Changes of NF-kB, p53, Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells: role of reactive oxygen species

Abstract

Aim: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process.

Methods: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms.

Results: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner.

Conclusion: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.

Figure 1

Effect of JTE-522 on cell growth in AGS cells. The cells were treated with various concentrations of JTE-522 for 72 h. The antiproliferative effect was measured by MTT assay. Results are the means ± SD from three independent determinations.

Figure 2

Electro micrographs of JTE-522-treated AGS cells. Control AGS cells (A), or treated with 1 mmol/L (B) JTE-522 for 72 h, were examined by EM as in "Materials and Methods". Magnification: × 4000

Figure 3

DNA ladder pattern formation of AGS cells. Cells treated with different concentrations of JTE-522 for 72 h and uhe formation of oligonucleosomal fragments was determined by 1.5% agarose gel electrophoresis. M, DNA markers; lanes 1-4, AGS cells treated with 0, 0.25, 0.50, 1 mmol/L of JTE-522

Figure 4

Activation of caspase-3 activities by JTE-522 in AGS cells for indicated time period. JTE-522 treatment (0.75 mmol/L) induced cleavage of Ac-DEVD-AMC and Ac-IETD-AMC, indicating activation of caspase-3 activity, respectively

Figure 5

Effect of JTE-522 on the generation of ROS. AGS cells were incubated for 6 h with JTE-522 (0.25-1 mmol/L) in the presence or absence of PDTC at 100 μmol/L. Lucigenic-associated chemiluminescence was mea-sured for 3 min with a lumirometer. (A). Lane 1: control; lane 2-5: AGS cells treated with 0.25, 0.5, 0.75, 1 mmol/L of JTE-522; lane 6: JTE-522 (1 mmol/L) +PDTC (100 μmol/L) ^cP < 0.01 vs control. Results are the means ± SD from three independent determinations.

Figure 6

Effect of JTE-522 on NF-κB binding activity and IkBα degradation. Cells were treated with JTE-522 for 6 h. Cells were harvested and EMSA was performed as described (A). Lane 1: control; lane 2-5: AGS cells treated with 0.25, 0.50, 0.75, 1 mmol/L of JTE-522. The identity of DNA-complexed pro-teins was confirmed by supershift assays using antibodies against p65 sub-unit of NF-κB (lane 6). Immunobolt analysis of IkBα of corresponding cytosilic supernatant (B). Representative results from four independent experiments.

Figure 7

P53, bcl-2 protein levels in AGS cells treated with JTE-522. Cell lysates were collected and processed at 6 h. The whole cellular protein was electrophoresed in SDS-PAGE gel, Western blot was performed using antibodies against p53, bcl-2. Beta actin was used as a lane-loading control. (1) control; (2) 0.25 mmol/L; (3) 0.5 mmol/L; (4) 0.75 mmol/L; (5) 1 mmol/L. Representative results from three independent experiments.