Tunicamycin Sensitizes Human Gastric Adenocarcinoma Cells To TRAIL-induced Apoptosis

Background & Objective : Tumor necrosis factor (TNF)– related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that seems to be a promising candidate for cancer therapeutics because of its selective cytotoxicity against malignancies. However, many tumors including gastric cancer remain resistant to treatment with TRAIL. Therefore how to sensitize cancer cells to TRAIL-induced apoptosis may be promising for TRAIL application. Tunicamycin, a naturally occurring antibiotic that induces ER stress by inhibiting the first step in the biosynthesis of N-linked oligosaccharides in cells, was reported to sensitize human prostate and colon cancer cells to TRAIL-induced apoptosis. To explore the effective approaches for overcoming resistance of gastric adenocarcinoma cells to TRAIL-induced apoptosis and find the theoretical basis for the clinical application of TRAIL, the current study examined the mechanism(s) of tunicamycin sensitizes human gastric adenocarcinoma cells to TRAIL-induced apoptosis.Method: SGC-7901 and BGC-823 gastric adenocarcinoma cells were treated with tunicamycin and TRAIL at varying doses. The levels of apoptosis were quantitated using propidium iodide staining in flow cytometry. With or without treatment with tunicamycin and TRAIL, the expression of caspase at protein level was measured using western blot analysis. Mitochondrial membrane potential was measured using JC-I by flow cytometry. The cell surface expression of TRAIL receptors before and after tunicamycin treatment were measured with flow cytometry. The expression of Bip at protein level was measured using western blot analysis after treatment with tunicamycin for different time periods.Results: Gastric adenocarcinoma cells were relatively resistant to tunicamycin-induced apoptosis. Surprisingly, we found that tunicamycin strongly enhanced TRAIL-induced apoptosis in a synergistic manner. BGC-823 cells appeared to be sensitive, whereas SGC-7901 cells were relatively resistant to the sensitization of TRAIL-induced apoptosis by tunicamycin. Tunicamycin promoted TRAIL-induced caspase activation, including caspase-8,caspase-9,caspase-3,PARP. Tunicamycin promoted TRAIL-induced reduction in mitochondrial membrane potential. Tunicamycin up-regulated cell surface expression of TRAIL-R2 in either BGC-823 or SGC-7901 cells. However, the changes were more pronounced in BGC-823 than in SGC-7901 cells. Tunicamycin moderately up-regulated expression of TRAIL-R1 in BGC-823 but not in SGC-7901 cells. In contrast, tunicamycin did not induce any change in the expression of TRAIL-R3, TRAIL-R4 in both cell lines. Exposure of the gastric adenocarcinoma cells.to tunicamycin resulted in the up-regulation of the ER chaperone protein Bip. The observation confirmed that treatment with tunicamycin initiated the UPR in both cell lines.Conclusions: Tunicamycin sensitizes human gastric adenocarcinoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL receptors, especially TRAIL-R2. BGC-823 cells appeares to be sensitive, whereas SGC-7901 cells are relatively resistant to the sensitization of TRAIL-induced apoptosis by tunicamycin. Tunicamycin-mediated sensitization is associatedwith increased activation of the caspase cascade. Tunicamycin-mediated sensitization is largely dependent on a mitochondrial-mediated apoptosis pathway. Tunicamycin-induced ER stress seems to play an important role in tunicamycin-mediated up-regulation of TRAIL receptors.