Uncouplers of oxidative phosphorylation sensitize resistant colon carcinoma cell lines (cc) to the cytotoxic ligand TRAIL.

Abstract

3582

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of proapoptotic factors (cytochrome c, Smac/DIABLO, HtrA2/Omi), which induce cell death. Mitochondria rapidly lose their transmembrane potential (Δ Ψm) and generate reactive oxygen species (ROS), both of which can contribute to dismantling of the cell. We have demonstrated that an uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), enhances the apoptosis-inducing capacity of TRAIL in TRAIL-resistant cc (RKO, HT29, SW480). This sensitization was inhibited by zVAD-fmk, indicating the requirement for caspase activation. In RKO, CCCP induced loss of ΔΨm without release of proapoptotic factors from the mitochondria. Overexpression of Bcl-2 did not prevent the initial loss of ΔΨm following CCCP treatment, but did prevent further decrease in ΔΨm and cell death following TRAIL+CCCP exposure. Treatment of RKO cells with TRAIL+CCCP resulted in 1) enhanced caspase-8 activation, cleavage of Bid and Bax conformational change, 2) release of cytochrome c, Smac/DIABLO and HtrA2/Omi into the cytosol, and 3) subsequent caspase-3 activation, PARP cleavage and apoptosis. Degradation of XIAP appeared critical, as it occurred only when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2/Omi, and cytochrome c release after TRAIL + CCCP treatment. Of interest, caspase-9 was activated to the same extent when cells were treated with TRAIL +/− CCCP. Overexpression of a mutant caspase-9 did not protect RKO cells from TRAIL+CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Since cytochrome c is a key factor in caspase-9 activation, non involvement of caspase-9 in the apoptotic pathway indicated that the major mitochondrial pro-apoptotic factors involved in apoptosis were Smac/DIABLO and/or HtrA2/Omi. In addition to loss in ΔΨm, CCCP induced a pronounced increase in ROS production, not prevented by overexpression of Bcl-2. In Bcl-2-expressing cells, ROS production was sustained while in control transfected cells, ROS production was disrupted, correlating with the further decrease in ΔΨm following TRAIL+CCCP treatment. Preincubation of RKO with antioxidants (N-acetyl-L-cysteine or GSH) completely abrogated TRAIL+CCCP-induced apoptosis, production of ROS and loss in ΔΨm. Results also demonstrated that caspase-8 was not cleaved after TRAIL+CCCP in the presence of N-acetyl-L-cysteine, indicating that TRAIL induced caspase-8 activation in a ROS-dependent manner. Data suggest that cellular resistance to TRAIL is modulated by the redox state and by mitochondrial-derived ROS. Supported by NCI awards CA 32613, CA 21765 and by ALSAC.