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.

MATERIALS AND 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.

CONCLUSION

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.