MEK and p38MAPK inhibitors potentiate TNF-alpha induced apoptosis in U937 cells.
BACKGROUND
TNF-alpha is one of the key inflammatory cytokines and it modulates various events through several pathways. U937 myelomonocytic leukemia cells are sensitive to TNF-alpha and about 20% of these cells undergo apoptosis within 6 hours after treatment. Co-treatment of these cells with actinomycin D or cycloheximide enhances TNF-alpha induced apoptosis, suggesting that some TNF-alpha-derived signals can augment apoptosis. We investigated whether mitosis-activating protein kinases (MAPKs) had an influence on TNF-alpha induced apoptosis.


METHODS
U937 cells were treated by TNF-alpha with or without MEK or p38MAPK inhibitors. Apoptosis was assessed morphologically by fluorescence microscopy and caspase-3 was studied by immunoblotting. Expression of apoptosis-inhibitory proteins was studied by RT-PCR whilst the activation of JNKs was investigated by detecting their phosphorylation.


RESULTS
TNF-alpha treatment induced apoptosis in about 23% of the cells, while pretreatment with a MEK inhibitor (PD98059) caused 69% of the cells to undergo apoptosis. The inhibition of p38MAPK by SB203580 scarcely enhanced apoptosis, although another p38MAPK inhibitor (PD169316) induced apoptosis in 37% of the cells. Simultaneous pretreatment of cells with PD98059 and PD169316 resulted in the highest level of TNF-alpha induced apoptosis and 90% of the cells underwent apoptosis after 6 hours. In cells pretreated with PD98059 plus PD169316, caspase-3 was completely cleaved at 6 hours and early induction of c-IAP2/HIAP 1 mRNA was not observed. JNKs showed rapid and extensive phosphorylation in these cells.


CONCLUSIONS
TNF-alpha induced apoptosis was potentiated by the inhibition of either MEK alone, or MEK plus p38MAPK, suggesting that the MAPK pathway may be a promising target for cancer therapy.
Nakada
S
S
Department of Molecular Genetics, Institute of DNA Medicine, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan.
Kawano
T
T
Saito-akita
S
S
Iwase
S
S
Horiguchi-Yamada
J
J
Ohno
T
T
Yamada
H
H
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D051034:Inhibitor of Apoptosis Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020298:U937 Cells; D048051:p38 Mitogen-Activated Protein Kinases
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D005419:Flavonoids; D007093:Imidazoles; D051034:Inhibitor of Apoptosis Proteins; D011506:Proteins; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C408604:2-(4-nitrophenyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazole; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one