Mcl-1-mediatd Regulation Of Endoplasmic Reticulum Stress-induced Apoptosis Of Gastric Adenocarcinoma Cells

Background & Objective: Treatment of metastatic gastric adenocarcinoma remains unsatisfactory. This is believed to be largely due to resistance of gastric adenocarcinoma cells to apoptosis induced by clinically available therapeutics. Recent studies have shown that, in addition to mitochondria, the endoplasmic reticulum (ER) plays an important role in apoptosis induced by many chemotherapeutic drugs, which triggers ER stress and subsequent activation of the unfolded protein response (UPR) that either protects the cell by alleviating the ER stress condition, or eliminates the cell by induction of apoptosis should ER stress proves irrevocable. Although the mechanism(s) of regulation of ER stress-induced apoptosis of gastric adenocarcinoma cells remains to be determined, it is known that induction of apoptosis by ER stress involves many of the same molecules that have important roles in other apoptotic cascades. Among them, Bcl-2 family proteins appear critical, as ER stress-induced apoptosis can be inhibited by over-expression of Bcl-2 or its anti-apoptotic homologs. In view of this, this project aimed to elucidate the role of Bcl-2 family members, in particular, the anti-apoptotic protein Mcl-1, in regulation of ER stress-induced apoptosis of gastric adenocarcinoma cells, and to identify potential targets for the treatment of gastric adenocarcinoma in combination with therapeutics that induce ER stress.Method: The gastric adenocarcinoma cell lines SGC-7901 and BGC-823 were treated with tunicamycin, a naturally occurring antibiotic that induces ER stress by inhibition of glycosylation, at varying doses for different periods. Apoptotic cells were quantitated by measurement of sub-G1 DNA content using the propidium iodide method in flow cytometry. Activation of caspase-8, -9, and -3, was assayed by Western blot analysis. The expression of Bcl-2 family proteins, including Bcl-2, Bcl-XL, and Mcl-1 was also monitored using Western blotting. Changes in Mitochondrial Membrane Potential (ΔΨ)m were studied by staining the cells with the cationic dye, JC-1, in flow cytometry. The expression of the Mcl-1 mRNA was examined by quantitative RT-PCR. Over-expression of Mcl-1 was achieved by transfection of cDNA encoding Mcl-1 cloned into the pcDNA3.0 (pcDNA3.0/Mcl-1) into cells. siRNA knockdown of Mcl-1 was achieved by transfection siGENOME SMARTpool Mcl-1 into cells. The efficiency of transfection was monitored by Western blot analysis.Results: While TM induced moderate levels of apoptosis in BGC-823 cells as shown by accumulation of sub-G1 DNA content, activation of caspase-8, -9, and -3, and reduction inΔΨ, SGC-7901 cells appeared more resistant to apoptosis induced by TM. This was associated with a marked increase in the expression of Mcl-1 after exposure to TM, which played a critical role in protection of the cells against TM-induced apoptosis, in that siRNA knockdown of Mcl-1 enhanced, whereas over-expression of Mcl-1 protected against, apoptosis induced by TM in the cells. In contrast, treatment with TM did not cause any notable changes in the expression of Bcl-2 and Bcl-XL. Up-regulation of Mcl-1 by TM in SGC-7901 cells is associated with an increase in the Mcl-1 mRNA levels, suggesting that a transcriptional mechanism(s) may be involved.Conclusions: Variations in sensitivities of cultured human gastric adenocarcinoma cells to ER stress-induced apoptosis is related to the different levels of Mcl-1 expression in response to ER stress. Gastric adenocarcinoma cells that express increased levels of Mcl-1 under ER stress are more resistant to ER stress-induced apoptosis. Therefore, targeting Mcl-1 may be useful in combination with chemotherapeutic drugs that induce ER stress in the treatment of gastric adenocarcinoma.