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. 2011 Jan 27:11:39.
doi: 10.1186/1471-2407-11-39.

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

Caroline Mm van Geelen  1 , Bodvael Pennarun, Phuong Tk Le, Elisabeth Ge de Vries, Steven de Jong
Affiliations

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

Caroline Mm van Geelen et al. BMC Cancer. .

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.

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Figures

Figure 1
Figure 1
Inefficient DISC formation in SW948-TR cells. A: Membrane expression of the TRAIL receptors DR4 and DR5 in SW948 and SW948-TR cells as determined by flow cytometry. Values are expressed as the mean fluorescence intensity (MFI) and are mean ± SE of at least three independent experiments. B: Analysis of the TRAIL-DISC in SW948 and SW948-TR. Cells were incubated for 30 min with Flag-tagged TRAIL and the assembled DISCs were immunoprecipitated and analyzed by Western blotting using antibodies to DR4, DR5, FADD, caspase 8 and c-FLIP. T = total cell lysates; C = control of immunoprecipitation, IP = immunoprecipitation (After a longer exposure DR5 bands were also detectable in the total cell lysates (T)). C: Western blot analysis comparing caspase 8 levels in SW948 and SW948-TR cells. One of at least three independent experiments is shown.
Figure 2
Figure 2
Important contributions of DR4 to rhTRAIL-induced apoptosis. A: Apoptosis assay in SW948. Cells were pre-incubated with 10 μg/ml antagonistic anti-DR4 (αDR4), anti-DR5 (αDR5) antibodies or IgG1 control for 1 h before 4-5 h rhTRAIL treatment (0.1 μg/ml). Values are mean ± SD of at least three independent experiments. B: rhTRAIL binding to DR4 and DR5 in SW948 cells. Cells were incubated with increasing concentrations of rhTRAIL before detection of accessible cell surface DR4 and DR5. Values are expressed as the mean fluorescence intensity (MFI) and are mean ± SE of at least three independent experiments. C: Apoptosis assay in SW948-TR. CHX (5 μg/ml) was combined or not with the blocking antibodies 1 h before TRAIL treatment as described in (A).
Figure 3
Figure 3
Sensitivity of SW948 and SW948-TR cells to agonistic DR4 and DR5 antibodies. A: Survival (%) of SW948 and SW948-TR after continuous incubation with agonistic DR4 antibody and agonistic DR5 antibody as measured by cytotoxicity assays. B: Apoptosis assay in SW948 and SW948-TR. Cells were pre-incubated with 5 μg/ml CHX for 1 h before incubation with various concentrations of agonistic DR4 antibody (DR4 Ab) and agonistic DR5 antibody (DR5 Ab) for 24 h.
Figure 4
Figure 4
Equivalent DISC protein recruitment, but not caspase 8 activation, following agonistic DR4 and DR5 antibody treatment. A: Analysis of the DR4 and DR5-DISC in SW948. Cells were incubated for 15 min with agonistic DR4 and DR5 antibodies before co-immunoprecipitation of the associated DISCs using protein G-agarose beads, and subsequent analysis by Western blotting using antibodies directed against DR4, DR5, FADD, caspase 8 and c-FLIP. Post = antibodies added after cell lysis; Stim = cells stimulated with the indicated antibody for 15 min. One representative of at least two independent experiments is shown. B: Time-dependent cleavage of pro-caspase 8 in SW948 cells treated with 50 nM agonistic DR4 and DR5 antibodies.
Figure 5
Figure 5
Sensitization of SW948-TR cells to rhTRAIL, agonistic DR4 and DR5 antibodies by MG-132. A: Apoptosis assay in SW948-TR after 17 h of incubation with MG-132 in combination with rhTRAIL, agonistic DR4 or DR5 antibody. B: The effect of MG-132 on DR4 (left figure) and DR5 (right figure) membrane expression as determined by flow cytometry. Receptor expression was detected as the average antigenic density of the whole cell population and resulted in a peak shift to the right. (1 = control; 2 = basal DR4 or DR5 membrane expression level; 3 = DR4 or DR5 expression after exposure to 10 μM MG-132 for 17 h). C: Western blot analysis of caspase 8 activation in SW948-TR after 17 h incubation with 1 μM MG-132 in combination with rhTRAIL (0.1 μg/ml), agonistic DR4 or DR5 antibody (50 nM).
Figure 6
Figure 6
Increase in caspase 8 levels following treatment of SW948-TR cells with IFN-γ. A: Effects of 48 h incubation with increasing concentrations of IFN-γ on caspase 8 levels in SW948 and SW948-TR. B: Effects of IFN-γ on DR4 (left figure) and DR5 (right figure) membrane expression as determined by flow cytometry. Receptor expression was detected as the average antigenic density of the whole cell population and resulted in a peak shift to the right. (1 = control; 2 = basal DR4 or DR5 membrane expression level; 3 = DR4 or DR5 expression after exposure to 1000 units (U)/ml IFN-γ for 48 h).
Figure 7
Figure 7
Stimulation of rhTRAIL- and agonistic DR4 antibody-induced but not agonistic DR5 antibody-induced apoptosis by IFN-γ. A: Survival (%) of SW948-TR cells after continuous incubation with rhTRAIL, agonistic DR4 or DR5 antibody in combination with different concentrations of IFN-γ. B: Apoptosis assay of SW948-TR after 48 h incubation with rhTRAIL, agonistic DR4 or DR5 antibody in combination with various concentrations of IFN-γ. C: Apoptosis assay in SW948-TR after 48 h incubation with 1000 U/ml IFN-γ. Cells were then incubated with 10 μg/ml antagonistic anti-DR4 (αDR4), anti-DR5 (αDR5) antibodies, both (αDR4/5) or IgG1 control for 1 h before 4-5 h rhTRAIL treatment (0.1 μg/ml). D: Western blot analysis of caspase 8 activation in SW948-TR cells. The cells were pre-incubated for 48 hours with 1000 U/ml IFN-γ, then either left untreated or exposed for 4-5 additional hours with rhTRAIL (0.1 μg/ml) before harvest. E: Western blot analysis of caspase 8 activation in SW948-TR cells. The cells were pre-incubated for 48 hours with 1000 U/ml IFN-γ, then either left untreated or exposed for 4-5 additional hours with agonistic DR4 antibody (50 nM) or agonistic DR5 antibody (50 nM) before harvest.
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