 0及5.000 0μmol/L;紫杉醇:0.001 0、0.010 0、0.100 0及1.000 0μmol/L)、不同时间(24、48及72 h)17-AAG、紫杉醇单药和联合处理(17-AAG:0.625 0μmol/L,紫杉醇:0.001 0、0.010 0、0.100 0及1.000 0μmol/L)后FRO细胞的增殖抑制率。2采用流式细胞仪检测17-AAG、紫杉醇单药及联合处理24 h后(17-AAG:0.625 0μmol/L、紫杉醇:0.100 0μmol/L;联合用药:17-AAG的浓度为0.625 0μmol/L,紫杉醇的浓度为0.100 0μmol/L)FRO细胞的细胞周期变化及凋亡率。3采用胱天蛋白酶-3(Caspase-3)和Caspase-9检测试剂盒检测17-AAG、紫杉醇单药及联合处理24 h后(17-AAG:0.625 0μmol/L、紫杉醇:0.100 0μmol/L;联合用药:17-AAG的浓度为0.625 0μmol/L,紫杉醇的浓度为0.100 0μmol/L)后FRO细胞中的Caspase-3和Caspase-9活性。空白对照组均不加任何药物,只加培养液。结果 1同时点空白对照组、各剂量17-AAG组/紫杉醇组/17-AAG联合紫杉醇组的增殖抑制率随浓度升高而逐渐升高,任2组比较差异均有统计学意义(P<0.05);各剂量17-AAG组/紫杉醇组/17-AAG联合紫杉醇组的增殖抑制率在24、28及72 h逐渐增高,任2组比较差异均有统计学意义(P<0.05);同时点同浓度情况下,17-AAG联合紫杉醇组的增殖抑制率均高于单独用药组(P<0.05)。各时点17-AAG与紫杉醇联合的q值均大于1.15,两者之间呈协同作用。2 17-AAG组、紫杉醇组及17-AAG联合紫杉醇组FRO细胞的凋亡率均明显高于空白对照组(P<0.05),且17-AAG联合紫杉醇组FRO细胞的凋亡率高于17-AAG组和紫杉醇组(P<0.05)。3 17-AAG组、紫杉醇组及17-AAG联合紫杉醇组FRO细胞的Caspase-3和Caspase-9活性均高于空白对照组(P<0.05);且17-AAG联合紫杉醇组细胞的Caspase-3和Caspase-9活性均高于17-AAG组和紫杉醇组相应指标(P<0.05)。结论 17-AAG和紫杉醇均可明显抑制FRO细胞的增殖并诱导细胞凋亡,联合用药有一定的协同效应,呈剂量依赖关系。

Objective To investigate the inhibitory effect of heat shock protein 90（HSP90） inhibitors of 17-propylene amino-17-demethoxy geldanamycin（17-AAG） combining with paclitaxel on human anaplastic thyroid cancer FRO cell line. Methods 1 The proliferation inhibition rates of FRO cells were detected by mmethyl thiazolyl tetrazolium（MTT） assay in different concentration groups（17-AAG： 0.312 5, 0.625 0, 1.250 0, 2.500 0, and 5.000 0 μmol/L; paclitaxel： 0.001 0, 0.010 0, 0.100 0, and 1.000 0 μmol/L; combination group, 17-AAG： 0.625 0 μmol/L, paclitaxel： 0.001 0, 0.010 0, 0.100 0, and 1.000 0 μmol/L） and at different time points（24, 48, and 72 hours）. 2 The change of cell cycle and apoptosis rates of FRO cells were detected in 17-AAG group（0.625 0 μmol/L）, paclitaxel group（0.100 0 μmol/L）, and combination group（17-AAG： 0.625 0 μmol/L, paclitaxel： 0.100 0 μmol/L） by flow cytometry at 24 hours after treatment. 3 Activity of Caspase-3 and Caspase-9 in FRO cells of 17-AAG group（0.625 0 μmol/L）, paclitaxel group（0.100 0 μmol/L）,and combination group（17-AAG： 0.625 0 μmol/L, paclitaxel： 0.100 0 μmol/L） was detected by Caspase-3 detection reagent box and Caspase-9 detection reagent box respectively. FRO cells of normal control group were treated without any drug,but culture solution. Results 1 The proliferation inhibition rates of FRO cells increased with the increase of concentration（17-AAG, paclitaxel, combination of 17-AAG and paclitaxel）, there was significant difference between any 2 groups（normal control group included）, P〈0.05. In addition, the proliferation inhibition rates of FRO cells in any concentration group（normal control group excluded） increased over time（24, 48, and 72 hours）, there was significant difference between any 2 time points（P〈0.05）. The proliferation inhibition rates of FRO cells in combination group were all higher than those of 17-AAG group and paclitaxel group in condition of same time point and same concentration（P〈0.05）. The q value of combination group was higher than 1.15 at 3 time points in all concentration, that meant 17-AAG could increase the efficiency of paclitaxel. 2 The apoptosis rate of FRO cells in normal control group was lower than those of 17-AAG group, paclitaxel group, and combination group（P〈0.05）, and apoptosis rate of FRO cells in combination group was higher than those of 17-AAG group and paclitaxel group（P〈0.05）. 3 Activity of Caspase-3 and Caspase-9 of FRO cells in normal control group were lower than those of 17-AAG group, paclitaxel group, and combination group（P〈0.05）, and activity of Caspase-3 and Caspase-9 of FRO cells in combination group were higher than those of 17-AAG group and paclitaxel group（P〈0.05）. Conclusions 17-AAG and paclitaxel can significantly inhibit the proliferation and induce the apoptosis of FRO cells. The combination of the two kinds of drugs may generate synergy, with dose-dependence effect.