Effects Of JTE-522 On Apoptosis In Human Gastric Adenocarinoma Cell Line AGS Cells

AIM: To investigate the effects of a selective COX-2 inhibitor, JTE-522 on human gastric adenocarcinoma cell line AGS cells and related molecular mechanism. METHODS: Cell culture MTT EMU ELISA assay flowcymetry EMSA lucigenin assay fluorometric assay and Western blot were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: Significantly inhibitory effect and morphological changes typical of apoptosis were observed after treatment with different dose of JTE-522, and these effects were in a concentration-dependent manner. EMSA and Western blot revealed that NF-kB activity was almost complete inhibited by preventing the degradation of IkB a , the levels of p53 showed a great increase and bcl-2 was downregulated following JTE-522 treatment. Their changes were in a dose dependent manner. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. Caspases 8 and 9 were activated as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrats in JTE-522-induced apoptosis. which were significantly inhibited by the caspases inhibitors. The decrease of mitochondrial membrane potential, the membrane translocation of Bax accompanying with cytosolic release of cytochrome c were detected at an early stage of apoptosis. Furthore, Bax translocation, cytochrome c release, and caspase 9 activation wereblocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: 1. JTE-522 can inhibit cell growth and induce apoptosis in human gastric adenocarcinoma cell line AGS cells; 2: JTE-522 can increase the levels of p53 protein, downregulate the levels of Bcl-2 and inhibit NF-kB activity by preventing the degradation of IkB a , which may as the upstream mediators of ROS; 3: Decrease the mitochondrial membrane potential Activation of caspase 8 by JTE-522 triggered the membrane translocation of Bax and cytosolic release of cytochrome C. The cytosolic release of cytochrome c forms complex with Apafl and caspase 9 and then activates caspase 9, which in turn activates caspase 3, leading to apoptosis.