JTE-522-induced apoptosis in human gastric adenocarcinoma [correction of adenocarcinoma] cell line AGS cells by caspase activation accompanying cytochrome C release, membrane translocation of Bax and loss of mitochondrial membrane potential.
OBJECTIVE
To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim).


METHODS
Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism.


RESULTS
JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO.


CONCLUSIONS
The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.
Li
Hong-Liang
HL
Department of Pharmacology, Jinan University Pharmacy College, Guangzhou 510632, Guangdong Province, China. tsam@jnu.edu.cn
Chen
Dan-Dan
DD
Li
Xiao-Hong
XH
Zhang
Hai-Wei
HW
Lü
Jun-Hua
JH
Ren
Xian-Da
XD
Wang
Cun-Chuan
CC
D000230:Adenocarcinoma; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001557:Benzenesulfonates / Q000494:pharmacology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D020287:In Situ Nick-End Labeling; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013274:Stomach Neoplasms; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein; D000096926:Benzenesulfonamides
D000590:Amino Acid Chloromethyl Ketones; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001557:Benzenesulfonates; D061945:Caspase Inhibitors; D020169:Caspases; D016861:Cyclooxygenase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D010080:Oxazoles; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C110453:4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide; D000096926:Benzenesulfonamides; C494826:BAX protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone