title: Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines.
abstract: The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.
authors.affiliation: Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. kamel.izeradjene@stjude.org
authors.forename: Kamel
authors.initials: K
authors.lastname: Izeradjene
authors.forename: Leslie
authors.initials: L
authors.lastname: Douglas
authors.forename: David M
authors.initials: DM
authors.lastname: Tillman
authors.forename: Addison B
authors.initials: AB
authors.lastname: Delaney
authors.forename: Janet A
authors.initials: JA
authors.lastname: Houghton
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000031:analogs & derivatives / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D019069:Cell Respiration / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D045304:Cytochromes c / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D010085:Oxidative Phosphorylation; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D014475:Uncoupling Agents / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein; D051028:bcl-2-Associated X Protein
chemical_list: D000975:Antioxidants; D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D014475:Uncoupling Agents; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D051028:bcl-2-Associated X Protein; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D045304:Cytochromes c; D020169:Caspases; D000111:Acetylcysteine