Zhou Fang, Li Yan-Hua, Wang Jian-Jun, Pan Jia, Lu Hong
Department of Oncology, Huaihe Hospital of Henan University, Kaifeng, P.R. China.
Tumour Biol. 2017 Jun;39(6):1010428317705748. doi: 10.1177/1010428317705748.
The study was designed to explore the mechanism of tunicamycin-induced endoplasmic reticulum stress in human esophageal cancer EC9706 cells and EC109 cells, as well as its effects on cell autophagy, apoptosis, and chemoresistance. Tunicamycin-induced endoplasmic reticulum stress model was established in EC9706 and EC109 cell lines. Western blotting was employed to detect the expression of endoplasmic reticulum stress iconic protein GRP78. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate the effect of different cisplatin and tunicamycin concentrations on survival rate of EC9706 cells and EC109 cells. Autophagy was monitored using monodansylcadaverin and apoptosis was detected by flow cytometry. Western blotting was used to detect the expressions of endoplasmic reticulum stress-related proteins (PERK, eIF2α, and CHOP), PI3K/Akt/mTOR signaling pathway-related proteins, autophagy-related proteins (LC3-I/LC3-II, Beclin-1, and p62), and apoptosis-related proteins (Bcl-2, Bax, and cleaved caspase-3). Tunicamycin led to increased expression of GRP78. With tunicamycin treatment, phosphorylation of PERK and eIF2α and CHOP expression increased. Meanwhile, the increase in cytolysosome was concentration and time dependent. With the increased tunicamycin concentration, there were increased expressions of Bax and cleaved caspase-3, decreased expression of Bcl-2, and lower phosphorylation of PI3K/Akt/mTOR signaling pathway-related proteins. Therefore, it can be concluded that the combination of tunicamycin and cisplatin could improve the sensitivity of EC9706 cells and EC109 cells to cisplatin; PI3K inhibitor BEZ235 could enhance cell autophagy and apoptosis and increase cell sensitivity to cisplatin.
本研究旨在探讨衣霉素诱导人食管癌EC9706细胞和EC109细胞内质网应激的机制及其对细胞自噬、凋亡和化疗耐药性的影响。在EC9706和EC109细胞系中建立衣霉素诱导的内质网应激模型。采用蛋白质免疫印迹法检测内质网应激标志性蛋白GRP78的表达。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法评估不同顺铂和衣霉素浓度对EC9706细胞和EC109细胞存活率的影响。使用单丹磺酰尸胺监测自噬，通过流式细胞术检测凋亡。采用蛋白质免疫印迹法检测内质网应激相关蛋白（PERK、eIF2α和CHOP）、PI3K/Akt/mTOR信号通路相关蛋白、自噬相关蛋白（LC3-I/LC3-II、Beclin-1和p62）以及凋亡相关蛋白（Bcl-2、Bax和裂解的caspase-3）的表达。衣霉素导致GRP78表达增加。经衣霉素处理后，PERK和eIF2α的磷酸化以及CHOP表达增加。同时，溶酶体的增加呈浓度和时间依赖性。随着衣霉素浓度的增加，Bax和裂解的caspase-3表达增加，Bcl-2表达降低，PI3K/Akt/mTOR信号通路相关蛋白的磷酸化水平降低。因此，可以得出结论，衣霉素和顺铂联合使用可提高EC9706细胞和EC109细胞对顺铂的敏感性；PI3K抑制剂BEZ235可增强细胞自噬和凋亡，并增加细胞对顺铂的敏感性。
