The tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-κB (NF-κB) has been implicated, but its exact role is controversial. We studied different cell lines displaying varying responses to TRAIL and found that TRAIL can activate NF-κB in all our cancer cell lines regardless of their TRAIL sensitivity. Inhibition of NF-κB via adenoviral expression of the IκB-α super-repressor only sensitized the TRAIL-resistant pancreatic cancer cell line Panc-1. Panc-1 cells harbor constitutively activated NF-κB, pointing to a possible role of preactivated NF-κB in protection from TRAIL. Furthermore, we could reduce X-linked inhibitor of apoptosis protein (XIAP) levels in Panc-1 cells by inhibition of constitutively activated NF-κB and sensitize Panc-1 cells to TRAIL by RNA interference against XIAP. These results implicate elevated XIAP levels caused by high basal NF-κB activity in TRAIL resistance and suggest that therapeutic strategies involving TRAIL can be abetted by inhibition of NF-κB and/or XIAP only in tumor cells with constitutively activated NF-κB. (Mol Cancer Res 2006;4(10):715–28)

Apoptosis or programmed cell death plays an important role in development, in tissue and immune homeostasis, and in the elimination of virus-infected cells and tumor cells (1-4). There are two general, interconnected apoptosis pathways. The extrinsic apoptotic pathway is initiated by binding of death ligands to their cognate membrane receptors that subsequently transduce the apoptotic signal. This pathway is complemented by the intrinsic pathway initiated by mitochondrial dysfunction as caused by intracellular stress triggered by cytotoxic drugs or irradiation. One of the ligands that can activate the extrinsic pathway by binding to its receptor(s) is the tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL; refs. 5-7). TRAIL is a transmembrane protein and cytotoxic ligand belonging to the TNF superfamily that also includes TNF-α and Fas ligand. Five distinct receptors for TRAIL have been identified to date. TRAIL-R1 (DR4; ref. 8) and TRAIL-R2 (DR5; refs. 5, 9, 10) contain a cytoplasmic death domain. Trimerization of the receptors upon TRAIL binding leads to recruitment of the adaptor protein Fas-associated death domain and procaspase-8 and procaspase-10 to the death domain, resulting in the formation of the death-inducing signaling complex and the initiation of the apoptotic pathway (11-13). TRAIL-R3 (DcR1; ref. 14), TRAIL-R4 (DcR2; ref. 15), and osteoprotegerin (16) cannot transduce apoptotic signals as they do not contain a cytosolic death domain or only possess a truncated version of the cytoplasmic tail, and their exact role remains to be identified. TRAIL has gained particular attention because recombinant soluble TRAIL can induce apoptosis in a wide range of human tumor cells, but not in most normal cells (17-20). Therefore, TRAIL bears potential as a cancer therapy agent. Nevertheless, many cancer cells respond only poorly to TRAIL treatment or are even completely resistant to TRAIL-induced apoptosis.

Evasion of apoptosis is a hallmark of cancer cells, and the gain of defects in cell death control is a basic event of carcinogenesis. Apoptosis is a tightly controlled process, and the apoptotic pathway is subjected to several levels of inhibitory regulation. These inhibitory, antiapoptotic proteins can intervene in the apoptosis pathway at different stages, and overexpression of these proteins has been associated with resistance to programmed cell death (21-24). At the death-inducing signaling complex level, c-Flip and caspase-8L have been reported to interfere with caspase-8 activation (25-27), whereas the antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL act at the stage of mitochondrial activation by inhibiting cytochrome c release. (28-30). At the caspase level, inhibitor of apoptosis protein (IAP) family members [c-IAP1, c-IAP2, survivin, NIAP, and X-linked IAP (XIAP)] can bind to caspase-9, caspase-7, and caspase-3 and prevent the activation of the caspase cascade (31-35). Among these IAP members, XIAP seems to be the most potent direct caspase inhibitor (31, 34) and has been shown to be overexpressed in tumor cells (36-39). The implication of XIAP overexpression in resistance to TRAIL-mediated apoptosis was recently shown for different cancer cell lines (40-43).

The nuclear factor-κB (NF-κB) is involved in the regulation of the transcription of many of these antiapoptotic proteins (44-47), including XIAP (48). Activation of NF-κB has been implicated in resistance to different chemotherapeutic agents as well as cytokines, such as TNF-α, TRAIL, and Fas ligand (49-53). Intriguingly, the respective receptors cannot only activate the apoptotic pathway but can also elicit a signal cascade, leading to NF-κB activation (23, 54-57). Therefore, it has been suggested that the apoptotic response depends on the balance of proapoptotic signals as mediated by caspases and antiapoptotic programmes controlled by NF-κB. Resistance to TNF-α has been frequently reported to be mediated by NF-κB activation, and inhibition of NF-κB sensitizes most cell types to TNF-α (49, 53, 58). In respect to TRAIL, the role of induced NF-κB is more controversial. Some reports suggest that TRAIL-mediated NF-κB activation has no effect on TRAIL-mediated apoptosis (23, 59), whereas other results implicate the transcription factor in an antiapoptotic role analogous to the TNF-α–induced pathways (50-52). To clarify whether inhibition of NF-κB activation is a useful strategy to overcome TRAIL resistance in cancer cells, we studied TRAIL-mediated apoptotic responses and NF-κB activation in various types of cancer cell lines. We could show that NF-κB induced by TRAIL itself cannot protect from TRAIL-induced apoptosis, whereas constitutive activated NF-κB gives rise to TRAIL resistance. Furthermore, we could identify XIAP as a target protein of constitutively active NF-κB contributing to TRAIL resistance in Panc-1 cells.

Apoptotic Response to TRAIL Varies among Different Cancer Cells

To evaluate the cell killing effect of TRAIL in various cell lines, cells were treated with 1 to 10 ng/mL of soluble recombinant TRAIL for 24 hours, and the apoptotic response was measured (Fig. 1A).

FIGURE 1.

Sensitivity of different cell lines to TRAIL. A and B. Cells were treated for 24 hours (A) or 7 hours (B) with TRAIL at the indicated doses, and the percentage of apoptotic cells was assessed by the standard DNA fragmentation assay according to Nicoletti. TRAIL-induced specific apoptosis was calculated by subtracting basal apoptosis of untreated cells from TRAIL-induced apoptosis. Points/columns, mean from three independent experiments done in triplicate; bars, SE.

FIGURE 1.

Sensitivity of different cell lines to TRAIL. A and B. Cells were treated for 24 hours (A) or 7 hours (B) with TRAIL at the indicated doses, and the percentage of apoptotic cells was assessed by the standard DNA fragmentation assay according to Nicoletti. TRAIL-induced specific apoptosis was calculated by subtracting basal apoptosis of untreated cells from TRAIL-induced apoptosis. Points/columns, mean from three independent experiments done in triplicate; bars, SE.

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The colon cancer cell line DLD-1 was found to be highly sensitive to TRAIL with nearly all cells killed when treated with 10 ng/mL of TRAIL for 24 hours, and still 23% of the cell population underwent apoptosis at the lowest dose of TRAIL (1 ng/mL). The colon cancer cell line HCT116 responded more moderately to TRAIL treatment. However, still a large number of cells (69%) were killed at the highest dose of TRAIL; in addition, at 1 ng/mL, an apoptotic response was seen (16%). The cervical cancer cell line HeLa was less sensitive; that is, 12% (1 ng/mL) to 50% (10 ng/mL) could be killed by TRAIL. The lung cancer cell line A549 and the pancreatic carcinoma cell line Panc-1 were relatively resistant to TRAIL-mediated cell death as only a modest apoptotic response was observed when challenged with the highest dose of TRAIL (14% and 17%, respectively), and almost no apoptosis (<6%) could be measured when treated with low doses of TRAIL (1 and 2 ng/mL, respectively). As previously reported for primary cells, human fibroblasts did not show any apoptotic response even at the highest dose of TRAIL.

Early apoptotic events could already be observed a few hours after stimulation with TRAIL. To evaluate whether the different apoptosis responses were also reflected in the initial onset of cell death, we measured apoptotic cell death at early time points (7 hours; Fig. 1B). We found no apoptotic response in human fibroblasts: A549 and Panc-1 cells (<2%). In HeLa, HCT116, and DLD-1 cells, around 30% to 50% of the cells underwent apoptosis after 7 hours when treated with 10 ng/mL. These data are in accordance with the results of the dose response experiments (Fig. 1A). Overall, the cancer cell lines tested showed different sensitivity to TRAIL-mediated cell death. DLD-1 represented the most sensitive cell line, and A549 and Panc-1 cells, aside from normal, primary human fibroblasts, were the most resistant ones. Next, we wanted to know whether different NF-κB activation levels could explain the differences seen in TRAIL responses.

TRAIL-Induced NF-κB Activation and Apoptosis

Many anticancer drugs are described to activate NF-κB that in turn antagonizes the apoptotic response. As TRAIL is reported to activate NF-κB, we investigated TRAIL-induced NF-κB responses. NF-κB DNA-binding activity was measured in electrophoresis mobility shift assays (EMSA). Nuclear extracts were prepared from cells treated with 10 ng/mL TRAIL for 1 hour (Fig. 2A). NF-κB was activated 1 hour after TRAIL treatment in all cell lines with the exception of primary fibroblasts. Although the levels of nuclear NF-κB after TRAIL treatment differed in the various cell lines, no correlation to TRAIL sensitivity could be observed, as verified by the quantification of the EMSA results shown on the right-hand side in Fig. 2A. Interestingly, within the panel of cell lines analyzed, Panc-1 is the only cell line with high basal NF-κB activity. Furthermore, NF-κB transcriptional activity was measured in a NF-κB–responsive luciferase reporter assay (Fig. 2B). In accordance with the EMSA results, all cell lines, except for the human fibroblasts, showed increased NF-κB activity after TRAIL stimulation (10 ng/mL, 1 hour). The strong basal NF-κB activity in Panc-1 cells [i.e., the NF-κB activity of untreated (medium) cells] is also shown in Fig. 2B.

FIGURE 2.

Activation of NF-κB by TRAIL stimulation. A. Induction of NF-κB DNA-binding activity shown by EMSA using consensus NF-κB oligonucleotides and nuclear extracts 0 and 1 hour after stimulation with TRAIL (10 ng/mL). Right, relative band intensity in the EMSA quantified by the Scion Image Software. B. Basal and TRAIL-induced NF-κB transcription activity shown by an NF-κB–dependent firefly luciferase reporter assay. Differences in transfection efficiency were normalized by cotransfection with a plasmid expressing Renilla luciferase from the ubiquitin promoter. Cells were treated with TRAIL (10 ng/mL) for 1 hour. Columns, mean of the relative luminescence of nontreated (medium) and TRAIL-treated cells of three independent experiments done in duplicate (Panc-1 basal activity; P < 0.001, against all other cell lines); bars, SE.

FIGURE 2.

Activation of NF-κB by TRAIL stimulation. A. Induction of NF-κB DNA-binding activity shown by EMSA using consensus NF-κB oligonucleotides and nuclear extracts 0 and 1 hour after stimulation with TRAIL (10 ng/mL). Right, relative band intensity in the EMSA quantified by the Scion Image Software. B. Basal and TRAIL-induced NF-κB transcription activity shown by an NF-κB–dependent firefly luciferase reporter assay. Differences in transfection efficiency were normalized by cotransfection with a plasmid expressing Renilla luciferase from the ubiquitin promoter. Cells were treated with TRAIL (10 ng/mL) for 1 hour. Columns, mean of the relative luminescence of nontreated (medium) and TRAIL-treated cells of three independent experiments done in duplicate (Panc-1 basal activity; P < 0.001, against all other cell lines); bars, SE.

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In conclusion, TRAIL is able to activate NF-κB, but this induced activation of NF-κB seems to be unable to protect cells from cell death. However, we wanted to know whether the induced NF-κB would afford at least a partial protection from apoptosis in some of the cells.

Inhibition of NF-κB by IκB-α Super-repressor and Its Effect on TRAIL-Induced Apoptosis

To further investigate the potential role of NF-κB in TRAIL-induced apoptosis, we analyzed cell death after specific inhibition of the NF-κB pathway. This was achieved by overexpression of a nondegradable form of the natural inhibitor of NF-κB, IκB-α (IκB-α super-repressor). This super-repressor that contains serine-to-alanine changes at amino acid residues 32 and 36, preventing phosphorylation and subsequent degradation, inhibits NF-κB activation (60). To achieve homogenous expression in all cell lines, we used an adenoviral system for transgene expression. An E1, E3–deleted adenoviral vector containing a cytomegalovirus promoter IκB-α super-repressor (IκB-SR) expression cassette (Ad.IκB-SR) was used. To achieve comparable transduction efficiencies in the different cell lines, cells were separately transduced with an adenoviral vector expressing enhanced green fluorescence protein (EGFP) at different multiplicities of infection (MOI). MOIs that achieved transduction rates of 70% to 90% and had no cytotoxic effects were used for all subsequent experiments. The expression of transgenic IκB-SR protein was verified by Western blotting. In all cell lines, the level of the transgene was substantially higher than the level of the endogenous IκB protein (data not shown).

Apoptosis assays were done 2 days after transduction with Ad.IκB-SR. As control for the effects of viral transduction or transgene overload on TRAIL-induced apoptosis, cells were transduced with an adenoviral vector expressing β-galactosidase (Ad.LacZ) at the same MOIs. No significant increase in cell death in response to TRAIL could be observed in DLD-1, HCT116, A549, HeLa, and human fibroblasts when NF-κB activation was blocked (Fig. 3A). However, Panc-1 cells expressing IκB-SR showed a significant increase in apoptosis, suggesting that blocking NF-κB sensitizes these cells to TRAIL-induced cell death. To test the functionality of the IκB-SR expression, DLD-1 cells were treated with TNF-α, as sensitization of DLD-1 cells to TNF-α by inhibition of NF-κB activation has been previously reported (58). In addition, we tested whether TRAIL-induced NF-κB could be blocked by Ad.IκB-SR in EMSAs. A clear sensitization to TNF-α was seen in DLD-1 cells transduced with Ad.IκB-SR compared with Ad.LacZ-transduced control cells (Fig. 3B), and the translocation of NF-κB to the nucleus after TRAIL treatment was completely blocked in Ad.IκB-SR–transduced cells (Fig. 3C). In conclusion, all cancer cell lines showed NF-κB activation after TRAIL stimulation, but blocking NF-κB activation only sensitized Panc-1 cells to TRAIL-mediated cell death.

FIGURE 3.

Blocking NF-κB activation by Ad.IκB-SR only sensitizes Panc-1 cells to TRAIL. Cells were transduced with Ad.IκB-SR and Ad.LacZ, respectively, or left untransduced. A. Apoptotic responses of cells treated with the indicated doses of TRAIL for 24 hours. Apoptosis was significantly enhanced only in Panc-1 cells when NF-κB was inhibited (P < 0.001). Columns, mean of three independent experiments done in triplicate; bars, SE. B. DLD-1 cells were sensitized to TNF-α (24 hours, 25 ng/mL) but not to TRAIL (24 hours, 1 ng/mL) by blocking NF-κB (P < 0.001). Columns, mean from three independent experiments done in triplicate; bars, SE. C. DLD-1 cells were treated with 10 ng/mL TRAIL for 1 hour, and nuclear extracts were harvested for EMSA. In IκB-SR–expressing cells, NF-κB activation after TRAIL stimulation was inhibited.

FIGURE 3.

Blocking NF-κB activation by Ad.IκB-SR only sensitizes Panc-1 cells to TRAIL. Cells were transduced with Ad.IκB-SR and Ad.LacZ, respectively, or left untransduced. A. Apoptotic responses of cells treated with the indicated doses of TRAIL for 24 hours. Apoptosis was significantly enhanced only in Panc-1 cells when NF-κB was inhibited (P < 0.001). Columns, mean of three independent experiments done in triplicate; bars, SE. B. DLD-1 cells were sensitized to TNF-α (24 hours, 25 ng/mL) but not to TRAIL (24 hours, 1 ng/mL) by blocking NF-κB (P < 0.001). Columns, mean from three independent experiments done in triplicate; bars, SE. C. DLD-1 cells were treated with 10 ng/mL TRAIL for 1 hour, and nuclear extracts were harvested for EMSA. In IκB-SR–expressing cells, NF-κB activation after TRAIL stimulation was inhibited.

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IκB-SR Blocks Constitutively Activated NF-κB in Panc-1 Cells

Inhibition of NF-κB sensitized Panc-1 cells to TRAIL treatment. The sensitizing effect was seen at the onset of apoptosis at early time points (10 ng/mL TRAIL, 7 hours) as well as in treatments with low doses of TRAIL (2.5 ng/mL, 24 hours; Fig. 4A). There was no evidence that NF-κB activation by TRAIL contributes to a resistant phenotype, because in sensitive cell lines (e.g., DLD-1), NF-κB was also found to be activated (Fig. 2). Interestingly, NF-κB inhibition did not sensitize A549, a cell line that is similar to Panc-1 in the apoptotic and NF-κB response to TRAIL (Figs. 1 and 2). As Panc-1 cells can be discriminated from the other cell lines by their high basal NF-κB activity (Fig. 2) and have been previously reported to harbor constitutively activated NF-κB (61, 62), we asked whether this might be correlated to the TRAIL resistance of Panc-1 cells and consequently allow for sensitization by NF-κB inhibition. To confirm that basal as well as activated NF-κB in Panc-1 cells can be inhibited by IκB-SR expression, we did EMSA analyses including untransduced Panc-1 cells and cells transduced with Ad.LacZ and Ad.IκB-SR, respectively, before TRAIL treatment (Fig. 4B). The EMSA showed nuclear NF-κB in untreated and Ad.LacZ-transduced Panc-1 cells and an induction 1 hour after TRAIL treatment. Transduction with Ad.IκB-SR completely abrogated basal NF-κB activation and blocked NF-κB activation after TRAIL treatment in Panc-1 cells. These results suggest that constitutively activated NF-κB can protect cells from TRAIL-induced apoptosis, whereas TRAIL-induced NF-κB is not capable of stopping the apoptotic fate.

FIGURE 4.

Expression of IκB-SR sensitizes Panc-1 cells to TRAIL and blocks basal activated NF-κB. A. Panc-1 transduced with Ad.IκB-SR showed significantly enhanced apoptosis over control cells. TRAIL stimulation was performed for 7 hours (10 ng/mL) or 24 hours (2.5 ng/mL). Columns, mean of three independent experiments done in triplicates (P < 0.001); bars, SE. Bottom, representative fluorescence-activated cell sorting profiles of DNA fragmentation assays. Apoptosis is measured as the sub-G1 fraction, representing cells with a DNA content <2n due to DNA fragmentation at late apoptotic stages. B. EMSA analysis showed that constitutive as well as TRAIL-induced NF-κB DNA-binding activity is completely inhibited in IκB-α-SR–expressing Panc-1 cells compared with untransduced and β-galactosidase expressing control cells. TRAIL treatment was done at 10 ng/mL for 1 hour. The NF-κB specificity of the DNA/protein complex band was confirmed by supershift analysis using a RelA/p65 antibody.

FIGURE 4.

Expression of IκB-SR sensitizes Panc-1 cells to TRAIL and blocks basal activated NF-κB. A. Panc-1 transduced with Ad.IκB-SR showed significantly enhanced apoptosis over control cells. TRAIL stimulation was performed for 7 hours (10 ng/mL) or 24 hours (2.5 ng/mL). Columns, mean of three independent experiments done in triplicates (P < 0.001); bars, SE. Bottom, representative fluorescence-activated cell sorting profiles of DNA fragmentation assays. Apoptosis is measured as the sub-G1 fraction, representing cells with a DNA content <2n due to DNA fragmentation at late apoptotic stages. B. EMSA analysis showed that constitutive as well as TRAIL-induced NF-κB DNA-binding activity is completely inhibited in IκB-α-SR–expressing Panc-1 cells compared with untransduced and β-galactosidase expressing control cells. TRAIL treatment was done at 10 ng/mL for 1 hour. The NF-κB specificity of the DNA/protein complex band was confirmed by supershift analysis using a RelA/p65 antibody.

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Preactivation of NF-κB Confers TRAIL Resistance onto DLD-1 Cells

As basal activated NF-κB might protect cells from TRAIL-induced apoptosis, we asked whether TNF-α–induced NF-κB might have the same effect in the TRAIL-sensitive cell line DLD-1. TNF-α is a potent inducer of NF-κB in DLD-1 (Fig. 5A), and NF-κB activation is sustained for at least 6 hours (data not shown). DLD-1 cells were pretreated with TNF-α (25 ng/mL) for 3.5 hours followed by TRAIL (10 ng/mL) for another 4 and 7 hours, respectively. TNF-α did not cause any apoptotic effects on its own (Fig. 5B). Pretreatment of DLD-1 with TNF-α led to a reduction in apoptotic cells in response to TRAIL compared with DLD-1 cells treated with TRAIL alone at both time points, although the effect was more pronounced at 7 hours (Fig. 5B). DLD-1 cells treated for only 0.5 hour with TNF-α (25 ng/mL) were not protected from TRAIL-induced apoptosis (Fig. 5B), suggesting that the TNF-induced NF-κB response requires sufficient time (at least 3.5 hours) to activate its antiapoptotic program that can protect from subsequent TRAIL challenges. To confirm that the protective effect was mediated by preactivated NF-κB, the experiment was done with DLD-1 cells transduced with Ad.IκB-SR or Ad.LacZ as control. Pretreatment with TNF-α gave rise to decreased sensitivity to TRAIL in β-galactosidase expressing cells, but not in IκB-SR expressing cells, showing that the protective effect is indeed mediated by NF-κB (Fig. 5C). IκB-SR expression had no effect on TNF sensitivity in these pretreatment experiments due to the short duration of the experiment.

FIGURE 5.

Preactivation of NF-κB mediates a protective effect to TRAIL-induced apoptosis in DLD-1 cells. A. TNF-α induced a strong NF-κB activation in DLD-1 cells as shown in the EMSA. B. DLD-1 cells pretreated with TNF-α (25 ng/mL) for 3.5 hours showed a decreased apoptotic response compared with cells treated with TRAIL alone. TRAIL stimulation was done for 4 and 7 hours at 10 ng/mL (left). Pretreatment for 0.5 hour with TNF-α (25 ng/mL) did not afford protection from TRAIL (10 ng/mL) for 4 hours (data not shown) and 7 hours (right). Columns, mean from three independent experiments done in triplicate (P < 0.001); bars, SE. C. Pretreatment with TNF-α (3.5 hours, 25 ng/mL) conferred resistance to β-galactosidase–expressing control cells but did not affect IκB-SR–expressing cells, indicating that the protective effect is mediated by NF-κB. TRAIL stimulation was done for 4 and 7 hours at 10 ng/mL. Columns, mean from two independent experiments done in duplicate (P < 0.05); bars, SE. D. Overexpression of p65 conferred resistance to DLD-1 cells. Cells were transfected with p65-EGFP and pN1-EGFP (control), respectively. Cells were analyzed by flow cytometry for vital EGFP-positive cells after TRAIL (7 hours, 10 ng/mL) treatment. Apoptosis rates were estimated in relation to EGFP-positive cells in untreated control samples and were calculated by subtracting % viable EGFP-positive cells from 100%. Three independent experiments were done (P < 0.001).

FIGURE 5.

Preactivation of NF-κB mediates a protective effect to TRAIL-induced apoptosis in DLD-1 cells. A. TNF-α induced a strong NF-κB activation in DLD-1 cells as shown in the EMSA. B. DLD-1 cells pretreated with TNF-α (25 ng/mL) for 3.5 hours showed a decreased apoptotic response compared with cells treated with TRAIL alone. TRAIL stimulation was done for 4 and 7 hours at 10 ng/mL (left). Pretreatment for 0.5 hour with TNF-α (25 ng/mL) did not afford protection from TRAIL (10 ng/mL) for 4 hours (data not shown) and 7 hours (right). Columns, mean from three independent experiments done in triplicate (P < 0.001); bars, SE. C. Pretreatment with TNF-α (3.5 hours, 25 ng/mL) conferred resistance to β-galactosidase–expressing control cells but did not affect IκB-SR–expressing cells, indicating that the protective effect is mediated by NF-κB. TRAIL stimulation was done for 4 and 7 hours at 10 ng/mL. Columns, mean from two independent experiments done in duplicate (P < 0.05); bars, SE. D. Overexpression of p65 conferred resistance to DLD-1 cells. Cells were transfected with p65-EGFP and pN1-EGFP (control), respectively. Cells were analyzed by flow cytometry for vital EGFP-positive cells after TRAIL (7 hours, 10 ng/mL) treatment. Apoptosis rates were estimated in relation to EGFP-positive cells in untreated control samples and were calculated by subtracting % viable EGFP-positive cells from 100%. Three independent experiments were done (P < 0.001).

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To further corroborate the role of preactivated NF-κB in TRAIL apoptosis, DLD-1 cells were transfected with a p65-EGFP fusion expression construct and a pN1-EGFP control plasmid. The EGFP expression allowed us to follow the fate of p65-expressing cells following TRAIL treatment. To this end, vital EGFP-positive cells were analyzed by fluorescence-activated cell sorting after TRAIL treatment (10 ng/mL, 7 hours). Cells overexpressing the NF-κB subunit p65 showed a higher survival rate and a decreased apoptosis rate, respectively, compared with cells transfected with pN1-EGFP (Fig. 5D). The reduction in the number of apoptotic cells was similar to the pretreatment with TNF-α. Taken together, these results suggest that although NF-κB induced by TRAIL itself is not decisive for apoptosis, preactivated or constitutive active NF-κB can modulate TRAIL sensitivity and thereby contribute to TRAIL resistance in Panc-1 cells.

Down-Regulation of XIAP Protein Levels by RNA Interference Increases TRAIL-Induced Apoptosis

XIAP is an important mediator of chemoresistance (63-66), and its transcription is regulated by NF-κB (48). XIAP is a member of the IAP family, and high levels of XIAP have been previously correlated to resistance to TRAIL-mediated apoptosis (67, 68). First, we examined the XIAP protein levels in the cell lines we had tested for TRAIL responses (Fig. 1). The Western blot and its quantification revealed that Panc-1 cells harbor the highest XIAP levels of all tumor cells tested (Fig. 6A). Furthermore, immunoblots and quantifications of XIAP following TNF-α treatment (7.5 hours, 25 ng/mL) and p65 overexpression in DLD-1 cells showed a modest induction (20-50%) of XIAP in response to these treatments, whereas short durations of TNF-α pretreatment (0.5 hour) failed to increase XIAP levels (Fig. 6B). These results point to an antiapoptotic role of NF-κB–dependent increases in XIAP in Panc-1 cells that could at least partially explain their TRAIL resistance. In addition, we analyzed the induction of another NF-κB target gene (i.e., Bcl-xL) and found it to be also up-regulated by TNF-α pretreatment (7.5 hours), indicating that not only XIAP but several antiapoptotic proteins might determine the response to TRAIL. To determine the influence of XIAP protein levels in TRAIL-mediated cell death in our cell lines and to assess its role in cells harboring constitutively activated NF-κB versus those that only show induced NF-κB activation, we knocked down XIAP protein levels by DNA-directed RNA interference (RNAi) via adenoviral (Ad.shRNA.XIAP)–mediated expression of small hairpin RNAs (shRNA) directed at XIAP. Forty-eight hours after viral transduction, XIAP expression was assessed by Western blot and found to be down-regulated to almost nondetectable levels (Fig. 6C; results for DLD-1 and Panc-1 cells are shown). To account for any effects of adenoviral infection and/or shRNA expression, an adenoviral vector (Ad.shRNA.EGFP) containing a shRNA construct against EGFP was used as control. Cells were then transduced with Ad.shRNA.XIAP or Ad.shRNA.EGFP, and 48 hours after transduction, cells were treated with 1 ng/mL TRAIL (DLD-1 and HCT116) or 2.5 ng/mL TRAIL (Panc-1, HeLa, and human fibroblasts) for another 24 hours, and apoptosis was assessed (Fig. 6D). No significant increase in apoptosis was seen in DLD-1, HCT116, HeLa, and human fibroblasts when XIAP was knocked down. Hence, XIAP does not represent a decisive block in TRAIL-mediated apoptosis in these cell lines. On the other side, the sensitivity of Panc-1 cells was considerably increased when XIAP protein levels were decreased by RNAi, suggesting that XIAP contributes, at least partially, to TRAIL resistance of Panc-1 cells, but not in the other cells investigated. Sensitization to TRAIL was observed at the onset of apoptosis at early time points (10 ng/mL TRAIL, 7 hours) as well as for treatment with low doses of TRAIL (2.5 ng/mL, 24 hours) for longer times (Fig. 7A).

FIGURE 6.

XIAP contributes to TRAIL resistance in cells with preactivated or constitutively activated NF-κB. A. Immunoblotting of the indicated cell lines revealed the different levels of XIAP protein (53 kDa). CuZnSOD (20 kDa) probing served as loading control. Right, a quantitative analysis of the XIAP protein bands. Signals of XIAP were quantified relative to CuZnSOD control protein bands. B. DLD-1 cells were treated with TNF-α for 0.5 or 7.5 hours and subjected to immunoblotting for XIAP, Bcl-xL, and CuZnSOD (left). Overexpression of p65 (p65-EGFP) also led to increased XIAP levels in DLD-1 cells compared with pN1-EGFP–transfected controls (right). Quantitative analyses of the protein bands were carried out with the Scion Image Software, and the results are in the diagrams on the right-hand side of the corresponding Western blots. Signals of XIAP protein bands were quantified relative to CuZnSOD control protein bands. C. Down-regulation of XIAP by RNAi. Cells were transduced with Ad.shRNA.XIAP or Ad.shRNA.EGFP (control). Adenoviral delivery of shRNA against XIAP lead to an efficient downregulation of the XIAP protein; DLD-1 and Panc-1 are shown. Down-regulation of XIAP was quantified using the Scion Image Software. Signals are expressed as percentage in respect to signals from negative controls (Ad.shRNA.EGFP). D. Apoptotic responses of cells treated with the indicated doses of TRAIL for 24 hours. Down-regulation of XIAP significantly sensitized Panc-1 to TRAIL. Columns, mean of three independent experiments done in triplicate (P < 0.001); bars, SE.

FIGURE 6.

XIAP contributes to TRAIL resistance in cells with preactivated or constitutively activated NF-κB. A. Immunoblotting of the indicated cell lines revealed the different levels of XIAP protein (53 kDa). CuZnSOD (20 kDa) probing served as loading control. Right, a quantitative analysis of the XIAP protein bands. Signals of XIAP were quantified relative to CuZnSOD control protein bands. B. DLD-1 cells were treated with TNF-α for 0.5 or 7.5 hours and subjected to immunoblotting for XIAP, Bcl-xL, and CuZnSOD (left). Overexpression of p65 (p65-EGFP) also led to increased XIAP levels in DLD-1 cells compared with pN1-EGFP–transfected controls (right). Quantitative analyses of the protein bands were carried out with the Scion Image Software, and the results are in the diagrams on the right-hand side of the corresponding Western blots. Signals of XIAP protein bands were quantified relative to CuZnSOD control protein bands. C. Down-regulation of XIAP by RNAi. Cells were transduced with Ad.shRNA.XIAP or Ad.shRNA.EGFP (control). Adenoviral delivery of shRNA against XIAP lead to an efficient downregulation of the XIAP protein; DLD-1 and Panc-1 are shown. Down-regulation of XIAP was quantified using the Scion Image Software. Signals are expressed as percentage in respect to signals from negative controls (Ad.shRNA.EGFP). D. Apoptotic responses of cells treated with the indicated doses of TRAIL for 24 hours. Down-regulation of XIAP significantly sensitized Panc-1 to TRAIL. Columns, mean of three independent experiments done in triplicate (P < 0.001); bars, SE.

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FIGURE 7.

Down-regulation of XIAP sensitizes Panc-1 cells to TRAIL by promoting processing of caspase-3. A. Panc-1 transduced by Ad.shRNA.XIAP showed significantly enhanced apoptosis over control cells with different concentrations of TRAIL and different time points. TRAIL stimulation was performed for 7 hours (10 ng/mL) or for 24 hours (2.5 ng/mL). Columns, mean of three independent experiments done in triplicates (P < 0.001); bars, SE. Bottom, representative fluorescence-activated cell sorting profiles of DNA fragmentation assays. Apoptosis was measured as the sub-G1 fraction, representing cells with a DNA content <2n due to DNA fragmentation in late apoptotic stages. B. Caspase-3 was processed more efficiently after TRAIL stimulation (24 hours, 2.5 ng/mL) in Panc-1 cells transduced with Ad.IκB-SR as well as in cells transduced with Ad.shRNA.XIAP. Western blot analysis against caspase-3 recognizing full-length caspase-3 (35 kDa), the cleaved intermediate form (20 kDa), and the active fragment (17 kDa). In addition, immunoblotting against procaspase-8 (55 kDa) was done. CuZnSOD (20 kDa) was used as loading control. C. A caspase-3 activity assay in Panc-1 cells that were transduced with either Ad.shRNA.EGFP or Ad.shRNA.XIAP and then treated with TRAIL (2.5 ng/mL) for 24 hours showed increased activity in the cells with the XIAP knockdown.

FIGURE 7.

Down-regulation of XIAP sensitizes Panc-1 cells to TRAIL by promoting processing of caspase-3. A. Panc-1 transduced by Ad.shRNA.XIAP showed significantly enhanced apoptosis over control cells with different concentrations of TRAIL and different time points. TRAIL stimulation was performed for 7 hours (10 ng/mL) or for 24 hours (2.5 ng/mL). Columns, mean of three independent experiments done in triplicates (P < 0.001); bars, SE. Bottom, representative fluorescence-activated cell sorting profiles of DNA fragmentation assays. Apoptosis was measured as the sub-G1 fraction, representing cells with a DNA content <2n due to DNA fragmentation in late apoptotic stages. B. Caspase-3 was processed more efficiently after TRAIL stimulation (24 hours, 2.5 ng/mL) in Panc-1 cells transduced with Ad.IκB-SR as well as in cells transduced with Ad.shRNA.XIAP. Western blot analysis against caspase-3 recognizing full-length caspase-3 (35 kDa), the cleaved intermediate form (20 kDa), and the active fragment (17 kDa). In addition, immunoblotting against procaspase-8 (55 kDa) was done. CuZnSOD (20 kDa) was used as loading control. C. A caspase-3 activity assay in Panc-1 cells that were transduced with either Ad.shRNA.EGFP or Ad.shRNA.XIAP and then treated with TRAIL (2.5 ng/mL) for 24 hours showed increased activity in the cells with the XIAP knockdown.

Close modal

XIAP interacts with caspase-9, caspase-7, and caspase-3, inhibiting further activation of these caspases (69). We therefore analyzed untransduced Panc-1 cells after TRAIL stimulation and cells transduced with Ad.IκB-SR and Ad.shRNA.XIAP, respectively, for the activation of caspase-3, a downstream effector caspase (Fig. 7B). RNAi against XIAP promoted processing of caspase-3 to the p17 active fragment following TRAIL treatment compared with controls. In IκB-SR expressing cells, processing to active caspase-3 (p17) is also promoted in comparison to the Ad.LacZ-transduced control cells. Furthermore, processing of procaspase-8 was slightly enhanced in IκB-SR and shRNA.XIAP expressing cells, which is most likely the result of enhanced activation of caspase-3 because procaspase-8 is a substrate for active caspase-3 (70-72). The immunoblot data could be confirmed by a caspase-3 activity assay showing a significant increase in caspase-3 activity after TRAIL treatment in cells treated with Ad.shRNA.XIAP compared with cells transduced with the control vector Ad.shRNA.EGFP (Fig. 7C). As XIAP contributes to TRAIL resistance in Panc-1 cell, and as NF-κB is reported to be involved in the regulation of XIAP transcription (48), we asked whether the sensitization effect observed in Ad.IκB-SR–transduced Panc-1 cells can be attributed to decreased XIAP levels as a result of inhibition of constitutively activated NF-κB.

Constitutively Activated NF-κB Causes High XIAP Protein Levels in Panc-1 Cells

We analyzed DLD-1 and Panc-1 cells that were transduced with Ad.IκB-SR for 48 hours, respectively, by Western blotting for XIAP protein expression (Fig. 8A). We found that XIAP protein levels decreased 48 hours after transduction to 42% of the protein level of the untransduced control in Panc-1 cells. Adenoviral transduction had no effect on XIAP protein levels as seen in the Ad.LacZ control. The observed decline in XIAP levels was not due to XIAP processing during apoptosis induced by inhibition of constitutively activated NF-κB, as blocking of caspase activity by zVAD.fmk did not prevent XIAP down-regulation. In contrast to Panc-1 cells, blocking NF-κB did not significantly influence XIAP protein levels in DLD-1 cells (Fig. 8A). These data point to basal activated NF-κB as the molecular lever that causes elevated XIAP levels and apoptosis resistance in Panc-1 cells.

FIGURE 8.

XIAP protein levels are down-regulated by inhibition of constitutively activated NF-κB in Panc-1 cells. A. Panc-1 and DLD-1 cells were transduced with Ad.LacZ, Ad.IκB-SR, or left untransduced for 48 hours. To inhibit protein degradation due to unspecific apoptosis, zVAD.fmk pancaspase inhibitor (20 μmol/L) was added to some of the samples. Western blot analysis was done against XIAP, IκB-α (41 kDa), and CuZnSOD. The tagged mutated IκB-SR has a lower mobility than the endogenous IκB-α. CuZnSOD was used as protein loading control. Right, quantitative analyses of the protein bands were carried out using Scion Image Software. Signals are expressed as percentage of signals from negative controls (Ad.LacZ; Ad.LacZ + zVAD.fmk). B. A Western Blot using an antibody directed against Smac/DIABLO shows that Ad.shRNA.XIAP did not alter the levels Smac/DIABLO in whole-cell extracts and mitochondria, respectively. COX4 and CuZnSOD were used as loading controls. C. Panc-1 and DLD-1 cells were transduced with Ad.LacZ, Ad.IκB-SR, Ad.shRNA.EGFP, or Ad.shRNA.XIAP respectively for 48 hours. Western blot analysis was done against c-Flip (55 kDa) and CuZnSOD. No changes in c-Flip levels could be detected.

FIGURE 8.

XIAP protein levels are down-regulated by inhibition of constitutively activated NF-κB in Panc-1 cells. A. Panc-1 and DLD-1 cells were transduced with Ad.LacZ, Ad.IκB-SR, or left untransduced for 48 hours. To inhibit protein degradation due to unspecific apoptosis, zVAD.fmk pancaspase inhibitor (20 μmol/L) was added to some of the samples. Western blot analysis was done against XIAP, IκB-α (41 kDa), and CuZnSOD. The tagged mutated IκB-SR has a lower mobility than the endogenous IκB-α. CuZnSOD was used as protein loading control. Right, quantitative analyses of the protein bands were carried out using Scion Image Software. Signals are expressed as percentage of signals from negative controls (Ad.LacZ; Ad.LacZ + zVAD.fmk). B. A Western Blot using an antibody directed against Smac/DIABLO shows that Ad.shRNA.XIAP did not alter the levels Smac/DIABLO in whole-cell extracts and mitochondria, respectively. COX4 and CuZnSOD were used as loading controls. C. Panc-1 and DLD-1 cells were transduced with Ad.LacZ, Ad.IκB-SR, Ad.shRNA.EGFP, or Ad.shRNA.XIAP respectively for 48 hours. Western blot analysis was done against c-Flip (55 kDa) and CuZnSOD. No changes in c-Flip levels could be detected.

Close modal

To exclude that RNAi against XIAP had an effect on Smac/DIABLO, a known XIAP antagonist, Smac/DIABLO protein levels were analyzed, but no differences in the protein status in whole-cell extract and mitochondrial fractions were found (Fig. 8B). The antiapoptotic protein c-Flip, another NF-κB target, has also been implicated in resistance to TRAIL-mediated apoptosis (25, 73). To investigate whether c-Flip levels were influenced when basal activated NF-κB was blocked, we carried out Western blots against c-Flip (Fig. 8C). No change in c-Flip levels could be observed in Ad.IκB-SR–transduced Panc-1 cells, indicating that c-Flip levels are not involved in the TRAIL resistance phenotype of Panc-1 cells. We could also exclude any influence of shRNA.XIAP and shRNA.EGFP expression on c-Flip protein level.

TRAIL is a promising agent in cancer treatment as it is a potent inducer of cell death in a wide range of tumor cells, but not in normal cells (7, 17-20, 74). The molecular determinants of the unresponsiveness of normal cells to TRAIL are still not fully understood. Although TRAIL possesses a good degree of tumor specificity, responses to TRAIL vary among tumor cells, and some are completely resistant (75). For the development of strategies that sensitize resistant tumors to TRAIL therapy, it is important to elucidate the mechanisms that modulate the sensitivity to TRAIL-induced apoptosis.

The transcription factor NF-κB has been implicated in resistance to chemotherapeutic treatment (44-47). Many anticancer drugs activate NF-κB in parallel to the apoptotic pathway, and inhibition of NF-κB sensitizes cells, although this often occurs in a cell type–specific manner (49, 53, 58). It has been shown that TRAIL can activate NF-κB mediated by TRAIL-R1, TRAIL-R2 (54, 56), and TRAIL-R4 (23, 76). The significance of NF-κB activation in TRAIL-induced apoptosis, however, is controversial, and sensitization to TRAIL-induced apoptosis by NF-κB inhibition seems to be cell type specific (45, 77). TRAIL-induced activation of NF-κB has been shown to either have no effect on survival (23, 59, 78) or exhibit antiapoptotic effects (50-52). In contrast to other studies, we did not focus on one type of tumor cells but analyzed tumor cell lines of different origin. TRAIL stimulation led to a rapid induction of NF-κB after 1 hour in all cells. Nevertheless, we did not find a correlation between the potential to activate NF-κB and sensitivity to TRAIL, suggesting that NF-κB activation by TRAIL is not able to modulate the apoptotic response. When we blocked NF-κB activation upon TRAIL signaling by overexpression of a mutated, nondegradable IκB-α protein IκB-SR, we could not sensitize the majority of our cell lines to TRAIL, suggesting that TRAIL-mediated NF-κB activation does not have a protective effect in TRAIL-induced apoptosis. However, we could sensitize the pancreatic carcinoma cell line Panc-1. In contrast to the other cell lines, Panc-1 cells possess constitutively activated NF-κB that was blocked by overexpression of IκB-SR. Our results suggest that constitutively activated NF-κB confers resistance to TRAIL, whereas TRAIL-induced NF-κB activation has no effect in this regard.

To further confirm these findings, we preactivated NF-κB before TRAIL treatment in DLD-1 cells, a cell line highly sensitive to TRAIL. This was achieved by pretreatment with TNF-α, a strong inducer of NF-κB and by overexpression of p65, a major component of the NF-κB complex. The activation of NF-κB, either by TNF-α or p65 overexpression, before TRAIL treatment reduced the sensitivity of DLD-1 cells to TRAIL. However, TNF pretreatment for only 0.5 hour failed to protect cells from cell death, indicating that the duration of NF-κB activation is critical to ensure that the antiapoptotic machinery is in place at the point of TRAIL stimulation. In this context, NF-κB induced by TRAIL itself might simply be too slow to stop the apoptotic cascade.

NF-κB mediates its antiapoptotic effect by the regulation of the transcription of a number of proteins with antiapoptotic functions, and their overexpression has been associated with resistance to anticancer drugs. In this context, XIAP has been identified as an antiapoptotic protein that is not only frequently up-regulated in tumors but also has a substantial potential to mediate resistance to various drugs and has therefore been suggested as a target in antitumor treatments (65, 66). As XIAP has been described to be NF-κB regulated (48), we were interested whether a potential NF-κB-XIAP axis is behind the TRAIL resistance and whether targeting XIAP and targeting NF-κB are comparable with regard to TRAIL sensitization. Interestingly, we found Panc-1 again to be the only cell line that was significantly sensitized to TRAIL by XIAP down-regulation. We could further clarify the correlation between basal activated NF-κB and XIAP protein levels by showing that blocking basal activated NF-κB reduces XIAP protein levels to <50% of its normal level. However, despite giving rise to some protection from TRAIL, preactivation of NF-κB by TNF-α and p65 overexpression in DLD-1 cells did not induce XIAP protein levels to the same extent as constitutive NF-κB does in Panc-1 cells and never afforded the same degree of protection, possibly pointing to additional, qualitative differences between induced and constitutive NF-κB. Interestingly, inhibition of basal activated NF-κB and targeting XIAP by RNAi sensitized Panc-1 cells to a similar extent, although XIAP was nearly completely down-regulated by the latter approach and only to about 50% by NF-κB inhibition. One possible explanation for this discrepancy is the existence of several NF-κB–dependent antiapoptotic proteins, although only a few seem to have importance in resistance mechanisms (40). Even partial inhibition of a combination of these factors, including XIAP by IκB-SR, could be as effective as complete knockdown of just one of the antiapoptotic proteins. To this end, we also addressed the role of c-Flip. c-FlipL, a caspase-8 inhibitor that has been shown to be transcriptionally regulated by NF-κB (79), has been correlated to TRAIL resistance in some cell lines (73, 80, 81). Here, we could show that there is no NF-κB–dependent up-regulation in Panc-1 cells; hence, c-Flip seems to have no role in TRAIL resistance in Panc-1 cells. Bcl-xL, another NF-κB antiapoptotic target gene, has been shown before to be involved in chemoresistance and TRAIL resistance in an NF-κB–dependent way (82-85). Inhibition of constitutive NF-κB by overexpression of IκB-SR inhibited expression of Bcl-xL (61, 83, 86, 87), and down-regulation of Bcl-xL protein sensitized Panc-1 to TRAIL-mediated apoptosis (88). In addition, we found Bcl-xL to be elevated by TNF-α pretreatment. Thus, Bcl-xL and XIAP together might constitute an NF-κB–driven apoptosis resistance mechanism in Panc-1 cells. Basal XIAP protein levels might not be predictive for the responsiveness of tumors to XIAP down-regulation alone. Although XIAP protein levels in Panc-1 cells exceeded those of the other tumor cell lines tested, all cell lines expressed XIAP at substantial levels. XIAP is also not the sole molecular determinant of apoptosis, as discussed above, and tumor cells differ in their molecular make-up with regard to the strength of proapoptotic and antiapoptotic pathways. Panc-1 cells, for example, show low expression levels of apoptosis-activating TRAIL receptors 1 and 2 (data not shown), and in such context (i.e., in cells with reduced proapoptotic potential), XIAP might gain relative importance. Hence, to fully predict tumor responses, both antiapoptotic and proapoptotic programs have to be assessed. Nevertheless, constitutively activated NF-κB is a common feature in tumors (62, 89-93), and activated NF-κB has been implicated in carcinogenesis, metastasis, and chemoresistance (92, 94-97). Because NF-κB activation has been associated with resistance to many anticancer drugs, targeting NF-κB to improve the outcome of cancer therapy has been frequently suggested. We could show that in TRAIL-based therapies targeting NF-κB only in tumors with basal activated NF-κB is a useful approach. Furthermore, we found that 5-fluorouracil responsiveness of Panc-1 cells could also be increased by NF-κB inhibition, pointing to a general principle that constitutively activated NF-κB is the main culprit in drug resistance, whereas induced NF-κB only plays a minor role in specific cases (e.g., TNF-α). Therefore, our results suggest that therapies targeting NF-κB, in particular in combination with TRAIL, should be limited to those cancers that exhibit high basal NF-κB. Alternatively, NF-κB target gene products (i.e., XIAP and Bcl-xL) could be blocked thereby affording a greater degree of specificity than blocking NF-κB.

Importantly, primary fibroblasts were not sensitized by targeting XIAP, providing an avenue of tumor-specific sensitization approaches that do not affect normal cells, which is an important goal in cancer therapy.

Cell Culture

The following cells were used in the study: DLD-1 and HCT116 human colon cancer cells, HeLa cervical cancer cells, A549 human non–small cell lung cancer cells, Panc-1 human pancreatic cancer cells, and primary human fibroblasts. Cells were grown in McCoy's medium (HCT116), RPMI 1640 (DLD-1), and DMEM (HeLa, A549, Panc-1, and human fibroblasts) supplemented with 10% FCS and 1% penicillin/streptomycin. The media and supplements were purchased from Invitrogen (Carlsbad, CA) and Biochrom (Berlin, Germany), respectively. Cells were electroporated in a Bio-Rad (Hercules, CA) Gene Pulser Xcell using an exponential decay pulse type in 0.2-cm cuvettes at 160 V and 1,000 μF.

Reagents and Antibodies

All chemicals if not otherwise stated were purchased from Sigma (St. Louis, MO). Human recombinant TRAIL was purchased from R&D Systems (Minneapolis, MN); human recombinant TNF-α was purchased from PeproTech (Rocky Hill, NY). Pancaspase inhibitor zVAD-fmk was from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-caspase-3 was purchased from Transduction Laboratories (Lexington, CA). Monoclonal anti-caspase-8 (1C12) was purchased from Cell Signaling Technology (Beverly, MA). Monoclonal anti-Smac/DIABLO was purchased from BD PharMingen (San Diego, CA). Polyclonal anti-CuZn superoxide dismutase (CuZnSOD) was purchased from The Binding Site (Birmingham, United Kingdom). Polyclonal anti-FlipL (Dave2) was purchased from Apotech (Lausen, Switzerland). Anti-NF-κB p65 (C-20) was purchased from Santa Cruz Biotechnology. Polyclonal anti-XIAP was purchased from Cell Signaling Technology. Peroxidase-conjugated secondary antibodies were anti-mouse and anti-rabbit and were purchased from Amersham Biosciences (Uppsala, Sweden). Anti-rat was purchased from Apotech, and anti-goat/sheep was purchased from Sigma.

Cytokine Treatment and Apoptosis Assay

For the detection of apoptotic cell death, standard DNA fragmentation assays according to Nicoletti et al. (98) were used. Cells were seeded 24 hours before treatment in 12-well plates, and TRAIL was added at 70% to 80% confluency. When TNF-α pretreatment was done, TNF-α was removed after 0.5 or 3.5 hours, and fresh medium was added to the TNF-α control, or TRAIL-containing medium was added to the TRAIL and TNF-α/TRAIL samples. At the stated time points, cells were harvested, including the medium by trypsinization, directly into BD Falcon fluorescence-activated cell sorting tubes and centrifuged at 1,300 rpm for 7 minutes at 4°C. After washing with PBS, Nicoletti buffer [sodium citrate 0.1% (w/v) containing 0.5% Triton X-100 (w/v) and propidium iodide 50 μg/mL] was added to the cell pellets, tubes were vortexed for 10 seconds at medium speed and left overnight in the dark (4°C). The fluorescence intensity was measured in a flow cytometer (FACSCalibur, Becton Dickinson, Heidelberg, Germany) and analyzed with the CellQuest software. Untreated cells were taken as reference to calculate specific apoptosis by subtraction of the basal apoptosis values from the levels of treated cells.

Survival Assay of EGFP-Positive Cells

DLD-1 cells were seeded in 12-well plates. Cells were transfected at 70% confluency with 2.5 μg of a plasmid expressing the NF-κB subunit p65 fused to EGFP (p65-EGFP) and 2 μg of a EGFP expressing control plasmid (pN1-EGFP). Transfections were carried out using Fugene (Roche, Basel, Switzerland) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were treated with 10 ng/mL TRAIL for 7 hours. Cells were then washed with PBS, harvested in PBS, and analyzed by flow cytometry for vital EGFP-positive cells. The mean percentage of EGFP-positive cells was calculated from four samples. Cell survival was estimated by subtracting the mean of TRAIL-treated samples from untreated samples.

Luciferase Assay

Cells (2 × 105) were seeded in 12-well plates. Next day, cells were transiently transfected with 2 μg of a firefly luciferase reporter plasmid containing a 3× NF-κB response element and 0.1 μg of a ubiquitin promoter Renilla luciferase control plasmid using Fugene according to the manufacturer's instructions. Twenty-four hours after transfection, cells were treated with 10 ng/mL TRAIL for 1 hour and harvested. Luciferase activity was measured in a luminometer (Berthold, Bad Wildbad, Germany) using the dual-luciferase reporter assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Firefly luciferase activities were normalized to Renilla luciferase levels.

Adenoviral Constructs and Adenoviral Transduction

NF-κB inhibition was done using an E1, E3–deleted adenoviral vector containing a cytomegalovirus promoter IκB-α super-repressor (IκB-SR) expression cassette (Ad.IκB-SR; ref. 99). The IκB-SR protein contains serine-to-alanine changes at amino acid residues 32 and 36, preventing phosphorylation and subsequent degradation by ubiquination of the IκB protein and consequently NF-κB activation (60). To account for adenoviral transduction and transgene expression, a control vector containing a cytomegalovirus β-galactosidase (LacZ) expression cassette was used (Ad.LacZ). The XIAP DNA-directed RNAi studies were done using an E1, E3–deleted adenovirus containing a shRNA expression cassette against XIAP under the control of the human RNA polymerase III U6 promoter (Ad.shRNA.XIAP). The following shRNA motif was used: 5′-GTGGTAGTCCTGTTTCAGC-3′. The double-stranded XIAP.shRNA oligo containing the motif was cloned into a modified pENTR/U6 plasmid (Invitrogen), and the expression cassette was further transferred into a promoterless adenoviral vector plasmid (pAd/PL-DEST; Invitrogen) using the Clonase enzyme (Invitrogen) according to the manufacturer's instructions. To control for effects of adenoviral transduction and RNAi, a control vector containing a motif against EGFP was used (Ad.shRNA.EGFP). Adenoviral vectors were produced as described before (99, 100, 101).

Adenoviral vector transductions were carried out in medium containing 2% FCS and 1% penicillin/streptomycin overnight, 24 hours after plating of the cells. On the next day, the virus-containing medium was replaced by fresh medium. To take into account adenoviral transduction efficiencies of the different cell lines, cells were transduced with an adenoviral vector encoding EGFP at MOIs of 20, 50 100, 200, 300, and 400, and EGFP expression was evaluated by fluorescence-activated cell sorting analysis. Finally, cells were transduced with the following MOI, achieving around 70% to 90% of transduction: A549, MOI 50; DLD-1, HCT116, and HeLa, MOI 100; human fibroblasts and Panc-1, MOI 300.

Cellular Protein Extraction and Western Blotting

For the preparation of cell extracts, cells were grown on 10-cm plates. After the indicated treatment, cells were washed twice with ice-cold PBS, harvested by scraping into PBS, and pelleted by centrifugation (5 minutes, 3,000 rpm, 4°C) in a microcentrifuge. For whole-cell extracts, cell pellets were lysed for 15 minutes on ice in NP40 protein extraction buffer [0.3% NP40, 20 mmol/L HEPES (pH 7.9), 0.42 mmol/L NaCl2, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 25% glycerol, 1 mmol/L DTT, and Protease Inhibitor Cocktail (Sigma; added fresh each time)]. The lysate was clarified by centrifugation (13,000 rpm, 5 minutes, 4°C). For the preparation of nuclear extracts, cell pellets were resuspended in Dignam A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L DTT, and Protease Inhibitor Cocktail] and lysed for 15 minutes on ice. After centrifugation (13,000 rpm, 1 minute, 4°C), intact nuclei were resuspended in Dignam C [20 mmol/L HEPES (pH 7.9), 0.42 mmol/L NaCl2, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 25% glycerol, 1 mmol/L DTT, and Protease Inhibitor Cocktail], lysed for 30 minutes on ice with gentle agitation. Lysates were finally clarified by centrifugation (13,000 rpm, 5 minutes, 4°C).

Protein concentrations were determined by Bradford protein assay (Bio-Rad). For Western blots, 50 μg protein/lane were separated on a denaturing 12.5% SDS-PAGE and then transferred onto a nitrocellulose membrane. Membranes were blocked with 4% nonfat dry milk in PBS containing 0.2% Tween 20 for 1 hour at room temperature and then incubated with the first antibody in blocking solution overnight (4°C). Washing steps were done in blocking solution and finally twice in PBS. After incubation with horseradish peroxidase–conjugated secondary antibodies, signals were detected using a standard enhanced chemiluminescence method and Hyperfilm from Amersham Biosciences. Quantification was done using Scion Image Software.

Caspase Activity Assay

Cells were lysed in assay buffer [100 mmol/L HEPES, 10% sucrose, 2 mmol/L DTT, 0.0001% NP40, and 0.1% CHAPS (pH 7.25)] for 10 minutes on ice. The protein concentration was measured and adjusted to 1 μg/μL with assay buffer; 100 μL of these crude lysates were transferred to a black 96-well plate and supplemented with 20 μmol/L of the caspase-3 substrate Ac-DEVD-AFC (Biomol, Hamburg, Germany) in assay buffer. After 5 minutes of incubation/equilibration, the change in fluorescence (excitation, 400 nm; emission, 508 nm) at 30°C using a Cytofluor 2000 was measured. Liberated AFC was measured at 37°C kinetically every 60 seconds, for 50 repeats The rate of AFC formation was used to calculate the caspase-3 activity in the extracts of untreated versus treated cells and expressed as delta-FU/min.

EMSA

A double-stranded oligonucleotide containing a consensus NF-κB–binding site (5′-AGTTGAGGGGACTTTCCCAGGC-3′; Santa Cruz Biotechnology) was end-labeled with γ-32P-ATP (Amersham) by T4-polynucleotide kinase (Invitrogen) and purified on spin columns (Amersham); 7 μg of nuclear extracts were incubated in DNA-binding buffer [20 mmol/L Tris (pH 7.8), 75 mmol/L NaCl, 5 mmol/L EDTA, 25% glycerol, 2.5 mmol/L DTT, 1 μL bovine serum albumin (1 μg/μL), and 1 μL of poly(dI-dC)oligo (1 μg/μL) in the presence of 10 fmol of the oligonucleotide (50,000 cpm)] for 30 minutes at room temperature in a total volume of 20 μL. Supershift experiments were carried out by preincubating nuclear extracts with 1 μL antibody (1 μg/μL) for 1 hour on ice before the addition of labeled DNA-oligo probe. DNA-protein complexes were resolved on 4% nondenaturing polyacrylamide gels in 0.5× Tris-borate-EDTA buffer (pH 8.3). Signals were visualized by exposing to Kodak X-ray film at −80°C.

Cell Fractionation

For the purification of mitochondria, we used the ApoAlert Cell Fractionation kit from BD Clontech (Mountain View, CA) according to the manufacturer's instructions. The Western blots were carried out as described with the antibodies that are provided with the kit.

Statistical Analysis

If not stated, three independent experiments were done in triplicate. Experimental values are expressed as mean ± SE. For significance analyses, Student's t tests were used, and P < 0.05 was considered significant and P < 0.001 as highly significant.

Grant support: Emmy-Noether grant from the Deutsche Forschungsgemeinschaft (Zw60/2-1, 2-2, 2-3, 2-4) and the Landesstiftung Baden-Wuerttemberg.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: S.J. Braeuer, C. Büneker, and A. Mohr contributed equally to this work.

Present address for R.M. Zwacka: National University of Ireland Galway, National Centre for Biomedical Engineering Science, University Road, Galway, Ireland.

We thank Dr. K. Fisher for his assistance with RNAi vectors, Drs. S. Kochanek and F. Kreppel for practical help with adenoviral vector production, H. Kasperczyk for technical help with EMSA analyses, Drs. S. Kochanek and L. Behrend for carefully reading the article, T. Dick for technical help, and Dr. B. Baumann (Department of Physiological Chemistry, University of Ulm) and Prof. K. Scharffetter-Kochanek (Department of Dermatology, University of Ulm) for reagents.

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