Li Juan, Wang Yi, Liu Liu, Yuan Yuan, Bao Yangyi
Department of Oncology, the Third Affiliated Hospital of Anhui Medical University, Hefei 230061, China.
Central Laboratory of Hefei Binhu Hospital, Hefei 230061, China.
Zhongguo Fei Ai Za Zhi. 2017 Feb 20;20(2):80-87. doi: 10.3779/j.issn.1009-3419.2017.02.02.
Tumor necrosis factor-related apoptosis-inducting ligand (TRAIL) can induce apoptosis of tumor cells, however, various of tumor cells may survive because of resistance to TRAIL-mediated apoptosis. This study is to observe the proliferation inhibition effect of TRAIL sensitized by thioridazine on PC9 cells through endoplasmic reticulum (ER) stress mediated up-regulation of death receptor 5 (DR5) and investigate its mechanism.
PC9 cells were treated with different concentrations of thioridazine and TRAIL alone or in combination. Cell proliferation was measured by MTT assay, and cell apoptosis and cell-surface DR5 were detected by flow cytometry. Western blotting was utilized to measure the expressions of ER stress-related proteins glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), p-PKR-like ER kinase (PERK), p-eukaryotic initiation factor-2α (eIF2α), activating transcription factor 4 (ATF4) and apoptosis-related proteins caspase-3, caspase-9, caspase-8, PARP, DR5.
Thioridazine inhibited the proliferation of PC9 cells in a dose-dependent manner (P<0.05). Thioridazine increased the inhibition and apoptosis of PC9 cells and up-regulated the expression of cell-surface DR5 induced by TRAIL. Flow cytometry showed that compared with TRAIL group, combination group of TRAIL and thioridazine increased cell apoptotic rates significantly (P<0.05). Western blotting indicated that compared with TRAIL group, expressions of Cleaved-caspase-8, Cleaved-PARP and DR5 increased significantly in combination group of TRAIL and thioridazine. The induction of DR5 and pro-apoptotic effect were mediated through activation of ER stress accompanying by increased synthesis of GRP78 and CHOP, which can be blocked by adding of ER stress inhibitor 4-PBA.
CONCLUSIONS: Thioridazine enhanced proliferation inhibition effect of TRAIL in PC9 cells may be facilitated through ER stress mediated upregulation of DR5. .
肿瘤坏死因子相关凋亡诱导配体（TRAIL）可诱导肿瘤细胞凋亡，然而，多种肿瘤细胞可能因对TRAIL介导的凋亡产生抗性而存活。本研究旨在观察硫利达嗪致敏的TRAIL通过内质网（ER）应激介导死亡受体5（DR5）上调对PC9细胞的增殖抑制作用，并探讨其机制。
PC9细胞分别单独或联合用不同浓度的硫利达嗪和TRAIL处理。通过MTT法检测细胞增殖，通过流式细胞术检测细胞凋亡和细胞表面DR5。利用蛋白质免疫印迹法检测内质网应激相关蛋白葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）、磷酸化蛋白激酶R样内质网激酶（PERK）、磷酸化真核翻译起始因子2α（eIF2α）、活化转录因子4（ATF4）以及凋亡相关蛋白半胱天冬酶-3、半胱天冬酶-9、半胱天冬酶-8、聚（ADP-核糖）聚合酶（PARP）、DR5的表达。
硫利达嗪以剂量依赖性方式抑制PC9细胞的增殖（P<0.05）。硫利达嗪增强了TRAIL对PC9细胞的抑制和凋亡作用，并上调了TRAIL诱导的细胞表面DR5的表达。流式细胞术显示，与TRAIL组相比，TRAIL与硫利达嗪联合组显著提高了细胞凋亡率（P<0.05）。蛋白质免疫印迹表明，与TRAIL组相比，TRAIL与硫利达嗪联合组中裂解的半胱天冬酶-8、裂解的PARP和DR5的表达显著增加。DR5的诱导和促凋亡作用是通过内质网应激的激活介导的，同时伴有GRP78和CHOP合成增加，添加内质网应激抑制剂4-苯基丁酸（4-PBA）可阻断这种作用。
硫利达嗪增强TRAIL对PC9细胞的增殖抑制作用可能是通过内质网应激介导DR5上调实现的。
