Background. TRAIL (TNF-related apoptosis inducing ligand) is a promising anti-cancer agent that induces apoptosis in multiple tumor cell types while sparing most normal cells. TRAIL engages the caspase-8-dependent, death receptor-mediated apoptotic pathway. We determined whether TRAIL also triggers the mitochondrial apoptotic pathway in SW480 human colon cancer cells that is regulated by anti-apoptotic Bcl-2. Materials/Methods. SW480 human colon cancer cells were stably transfected with PC3-bcl-2 plasmid (SW480/Bcl-2) or vector alone (PC3-neo). Bcl-2 overexpressing cells (SW480/Bcl-2) and SW480/neo cells were incubated with recombinant human TRAIL (0-200 ng/mL; Calbiochem) and apoptosis (FITC-annexin V labeling; 6 h) was analyzed in the presence or absence of a caspase-9 inhibitor (z-LEHD-fmk). Cell fractionation was performed; cytochrome oxidase IV (Molecular Probes) was used a mitochondrial marker. The effect of TRAIL (15 hr) upon Bid truncation, caspase-8 and –3 cleavage, and cytosolic cytochrome c (antibodies from BD PharMingen) and Smac/Diablo (Upstate Biotechnology) release were determined. Clonigenic cell survival was determined after a 7d incubation with TRAIL or no drug. Results. Bcl-2 overexpressing SW480 cells were partially protected (3-fold reduction) from a dosage-dependent induction of apoptosis by TRAIL. Bcl-2 blocked TRAIL-mediated caspase –3 cleavage, but had no effect upon caspase-8 nor Bid truncation. The caspase-9 inhibitor (z-LEHD-fmk) inhibited TRAIL-induced ( ng/ml) apoptosis (3-fold) in SW480/neo cells. Bcl-2 overexpression attenuated Bax translocation to the mitochondria and the cytosolic release of cytochrome c and Smac/Diablo. SW480/Bcl-2 cells showed a significant increase in clonigenic survival compared to SW480/neo cells. Conclusion. TRAIL engages the mitochondrial apoptotic pathway in SW480 cells that is regulated by Bcl-2 and caspase-9. Bcl-2 inhibited Bax translocation, release of pro-apoptotic mitochondrial proteins, and effector caspase-3 cleavage. These data indicate that Bcl-2 is an important modulator of the therapeutic efficacy of TRAIL in SW480 colon carcinoma cells.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]