Thioridazine Sensitizes Apoptotic Effect Of Trail In Human Lung Cancer PC9 Cells Through ER Stress Mediated Up-regulation Of DR5

Objective: TRAIL can induce apoptosis of tumor cells,however,various of tumor cells may survive because of resistance to TRAIL-mediated apoptosis.It is also one of the focuses of current research on TRAIL to promote the cells apoptosis of TRAIL by the combination of TRAIL treatment with traditional chemotherapy or epigenetic regulatory agents.Thioridazine as a dopamine receptor antagonist,is widely used in the treatment of schizophrenia,recently has been shown to have antiproliferative activity on tumor cells.This study is to observe the proliferation inhibition effect of TRAIL sensitized by thioridazine on PC9 cells through ER stress mediated up-regulation of DR5 and investigate its underlying mechanism.Methods:After cells were treatedwith different concentrations of thioridazine,cell proliferation was measured by MTT assay,and morphologic observation of PC9 cells was observed by microscope.Cell apoptosis and cell-surface DR5 were detected by flow cytometry.western blot was utilized to measure the expressions of ER stress related proteins GRP78,CHOP,p-PERK,p-e IF2?,ATF4 and apoptosis related proteins caspase-3,caspase-9.After treated with different dose of thioridazine and different dose of TRAIL,cell viability was measured by MTT assay and combination index of thioridazine and TRAIL were calculated to confirm the drug concentration.Cell-surface DR5 was detected by flow cytometry after treated with thioridazine or combined with ER stress inhibitor 4-PBA.Cell apoptosis was also detected by flow cytometry and western blot was utilized to measure the expressions of caspase-8,PARP and DR5 after treated with TRAIL and/or thioridazine and/or 4-PBA.Results: Thioridazine inhibited the proliferation of PC9 cells in a dose-dependent manner(P<0.05)and had the ability to induce ER stress.Thioridazine increased the inhibition and apoptosis of PC9 cells induced by TRAILand up-regulated the expression of cell-surface DR5,which can be inhibited by 4-PBA.Flow cytometry showed that cell apoptotic rates of combination group of TRAIL and thioridazine and TRAIL group were(32.6±4.7)% and(3.6±1.3)%compared with TRAIL group,combination group increased cell apoptotic rates significantly(P<0.05).While compared with combination group,the cell apoptotic rates of combination group after adding 4-PBA was(17.3±2.9)% and reduced significantly(P<0.01).western blot 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 upregulation of DR5 and enhanced TRAIL-mediated apoptosis induced by thioridazine were mediated through activation of ER stress accompanying by increased synthesis of GRP78 and CHOP,which can be blocked by the 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.