title: Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis is inhibited by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA 14-1, in human colon cancer cells.
abstract: OBJECTIVE
  Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that induces apoptosis in multiple tumor cell types while sparing most normal cells. We determined the effect of ectopic Bcl-2 expression on TRAIL-induced apoptosis and whether the small molecule Bcl-2 inhibitor, HA14-1, could increase TRAIL sensitivity.
  
  
  METHODS
  SW480 human colon cancer cells were stably transfected with the PC3-Bcl-2 plasmid or vector alone. Cells were incubated with recombinant human TRAIL +/- HA14-1 or caspase-9 inhibitor (Z-LEHD-FMK). Apoptosis was analyzed by Annexin V-fluorescein isothiocyanate labeling and DNA fragmentation factor 45 (DFF45) cleavage. Clonigenic survival was also studied. Caspase activation was determined by immunoblotting or colorimetric assay. The cytosolic expression of Bid, Bax, and XIAP and release of cytochrome c and Smac/DIABLO were determined by immunoblotting.
  
  
  RESULTS
  Bcl-2 overexpression partially protected SW480 cells from a dose-dependent induction of apoptosis by TRAIL, as did a caspase-9 inhibitor, and increased their clonogenic survival. Bcl-2 overexpression attenuated TRAIL-induced cleavage of caspase-8, indicating its activation upstream and downstream of mitochondria, as well as cleavage of Bid and caspase-3. Bcl-2 inhibited TRAIL-induced Bax translocation, cytosolic release of cytochrome c and Smac/DIABLO, and the downstream cleavage of XIAP and DFF45. Coadministration of HA14-1 and TRAIL increased apoptosis in SW480/Bcl-2 cells by restoring Bax redistribution and cytochrome c release.
  
  
  CONCLUSIONS
  Bcl-2 confers apoptosis resistance to TRAIL by inhibiting a mitochondrial amplification step and by inactivating downstream XIAP in SW480 cells. HA14-1 reversed Bcl-2-mediated TRAIL resistance, suggesting a novel strategy for increasing TRAIL sensitivity in Bcl-2-overexpressing colon cancers.
authors.affiliation: Divisions of Oncology, Gastroenterology and Hepatology, Mayo Clinic and Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA. sinicrope.frank2mayo.edu
authors.forename: Frank A
authors.initials: FA
authors.lastname: Sinicrope
authors.forename: Robert C
authors.initials: RC
authors.lastname: Penington
authors.forename: Xi Ming
authors.initials: XM
authors.lastname: Tang
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D001578:Benzopyrans / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism; D053453:Caspase 9; D061945:Caspase Inhibitors; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay; D051636:X-Linked Inhibitor of Apoptosis Protein; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
chemical_list: D017304:Annexin A5; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C494772:BAK1 protein, human; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D001578:Benzopyrans; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C412403:DIABLO protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009570:Nitriles; D009842:Oligopeptides; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; C109664:caspase-activated DNase inhibitor; C456800:ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; D045304:Cytochromes c; C506433:CASP9 protein, human; D053453:Caspase 9