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The findings suggest that ethanol-induced apoptosis of insulin-stimulated neuronal cells can be reduced by activating PI3 K and inhibiting pro-apoptosis gene expression and intracellular signaling through non-insulin-dependent pathways.

Tumor necrosis factor (TNF) is a potent activator of the nuclear factor-B (NF-B) pathway that leads to upregulation of anti-apoptotic proteins. Hence, TNF induces apoptosis in the presence of inhibitors of protein or RNA synthesis. We report that a novel triterpenoid, 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) inhibits NF-B-mediated gene expression at a step after translocation of activated NF-B to the nucleus. This effect appears specific for the NF-B pathway as CDDO does not inhibit gene expression induced by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). CDDO in combination with TNF caused a dramatic increase in apoptosis in ML-1 leukemia cells that was associated with activation of caspase-8, cleavage of Bid, translocation of Bax, cytochrome c release, and caspase-3 activation. Experiments with caspase inhibitors demonstrated that caspase-8 was an initiator of this pathway. TNF also induced a transient activation of c-Jun N-terminal kinase (JNK), which upon addition of CDDO was converted to a sustained activation. The activation of JNK was also dependent on caspase-8. Sustained activation of JNK is frequently pro-apoptotic, yet inhibition of JNK did not prevent Bax translocation or cytochrome c release, demonstrating its lack of involvement in CDDO/TNF-induced apoptosis. Apoptosis was acutely induced by CDDO/TNF in every leukemia cell line tested including those that overexpress Bcl-x L , suggesting that the mitochondrial pathway is not required for apoptosis by this combination. These results suggest that the apoptotic potency of the CDDO/TNF combination occurs through selective inhibition of NF-B-dependent anti-apoptotic proteins, bypassing potential mitochondrial resistance mechanisms, and thus may provide a basis for the development of novel approaches to the treatment of leukemia. Tumor necrosis factor (TNF)1 induces a broad range of cellular effects including inflammatory responses, NF-B activation, and apoptosis (for review, see Ref. 1). In many systems, the apoptotic potential of TNF is only realized when cells are co-treated with the protein synthesis inhibitor cycloheximide (CHX). This effect of CHX may be caused by inhibition of the translation of NF-B-dependent anti-apoptotic proteins such as TRAF1/2 and c-IAP1/2 (2). Apoptosis induced by TNF is initiated at the membrane where engagement of the tumor necrosis factor receptor results in the recruitment of TRADD and then FADD. A conserved sequence in FADD called the death effector domain serves as a docking site for procaspase-8, which upon activation initiates apoptosis by either of two distinct routes. The first pathway involves caspase-8-directed cleavage of Bid to its active form, tBid, resulting in translocation to the mitochondria and release of cytochrome c into the cytoplasm (3, 4). The released cytoplasmic cytochrome c interacts with caspase-9 and Apaf-1 in the presence of dATP to create the "apoptosome" that serves to cleave and activate caspase-9 (5). Caspase-9 then activates caspase-3, which carries out the exe...

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