JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release, membrane translocation of Bax and loss of mitochondrial membrane potential

Figure 5 Effect of JTE522 on the activation of caspase, cytochrome C release and membrane translocation of Bax. Figure 5A: AGS cells were treated with: RPMI 1640 medium for control (a) or 0.1 μmol/L staurosporine (b) or 1 mmol/L JTE-522 (c). After 24 h, the cells were collected and suspended in mitochondria isolation buffer. Cytosol (C) and pellet (P) fractions were separated and subjected to Western blotting as described in Materials and Methods. Cytochrome C (cyto c), cytochrome C oxidase subunit IV (Cyt oxi); Figure 5B: Effect of JTE522 on the changes of cytosolic cytochrome C release and procaspase 3 (casp-3); Figure 5C: Effect of caspase inhibitors on cytochrome C release and translocation of Bax. AGS cells were treated with 50 μmol/L Z-VAD.fmk (a) for 1 h or 0.1 μmol/L staurosporine (b) or 1 mmol/L JTE-522 (c) or without any treatment. The cytosol was prepared as in (A) and analyzed by Western blotting with indicated antibodies, Casp 3 (procaspase 3), Casp 8 (procaspase 8); Figure 5D: AGS cells were cultured at RPMI 1640 medium or treated with caspase inhibitors: Figure 5D1: Effect of caspase inhibitor on the activity of caspase 9: 1: control; 2: Z-VAD.fmk + 1 mmol/L JTE-522; 3: Ac-IETD-CHO + 1 mmol/L JTE-522; 4: 1 mmol/L JTE-522 for 24 h; Figure 5D2: Effect of caspase inhibitor on caspase 8 cleavage, cytochrome c release: 1: control (a); 2: Z-VAD.fmk + 1 mmol/L JTE-522 (b); 3: Ac-IETD-CHO + 1 mmol/L JTE-522 (c); 4: Ac-LEHD-CHO + 1 mmol/L JTE-522 (d). Cells were collected and analyzed for caspase 8 cleavage, cytochrome C release, or caspase-like acitivity as described in Materials and Methods.