竹节香附素A对耐奥沙利铂肠癌细胞株的作用及机制研究  

Effect and mechanism of raddeanin A on oxaliplatin-resistance colorectal cancer cell line

作　　者：孟春芹 刘先勇 MENG Chunqin;LIU Xianyong(Department of Integrated Chinese Medicine and Western Medicine,Affiliated Jiangning Hospital of Nanjing Medical University,Nanjing,Jiangsu 211100,China)

机构地区：[1]南京医科大学附属江宁医院中西医结合科,南京211100

出　　处：《重庆医学》2021年第11期1806-1811,共6页Chongqing medicine

基　　金：2019江苏省双创博士项目(JNYYSC201903);2019年南京医科大学科研发展基金(NMUB2019238);2019年康达学院科研发展基金(KD2019KYJJZD023)。

摘　　要：目的探讨竹节香附素A(RA)对人结肠癌耐奥沙利铂细胞株HCT116/L-OHP的逆转作用及其可能机制。方法四甲基偶氮唑蓝(MTT)实验检测细胞增殖情况;流式细胞术检测细胞凋亡率;逆转录聚合酶链反应(RT-PCR)检测caspase-3、聚腺苷二磷酸核糖聚合酶(PARP)、耐多药基因1(MDR1)等基因的表达;免疫荧光试验及蛋白免疫印迹(WB)检测Cleaved-caspase-3、Cleaved-PARP、P-gp、IκB-α、P65蛋白的表达情况。结果RA可增加HCT116/L-OHP细胞株对奥沙利铂的敏感性,两药联合后的半数抑制浓度(IC50)从原来的(234.12±6.13)μg/mL降低至(140.45±2.25)μg/mL,耐药逆转倍数约为1.67,差异有统计学意义(P<0.05);流式细胞术显示,单用奥沙利铂时,细胞凋亡率为20.5%,而联合RA后,细胞凋亡率增加至34.4%,差异有统计学意义(P<0.01);RT-PCR及WB结果显示,凋亡关键因子caspase-3、PARP在联合组中表达较单用奥沙利铂组增加;耐药蛋白基因MDR-1及其编码蛋白P-gp在联合组中的表达较单药奥沙利铂组减少;此外,WB结果还显示,核因子-κB(NF-κB)信号通路中IκB-α、P65在联合组中的表达较单药组减少。结论RA可增加结肠癌耐奥沙利铂细胞株对奥沙利铂的敏感性可能与调节NF-κB信号通路减少P-gp的表达有关。Objective To investigate the reversal effect of raddeanin A(RA)on oxaliplatin-resistance colorectal cancer cell line(HCT116/L-OHP)and its possible mechanism.Methods The MTT assay was used to detect the cell proliferation;the flow cytometry(FCM)was used to detect the cellular apoptosis rate.The expressions of caspase-3,PARP and MDR1 were tested by real-time fluorescence quantitative polymer chain reaction(RT-PCR),and the expressions of Cleaved-caspase-3,Cleaved-PARP,P-gp,IκB-αand p65 proteins were detected by Western blot(WB)and immunofluorescence analysis(IF).Results RA could increase the sensitivity of HCT116/L-OHP to oxaliplatin,the IC50 value after two drugs combination was decreased from(234.12±6.13)μg/mL to(140.45±2.25)μg/mL,the resistance reversal multiple was about 1.67,and the difference was statistically significant(P<0.05).The flow cytometry showed that in oxaliplatin single use,the apoptosis rate was 20.5%,when the combination use with RA,the cell apoptosis rate was increased to 34.4%,and the difference was statistically significant(P<0.01);the RT-PCR and WB results showed that the expressions of apoptosis key factors caspase-3 and PARP in the combination group were increased compared with the oxaliplatin single use group,the expressions of drug-resistant protein gene MDR-1 and its coding protein P-g in the combination group were decreased compared with the oxaliplatin single use group.In ad-dition,the WB results also showed that the IκBαand p65 expressions in NF-κB signaling pathway in the combination group were decreased compared with the single drug group.Conclusion RA can increase the sensitivity of oxaliplatin-resistance colorectal cancer cell line to oxaliplatin,which is possibly related with regulating the NF-κB signaling pathway and decreasing the P-gp expression.

关 键 词：竹节香附素A HCT116/L-OHP细胞株 结肠肿瘤 奥沙利铂 耐药 

分 类 号：R735.3[医药卫生—肿瘤]