title: Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells.
abstract: The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials.
authors.affiliation: Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Department of Medicine, Genetics, and Pharmacology, Abramson Cancer Center, School of Medicine, University of Pennsylvania, Philadelphia 19104, USA.
authors.forename: Seok-Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Kunhong
authors.initials: K
authors.lastname: Kim
authors.forename: Jae G
authors.initials: JG
authors.lastname: Kwagh
authors.forename: David T
authors.initials: DT
authors.lastname: Dicker
authors.forename: Meenhard
authors.initials: M
authors.lastname: Herlyn
authors.forename: Anil K
authors.initials: AK
authors.lastname: Rustgi
authors.forename: Youhai
authors.initials: Y
authors.lastname: Chen
authors.forename: Wafik S
authors.initials: WS
authors.lastname: El-Deiry
mesh_terms: D000230:Adenocarcinoma; D000970:Antineoplastic Agents / Q000494:pharmacology*; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D004947:Esophagus / Q000166:cytology; D058851:GPI-Linked Proteins; D006801:Humans; D008175:Lung Neoplasms; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics* / Q000627:therapeutic use; D053320:Receptors, Tumor Necrosis Factor, Member 10c; D011994:Recombinant Proteins / Q000235:genetics / Q000633:toxicity*; D053319:Tumor Necrosis Factor Decoy Receptors
chemical_list: D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D058851:GPI-Linked Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D053320:Receptors, Tumor Necrosis Factor, Member 10c; D011994:Recombinant Proteins; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; C108661:TNFRSF10C protein, human; D053319:Tumor Necrosis Factor Decoy Receptors; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases