Gu Wen, Yin Hubin, Liu Yan, Gou Xin
Department of Urology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Department of Urology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Jun;33(6):772-777.
Objective To investigate the mechanism by which epirubicin (EPI) induces autophagy and the mechanism by which tea polyphenol (TP) regulates EPI-induced autophagy and apoptosis in T24 bladder cancer cells. Methods T24 cells weredivided into control group, EPI group, TP group and TP plus EPI group. Eight hours after corresponding treatments in different groups, transmission electron microscopy (TEM) was used to observe the image of autophagosomes. The expressions of autophagy-related protein LC3II and p62 in the cells were detected by Western blotting. Apoptotic cells were evaluated after EPI-treatment for 24 hours by flow cytometry combined with annexin V-FITC/PI staining. Western blotting was performed to determine the levels of cleaved-caspase-3 (c-caspase-3) and cleaved-PARP (c-PARP). LC3II was again tested by Western blotting 8 hours after T24 cells were treated with EPI added with autophagy pathway inhibitor chloroquine and 3-methyladenine, and moreover, the levels of LC3II and p-JNK were detected by Western blotting after T24 cells were treated with EPI combined with TP or the JNK inhibitor SP for 8 hours. Results The amount of autophagosomes and the level of LC3IIin TP plus EPI group were much lower than those in EPI group. SP reduced the level of LC3II induced by EPI. EPI increased p-JNK in a time-dependent manner. TP combined with EPI reduced the activity of JNK pathway. The apoptosis rate and the levels of c-caspase-3 and c-PARP in TP plus EPI group were much higher than those in EPI group. Conclusion TP inhibits autophagy through JNK pathway to enhance EPI-induced apoptosis in T24 bladder cancer cell line.
目的 探讨表柔比星（EPI）诱导自噬的机制以及茶多酚（TP）调控EPI诱导T24膀胱癌细胞自噬和凋亡的机制。方法 将T24细胞分为对照组、EPI组、TP组和TP+EPI组。不同组进行相应处理8小时后，采用透射电子显微镜（TEM）观察自噬体图像。通过蛋白质免疫印迹法检测细胞中自噬相关蛋白LC3II和p62的表达。EPI处理24小时后，采用流式细胞术结合膜联蛋白V-FITC/PI染色评估凋亡细胞。通过蛋白质免疫印迹法检测裂解的半胱天冬酶-3（c-caspase-3）和裂解的聚（ADP-核糖）聚合酶（c-PARP）的水平。用自噬途径抑制剂氯喹和3-甲基腺嘌呤处理T24细胞后，再用EPI处理8小时，通过蛋白质免疫印迹法再次检测LC3II；此外，T24细胞用EPI联合TP或JNK抑制剂SP处理8小时后，通过蛋白质免疫印迹法检测LC3II和磷酸化JNK（p-JNK）的水平。结果 TP+EPI组的自噬体数量和LC3II水平均明显低于EPI组。SP降低了EPI诱导的LC3II水平。EPI以时间依赖性方式增加p-JNK。TP与EPI联合降低了JNK途径的活性。TP+EPI组的凋亡率以及c-caspase-3和c-PARP水平均明显高于EPI组。结论 TP通过JNK途径抑制自噬，增强EPI诱导的T24膀胱癌细胞系凋亡。
