Acetaminophen induces apoptosis of C6 glioma cells by activating the c-JunNH2-terminal protein kinase-related cell death pathway

Acetaminophen (AAP), a widely used analgesic drug, can damage various organ s when taken in large doses. In this study, we investigate whether AAP caus es cell damage by altering the early signaling pathways associated with cel l death and survival. AAP caused time- and concentration-dependent apoptosi s and DNA fragmentation of C6 glioma cells used as a model. AAP activated c -Jun N-terminal protein kinase (JNK) by 5.3-fold within 15 min. The elevate d JNK activity persisted for up to 4 h before it returned to the basal leve l at 8 h. In contrast, activities of other mitogen-activated protein (MAP) kinases and the level of Akt phosphorylation in the cell survival pathway r emained unchanged throughout the treatment. Wortmannin, an inhibitor of pho sphatidylinositol-3 kinase, or SB203580, an inhibitor of p38 MAP kinase, di d not reduce AAP-induced toxicity, indicating that these enzymes do not pla y a major role in cell toxicity. AAP-induced apoptosis was preceded by the sequential elevation of the pro-apoptotic Bax protein, cytochrome c release , and caspase-3 activity. Treatment with caspase inhibitor benzyloxycarbony l-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) significantly reduced AA P-induced caspase-3 activation and cytotoxicity. Transfection of cDNA for t he dominant-negative mutant JNK-KR or stress-activated protein kinase kinas e-1 Lys --> Arg mutant (SEK1-KR), an immediate upstream kinase of JNK, sign ificantly reduced AAP-induced JNK activation and cell death rate. The noncy totoxic analog of AAP, 3-hydroxyacetanilide, neither increased JNK activity nor caused apoptosis. Pretreatment with YH439, an inhibitor of CYP2E1 gene transcription, markedly reduced CYP2E1 mRNA, protein content, and activity , as well as the rate of AAP-induced JNK activation and cell death. These d ata indicate that AAP can cause cell damage by activating the JNK-related c ell death pathway, providing a new mechanism for AAP-induced cytotoxicity.