The Role And Regulation Of Mcl-1 In Endoplasmic Reticulum Stress-induced Apoptosis In Gastric Adenocarcinoma Cell

Background & Objective: Gastric adenocarcinoma is a common tumor in China and the treatment remains unsatisfactory. This is believed to be due to resistance of gastric adenocarcinoma cells to apoptosis. It is known that, in addition to traditional extrinsic and mitochondrial-dependent apoptosis pathway, the endoplasmic reticulum(ER) plays an important role in cell apoptosis. Because the insufficient blood supply, poor nutrition and drug effects and other factors, tumor cells exists endoplasmic reticulum stress. Stress is the behavior of cell self-protection,but when a stress is so strong or persistent that ER dysfunction cannot be corrected, apoptotic cell death is initiated. Recent years it is found that Bcl-2 family proteins involved in the regulation of ER stress-induced apoptosis.Mcl-1 is an significant anti-apoptotic family members, involved in the mitochondrial pathway of apoptosis, while also play an important role in ER stress. The expression of Mcl-1 gene is regulated at multiple levels and important transcription factor E2F-1 can regulated transcripitional expression of Mcl-1. For this reasons, we take gastric adenocarcinoma cells as our research object, build a model of ER stress by tunicamycin(TM). This project aimed to elucidate the role of Bcl-2 family members, in particular, the mechanism of regulation of anti-apoptotic protein Mcl-1, in ER stress-induced apoptosis of gastric adenocarcinoma cells, and to provide a new clinical target for the treatment of gastric adenocarcinoma.Method: SGC-7901 and BGC-823 gastric adenocarcinoma cells and HEK-293 human embryonic kidney epithelial cell were treated with tunicamycin at varying doses for different time periods. Apoptotic cells were quantitated by measurement of sub-G1 DNA content using the propidium iodide method in flow cytometry. The activation of caspase-9,3 and protein expression levels of GRP78,Bcl-2,Mcl-1 were detected by Western blot. Changes in Mitochondrial Membrane Potential (ΔΨm) were studied by staining the cells with JC-1 using flow cytometry. The Changes of the Mcl-1 mRNA level was examined by quantitative RT-PCR. cDNA (pcDNA-E2F-1-Flag) transfection was employed to over-express E2F-1 in cells. Western blot was used to detect the transfection efficiency and the change of Mcl-1expression level.Results: ER stress-induced apoptosis in gastric adenocarcinoma cells treated with tunicamycin was in a dose- and time-dependent manner. Both cells were resistant to tunicamycin-induced apoptosis compared HEK-293 cell. And SGC-7901 cell showed a stronger resistance to apoptosis induced by tunicamycin. This was associated with up-regulation of Mcl-1 at both the protein and mRNA levels.While, treatment with TM did not cause any notable changes in the expression of Bcl-2. Intriguingly, the transcription factor E2F-1 known to repress transcription of Mcl-1 was decreased by exposure to TM. Over-expression of E2F-1 attenuated up-regulation of Mcl-1 in gastric adenocarcinoma cells under ER stress. The decline of Mc1-1 expression was more obvious if the concentration of plasmid was increased.Conclusions: Human gastric adenocarcinoma cell lines are relatively resistant to ER stress -induced apoptosis. This was associated with up-regulation of Mcl-1 at both the protein and mRNA levels. This shows that Mcl-1 plays the role of tolerance in ER stress-induced apoptosis in gastric adenocarcinoma. This is, at least in part, due to regulation of E2F-1 in the cells upon ER stress.