Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis.
We investigated intracellular signalling pathways for apoptosis induced by epigallocatechin-3-gallate (EGCG) as compared with those induced by a toxic chemical substance (etoposide, VP16) or the death receptor ligand [tumour necrosis factor (TNF)]. EGCG as well as VP16 and TNF induced activation of two apoptosis-regulating mitogen-activated protein (MAP) kinases, namely c-Jun N-terminal kinase (JNK) and p38 MAP kinase, in both human leukaemic U937 and OCI-AML1a cells. In U937 cells, the apoptosis and activation of caspases-3 and -9 induced by EGCG but not VP16 and TNF were inhibited with SB203580, a specific inhibitor of p38, while those induced by EGCG and VP16 but not TNF were inhibited with SB202190, a rather broad inhibitor of JNK and p38. In contrast, the EGCG-induced apoptosis in OCI-AML1a cells was resistant to SB203580 but not to SB202190. Unlike TNF, EGCG did not induce the activation of nuclear factor-kappaB but rather induced the primary activation of caspase-9. N -Acetyl-L-cysteine (NAC) almost completely abolished apoptosis induced by EGCG under conditions in which the apoptosis induced by VP16 or TNF was not affected. The JNK/p38 activation by EGCG was also potently inhibited by NAC, whereas those by VP16 and TNF were either not or only minimally affected by NAC. In addition, dithiothreitol also suppressed both apoptosis and JNK/p38 activation by EGCG, and EGCG-induced activation of MAP kinase kinase (MKK) 3/6, MKK4 and apoptosis-regulating kinase 1 (ASK1) was suppressed by NAC. Dominant negative ASK1, MKK6, MKK4 and JNK1 potently inhibited EGCG-induced cell death. EGCG induced an intracellular increase in reactive oxygen species and GSSG, both of which were also inhibited by NAC, and the decreased synthesis of glutathione rendered the cell susceptible to EGCG-induced apoptosis. Taken together these results strongly suggest that EGCG executed apoptotic cell death via an ASK1, MKK and JNK/p38 cascade which is triggered by NAC-sensitive intracellular oxidative events in a manner distinct from chemically induced or receptor-mediated apoptosis.
Department of Haematology, Research Institute, International Medical Centre of Japan, 1-21-1, Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
Koichi
K
Saeki
Norihiko
N
Kobayashi
Yuko
Y
Inazawa
Hong
H
Zhang
Hideki
H
Nishitoh
Hidenori
H
Ichijo
Kumiko
K
Saeki
Mamoru
M
Isemura
Akira
A
Yuo
D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D016923:Cell Death; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005419:Flavonoids; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D000975:Antioxidants; D004791:Enzyme Inhibitors; D005419:Flavonoids; D016328:NF-kappa B; D019384:Nucleic Acid Synthesis Inhibitors; D010636:Phenols; D011108:Polymers; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D005047:Etoposide; D002392:Catechin; C045651:epigallocatechin gallate; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; D020930:MAP Kinase Kinase Kinases; C482858:MAP3K5 protein, human; D014568:Urokinase-Type Plasminogen Activator; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D005978:Glutathione; D010100:Oxygen