title: Modulation of TRAIL resistance in colon carcinoma cells: different contributions of DR4 and DR5.
abstract: BACKGROUND
  rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5). Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies.
  
  
  METHODS
  Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane.
  
  
  RESULTS
  SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN-gamma could also increase DR5-mediated apoptosis in SW948-TR.
  
  
  CONCLUSIONS
  These results highlight a critical difference between DR4- and DR5-mediated apoptotic signaling modulation, with possible implications for future combinatorial regimens.
authors.affiliation: Departments of Medical Oncology and Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
authors.forename: Caroline Mm
authors.initials: CM
authors.lastname: van Geelen
authors.forename: Bodvael
authors.initials: B
authors.lastname: Pennarun
authors.forename: Phuong Tk
authors.initials: PT
authors.lastname: Le
authors.forename: Elisabeth Ge
authors.initials: EG
authors.lastname: de Vries
authors.forename: Steven
authors.initials: S
authors.lastname: de Jong
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000187:drug effects / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D061988:Proteasome Inhibitors; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000819:agonists / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000627:therapeutic use*
chemical_list: D000911:Antibodies, Monoclonal; D053418:Death Domain Receptor Signaling Adaptor Proteins; D004791:Enzyme Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde