BMC Cancer. 2011 Jan 27;11:39.

Modulation of TRAIL resistance in colon carcinoma cells: different contributions of DR4 and DR5.External

van Geelen, C. M., Pennarun, B., Le, P. T., de Vries, E. G., de Jong, S.,
--- - Departments of Medical Oncology and Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
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.
PMID: 21272366External
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
In vivo interaction
  Endogenous
expression
Overexpression DD1 DD2 Reference
Family DD1 DD2 Method Species Region Species Region
DD DR4 Link FADD Co-immunoprecipitation SW948 21272366
DD DR5 Link FADD Co-immunoprecipitation SW948 21272366
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
Functional Role
Family DD1 DD2 Method Death-related Death-unrelated Reference
DD DR4 Link FADD Immunofluorescence staining Apoptosis (DISC) 21272366
DD DR5 Link FADD Immunofluorescence staining Apoptosis (DISC) 21272366
(Link: click this icon to show interactions only between the two corresponding DDs)