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The Journal of Immunology, 2008, 180, 8030 -8039
Copyright © 2008 by The American Association of Immunologists, Inc.
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Proteasome Inhibitors Enhance TRAIL-Induced Apoptosis through the Intronic Regulation of DR5: Involvement of NF-{kappa}B and Reactive Oxygen Species-Mediated p53 Activation1

Jun-Jie Chen, Chia-Wei Chou, Yu-Fan Chang and Ching-Chow Chen2

Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Manipulation of TRAIL receptor 2 (DR5) pathway is a promising therapeutic strategy to overcome TRAIL-resistant lung cancer cells. Preclinical studies have shown that proteasome inhibitors enhance TRAIL-induced apoptosis in lung cancer cells, but the underlying mechanism has not been fully elucidated. In this study, we demonstrated the enhancement of TRAIL-mediated apoptosis in human alveolar epithelial cells by proteasome inhibitors that up-regulate DR5 expression. This effect was blocked by DR5-neutralizing Ab. Using reporter assay, we demonstrated that the p53 and NF-{kappa}B elements on the DR5 first intron region were involved in proteasome inhibitor-induced DR5 expression. Both p53 small interfering RNA and NF-{kappa}B inhibitor suppressed DR5 expression, strengthening the significance of p53 and NF-{kappa}B in DR5 transcription. The protein stability, Ser392 phosphorylation and Lys373/Lys382 acetylation of p53 were enhanced by MG132. In addition to p53, I{kappa}B{alpha} degradation and NF-{kappa}B translocation was also observed. Moreover, the binding of p53 and p65 to the first intron of DR5 was demonstrated by DNA affinity protein-binding and chromatin immunoprecipitation assays. Intracellular reactive oxygen species (ROS) generation after MG132 treatment contributed to p53, but not p65 nuclear translocation and DNA-binding activity. ROS scavenger dramatically inhibited the apoptosis induced by proteasome inhibitors plus TRAIL. The p53-null H1299 cells were resistant to proteasome inhibitor-induced DR5 up-regulation and enhancement of TRAIL-induced apoptosis. These findings reveal that proteasome inhibitor-mediated NF-{kappa}B and ROS-dependent p53 activation are contributed to intronic regulation of DR5 transcription, and resulted in the subsequent enhancement of TRAIL-induced apoptosis in human lung cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ligand TRAIL, also called APO-2L, is a member of the TNF family that induces apoptotic cell death by interacting with its transmembrane receptors, such as death receptor DR5. Ligation with TRAIL results in DR5 trimerization and intracellular death domain clustering, leading to the formation of death-inducing signaling complex. This complex recruits the Fas-associated death domain adaptor molecule, and subsequently activates downstream caspases (1, 2). In addition to DR5, TRAIL also binds to the decoy receptors DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4), which contain truncated death domain or no cytoplasmic death domain, and therefore, is unable to induce apoptosis (3). TRAIL selectively induces apoptosis in a variety of malignant cells with little or no toxicity on nontransformed cells due to the presence of decoy receptors (4). Recent studies have shown that cancer cells resistant to TRAIL can be sensitized by cotreatment with classical chemotherapeutic drugs. This synergism is partially due to the ability of chemotherapeutic drugs to induce DR5 expression (5). Hence, the sensitization of TRAIL-induced apoptosis by up-regulation of DR5 becomes one of the most potential strategies for cancer therapy.

Proteasome inhibitors are a promising class of drugs inducing apoptosis in cancer cells by inhibiting the ubiquitin-proteasome pathway and blocking the degradation of intracellular proteins (6). There are several postulated mechanisms, such as accumulation of p53, expression of cyclin-dependent kinase inhibitors p21 and p27, activation of stress-activated protein kinases, and blockade of transcription factor NF-{kappa}B (7). These effects may contribute to the anti-inflammation and antitumor activity. PS-341 (bortezomib) has been shown to induce cell apoptosis through production of reactive oxygen species (ROS)3 and release of mitochondrial proteins (8), and was approved by the U.S. Food and Drug Administration for the treatment of refractory multiple myeloma. Notably, preclinical data demonstrate that PS-341 synergizes with chemotherapeutic agents to overcome drug resistance that occurred in hematologic malignancies and some solid tumors (9). PS-341 also sensitized TRAIL-induced apoptosis in non-small cell lung cancer (10). MG132 has been reported to induce DR5 expression through C/EBP homologous protein (CHOP) up-regulation, and lactacystin enhanced Fas-mediated apoptosis by induction of Fas and Fas ligand in human glioma cells (11, 12). These studies imply that proteasome inhibitors enhance apoptosis through up-regulation of death receptor. However, the underlying mechanism is still unclear. Recent studies showed the intronic regulation of decoy receptor DcR2 and DR4 expression by p53, and p53-responsive element has also been identified in the first intron region of DR5 (13, 14, 15). With this regard, the purpose of this study is to investigate proteasome inhibitor-mediated intronic regulation of DR5 induction and enhancement of TRAIL-induced apoptosis.

In this study, TRAIL-resistant alveolar epithelial cells with a low level of DR5 were used. We find that proteasome inhibitors render cells more susceptible to the cytotoxic activity of TRAIL and up-regulate DR5 expression. Intronic regulation of DR5 is revealed, and the enhanced binding of p53 and p65 to DR5 intron by MG132 is demonstrated. We further identify that ROS production in response to MG132 mediates p53 translocation and activation, leading to the up-regulation of DR5 gene expression and enhancement of the TRAIL-mediated apoptosis in human lung cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

TRAIL protein was obtained by GST fusion protein purification. Rabbit polyclonal Ab specific for DR5 was purchased from Imgenex. Goat polyclonal Ab specific for actin, rabbit polyclonal Ab specific for caspase-3, I{kappa}B-{alpha}, I{kappa}B kinase (IKK){alpha}, p65, p50, and proliferating cell nuclear Ag, and mouse mAb specific for p53 were purchased from Santa Cruz Biotechnology. Rabbit polyclonal Abs specific for the Ser392 form of p53 were purchased from New England Biolabs. Antiphosphorylated IKK and anti-acetylated p53 (Lys373/Lys382) Ab were from Upstate Biotechnology. RPMI 1640, FCS, penicillin, and streptomycin were obtained from Invitrogen. HRP-labeled donkey anti-goat or anti-rabbit secondary Ab and the ECL detection reagent were from Amersham Biosciences. Protein A-Sepharose, streptavidin, cycloheximide, N-acetylcysteine (NAC), and glutathione (GSH) were from Sigma-Aldrich. Proteasome inhibitor 1 (PSI-1), lactacystin, MG132, and sodium butyrate were from Calbiochem. The luciferase assay kit was from Promega. SuperFect and plasmid purification kit were from Qiagen. DR5-neutralizing Ab was purchased from Diaclone.

Plasmids

The DR5 promoter intron DNA construct in PGL3 plasmid was a gift from T. Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan). The site mutation in NF-{kappa}B and the p53 binding site of DR5 promoter intron constructs were provided by S. B. Gibson (University of Manitoba, Winnipeg, Manitoba, Canada).

Cell culture

The human alveolar epithelial cell lines NCI-H292 and A549, the p53-null epithelial cell line H1299, and the colon cancer cell line HCT116 were obtained from the American Type Culture Collection and cultured in the RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2. The cells were subcultured in 12-well plates for transfection experiments, in 10-cm dishes for cell extract preparations and chromatin immunoprecipitation (ChIP) experiments, or in 15-cm dishes for nuclear extraction.

Flow cytometry analysis

Progression of cells through the cell cycle and cell apoptosis were examined using flow cytometry. Cells were harvested by trypsinization, fixed in 70% ethanol at 4°C overnight, and washed once with PBS. After centrifugation, the cells were incubated for 30 min at room temperature in 0.5 ml of the phosphate-citric acid buffer (0.2 M NaHPO4, 0.1 M citric acid (pH 7.8)). Cells were centrifuged and resuspended in 100 µl of propidium iodide solution (1% Triton X-100, 80 µg/ml propidium iodide, and 0.1 µg/ml DNase-free RNase A). The cells were incubated at room temperature for 30 min in the dark, and the DNA content was analyzed using the FACScan and CellQuest software (BD Biosciences).

MTT assay

Cells were incubated with proteasome inhibitors alone or cotreated with TRAIL for 24 h. Cells were washed with PBS and then added 100 µl of medium containing MTT at a final concentration of 5 mg/ml. After the loading of MTT at 37°C incubator for 4 h, the medium was replaced with 100 µl of DMSO for 30 min at room temperature, and the 96-well plate was read by an ELISA reader (550 nm) to get the absorbance density values.

RNase protection assay

A RiboQuant MultiProbe RNase Protection assay system was used according to the manufacturer’s instructions (BD Pharmingen). Briefly, human APO3c probe set containing DNA templates for caspase-8, Fas, Fas ligand, decoy receptors DcR1 and DcR2, DR5, DR4, TRAIL, L32, and GADPH was used for T7 RNA-polymerase direct synthesis of antisense RNA probes. The probes were hybridized with 20 µg of RNA isolated from NCI-H292. Samples were then digested with RNase to remove ssRNA. Remaining probes were resolved on denaturing 5% polyacrylamide gels.

Preparation of total cell lysates and nuclear extracts

After pretreatment with various inhibitors for 30 min, cells were incubated with MG132 for the indicated time. Cells were then rapidly washed with PBS and lysed with ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 1 mM EGTA, 1 mM NaF, 150 mM NaCl, 1 mM PMSF, 5 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM Na3VO4, 10 mM β-glycerophosphate, 5 mM Na4P2O7, and 1% Triton X-100), as previously described (16).

Nuclear extracts were isolated as described previously. Briefly, cells were washed with ice-cold PBS and pelleted, then the cell pellet was resuspended in a hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 mM NaF, and 1 mM Na3VO4) and incubated for 15 min on ice, then lysed by the addition of 0.5% Nonidet P-40 followed by vigorous vortexing for 10 s. The nuclei were pelleted and resuspended in extraction buffer (20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 mM NaF, and 1 mM Na3VO4), and the tube was vigorously shaken at 4°C for 15 min on a shaking platform. The nuclear extracts were then centrifuged, and the supernatants were aliquoted and stored at –80°C.

Western blot analysis

Following treatment with PSI-1, lactacystin, or MG132, total-cell lysates or nuclear extracts were prepared and subjected to SDS-PAGE using 10% polyacrylamide gels. The proteins were transferred to a nitrocellulose membrane, which was then incubated successively at room temperature for 1 h with 0.1% milk in TTBS (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05% Tween 20) for 1 h with indicated primary Abs, and for 30 min with HRP-labeled secondary Ab. After the each incubation, the membrane was washed extensively with TTBS. The immunoreactive bands were detected using ECL detection reagent and visualized using Hyperfilm-ECL (Amersham). Quantitative data normalized with internal control were obtained using the computing densitometer and ImageQuant software (Molecular Dynamics).

RT-PCR

Total RNA was isolated from NCI-H292 cells using TRIzol reagent (Invitrogen). The reverse transcription reaction was performed using 2 µg of total RNA that was reverse transcribed into cDNA using oligo(dT) primer, then amplified for 25 cycles using two oligonucleotide primers derived from the published DR5 sequence 5'-AAGACCCTTGTGCTCGTTGTC-3' and 5'-GACACATTCGATGTCACTCCA-3' and two oligonucleotide primers from the β-actin sequence 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTTGCGGGGACGATGGAGGG-3'. Each PCR cycle was conducted for 30 s at 94°C, 30 s at 65°C, and 1 min at 70°C. The PCR products were subjected to electrophoresis on a 1% agarose gel. Quantitative data were obtained using the computing densitometer and ImageQuant software (Amersham Biosciences).

Transient transfection and luciferase activity assay

NCI-H292 cells, grown in 12-well plates, were transfected with the human DR5 promoter intron, site mutation in p53 and NF-{kappa}B binding site of DR5 promoter intron constructs luciferase plasmid using SuperFect (Qiagen), according to the manufacturer’s recommendations. Briefly, reporter DNA (1 µg) and β-galactosidase DNA (0.5 µg) were mixed with 0.75 µl of SuperFect in 0.9 ml of serum-free RPMI 1640. The plasmid pRK containing the β-galactosidase gene driven by the constitutively active SV40 promoter was used to normalize the transfection efficiency.

After a 10- to 15-min incubation at room temperature, the mixture was applied to the cells and 0.1 ml of FCS was added 8 h later. Twenty-four hours after transfection, the cells were treated with various inhibitors as indicated for 30 min, and then MG132 was added for 24 h. Cell extracts were prepared, and luciferase and β-galactosidase activities were measured. The luciferase activity was normalized to the β-galactosidase activity.

Small interfering RNA (siRNA) synthesis and transfection

Complementary siRNA oligonucleotides targeting p53 or p50 were purchased from Dharmacon RNA Technologies. These siRNA were transfected using TransIT-TKO reagent (Mirus Bio), according to the manufacturer’s protocol.

DNA affinity protein-binding assay (DAPA)

To generate the biotinylated double-stranded oligonucleotides containing the p53 and NF-{kappa}B binding sites in the human DR5 intron, PCRs were performed by the forward primer 5'-TGGAGAGGGCAGGGTAGAGA-3' and reverse primer 5'-TCACGCAGCTTACTCGG GAA-biotin-3'. Binding of transcription factors to the DR5 intron was assayed as previously described (16). The nuclear extract (400 µg) was precleared at room temperature for 1 h with 20 µl of the 4% streptavidin-coated beads (Sigma-Aldrich) mixed with 50% slurry to reduce nonspecific binding. Precleared nuclear extract was incubated with 2 µg of biotinylated DNA oligonucleotides and 20 µl of 4% streptavidin-agarose beads with 50% slurry in 400 µl of PBS at room temperature for 1 h with shaking. Beads were then collected by centrifugation at 2000 rpm for 2 min and washed with cold PBS three times. DNA-protein complexes bound to the beads were eluted with 30 µl of Laemmli sample buffer. Nuclear proteins were denatured by putting on dry bath at 95°C for 5 min and subjected to SDS-PAGE. Western blot analysis probed with specific anti-p53 or anti-p65 Ab was performed as described. The quantitative data were obtained using the computing densitometer and ImageQuant software (Molecular Dynamics).

ChIP assay

ChIP assay analysis was performed as previously described (16). DNA immunoprecipitated by p53 and p65 Abs was purified. The DNA was then extracted with phenol-chloroform. The purified DNA pellet was resuspended in H2O and subjected to PCR. To amplify the regions the DR5 intron, PCR was performed with the following primer pairs 5'-TGGAGAGGGCAGGGTAGAGA-3' and reverse primer 5'-TCACGCAGCTTACTCGG GAA-3'. PCR products were then resolved by 1.5% agarose-ethidium bromide gel electrophoresis and visualized by UV light.

Statistical analysis

Data were analyzed using Student’s t test. Values for p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Proteasome inhibitors enhancing TRAIL-induced apoptosis in NCI-H292 were abolished by DR5-neutralizing Ab

The effect of MG132 or PSI-1 on TRAIL-mediated apoptosis in NCI-H292 cells was examined by flow cytometry and cells with sub-G1 DNA content were measured as apoptotic cells. MG132, PSI-1, or TRAIL alone weakly induced apoptosis in NCI-H292 cells (Fig. 1A). Combination of MG132 or PSI-1 with TRAIL markedly increased the sub-G1 population from 2.8% and 2.9% to 35.7% and 43.7%, respectively. Moreover, caspase-3 activation (Fig. 1C) and decrease in cell viability (Fig. 1D) were also observed. These results suggested that combination with proteasome inhibitors can enhance TRAIL-induced apoptosis in NCI-H292 cells. To elucidate the possible mechanism of this synergistic effect, RNase protection assay was used to examine the apoptosis-related gene expression. As shown in Fig. 1B, MG132 dramatically induced DR5 but not DR4 and TRAIL mRNA expression. Hence, we further investigated the relationship between proteasome inhibitor-induced DR5 expression and the enhancement of TRAIL-mediated apoptosis. The caspase-3 activation was mostly abolished by the addition of DR5-neutralizing Ab (Fig. 1C), as is the decrease in cell viability (Fig. 1D). These results indicated that proteasome inhibitors up-regulate DR5 expression and enhance TRAIL-induced apoptosis.


Figure 1
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  		FIGURE 1. Proteasome inhibitors plus TRAIL-induced apoptosis was abolished by DR5-neutralizing Ab in NCI-H292 cells. A, NCI-H292 cells were treated with 100 ng/ml TRAIL in either absence or presence of 1 µM MG132 or PSI for 24 h. Cells were stained with propidium iodide and analyzed in the sub-G1 fraction by FACS. B, RNase-protected probes hybridized with total RNA from NCI-H292 cells treated with 1 µM MG132 for 8 or 16 h were subjected to RNase protection assay as described in Materials and Methods, GADPH and L32 were shown as internal controls. C, NCI-H292 cells were treated with 100 ng/ml TRAIL in either absence or presence of 1 µM MG132 or PSI or DR5-neutralizing Ab for 24 h, then whole-cell lysates were prepared and subjected to Western blotting using Ab against pro-caspase-3 or actin. D, MTT assay was performed as described in Materials and Methods for cells in C. The cell viability was compared with control cells (basal). Results were expressed as mean ± SEM of three independent experiments performed in triplicate. *, p < 0.05 as compared with basal.

 
Proteasome inhibitors induced DR5 expression at a transcriptional level

The increase in DR5 mRNA expression was confirmed by semiquantitative RT-PCR. DR5 mRNA level was significantly increased after treatment with MG132 for 4 h, and lasted for 24 h (Fig. 2A, lanes 3–6). The protein expression was further examined. The DR5 protein expression was increased significantly at 8 h and maximally at 24 h after MG132 treatment (Fig. 2B, lanes 6–8). To examine whether the synergism is a general phenomenon, three different proteasome inhibitors, MG132, PSI-1, and lactacystin were used. We found that all these inhibitors were capable to induce DR5 expression in a dose-dependent manner in both NCI-H292 and A549 cells (Fig. 2C). In addition, both transcriptional (actinomycin D) and translational (cycloheximide) inhibitors were found to block the MG132-induced DR5 expression (Fig. 3A, lanes 3 and 4), indicating the regulation at transcriptional level.


Figure 2
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  		FIGURE 2. Proteasome inhibitors stimulate DR5 expression in NCI-H292 and A549 epithelial cells. A, NCI-H292 cells were treated with 1 µM MG132 for the indicated time, then mRNA was harvested and RT-PCR was performed. B, Whole-cell lysates were prepared and subjected to Western blotting using Abs specific for DR5 or actin as described in Materials and Methods. Fold induction of DR5 mRNA or protein expression normalized with actin or β-actin was quantified using ImageQuant. C, Cells were treated with various concentrations of MG132, PSI-1, or lactacystin for 16 h. Whole-cell lysates were prepared and subjected to Western blotting using Ab specific for DR5 or actin. Results are representative of three independent experiments.

 

Figure 3
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  		FIGURE 3. Involvement of p53 and NF-{kappa}B in the proteasome inhibitor-induced transcriptional activity and expression of DR5 in NCI-H292 cells. A, NCI-H292 cells were pretreated with 200 ng/ml actinomycin D (Act D) or 500 nM cycloheximide (CHX) for 30 min before incubation with 1 µM MG132 for 16 h. Whole-cell lysates were prepared and subjected to Western blotting using Ab specific for DR5 or actin. Fold induction of DR5 normalized with actin was quantified using ImageQuant. B, Cells were transfected with the luciferase reporter plasmid containing the DR5 promoter and first intron region (wild type), or containing p53 or NF-{kappa}B site mutation, then treated with 1 µM MG132 for 24 h. Luciferase activity was measured as described in Materials and Methods. Data were normalized with β-galactosidase activity and expressed as mean ± SEM of three independent experiments performed in triplicate. *, p < 0.05 as compared with wild-type cells. C and D, NCI-H292 cells were transfected with 12.5, 25, or 50 nM p53 siRNA (C, lanes 3–5) or 50 nM p53 siRNA (D, lanes 3 and 5). After 48 h, cells were incubated with 1 µM MG132 or PSI-1 for 16 h. Whole-cell lysates were prepared and subjected to Western blot analysis using Ab specific for anti-p53 or anti-DR5. Results are representative of three independent experiments. E, Cells pretreated with helenalin for 30 min were incubated with 1 µM MG132 or PSI-1 for 16 h. Whole-cell lysates were prepared and subjected to Western blotting using Ab specific for DR5, I{kappa}B{alpha}, IKK{alpha}, or actin.

 
Proteasome inhibitors induced DR5 expression involving p53 and NF-{kappa}B

To elucidate the mechanism of the proteasome inhibitor-induced DR5 expression, cells were transfected with the human DR5 promoter reporter (intronless). MG132 only showed a weakly induction of promoter activity (data not shown). However, the luciferase reporter containing the DR5 promoter and first intron region (–1124/+736) showed 3-fold increase in activity following MG132 treatment (Fig. 3B). To further identify which cis-acting element in the DR5 intron region was involved, cells were transfected with the DR5 promoter mutated at putative p53 or NF-{kappa}B site. Our results showed an attenuation of the MG132-induced DR5 luciferase activity with either mutation at p53 or NF-{kappa}B site (Fig. 3B), demonstrating that p53 and NF-{kappa}B elements are responsible for DR5 transcription. To further confirm the role of p53, siRNA of p53 was used. As shown in Fig. 3C, the expression of p53 as well as MG132-induced DR5 expression was attenuated by p53 siRNA in a dose-dependent manner. PSI-1-induced DR5 expression was also inhibited by p53 siRNA (Fig. 3D). Because DR5 promoter activity is also blocked by the mutation of NF-{kappa}B binding site, the exact role of NF-{kappa}B in the DR5 expression was also examined. The DR5 expression and I{kappa}B{alpha} degradation induced by MG132 and PSI-1 were inhibited by helenalin (a NF-{kappa}B inhibitor), which has been demonstrated to inhibit p65 binding (17). However, the level of IKK{alpha} was not affected (Fig. 3E). In contrast, siRNA of p50 had no effect on MG132-induced DR5 expression (data not shown), implying that p65 but not p50 may involve in the MG132-induced DR5 expression.

Proteasome inhibitors activate p53 and NF-{kappa}B signaling pathway

The effect of MG132 on p53 activation was examined. As shown in Fig. 4A, increased expression of p53 was seen in response to MG132 stimulation after 2 h of treatment and sustained for 16 h. Its translocation from cytosol to the nucleus was also seen (Fig. 4C). Similarly, p53 translocation was also seen in the cells treated with PSI-1 (Fig. 4B). Posttranslational modification of p53 including phosphorylation and acetylation at C-terminal is closely related to its transcriptional activation (18). Therefore, we examined its phosphorylation on Ser392 and acetylation on Lys373/Lys382. Consequently, nuclear p53 phosphorylation on Ser392 and acetylation on Lys373/Lys382 were seen upon MG132 stimulation (Fig. 4C). To explore whether MG132 can modulate p53 turnover, we examined the decay rate of p53. After treatment with MG132 for 4 h, cells were treated with cycloheximide for the indicated time. The degradation of p53 was greatly reduced in the presence of MG132 (Fig. 4D). Thus, MG132-mediated p53 stabilization resulted from the decrease in its degradation.


Figure 4
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  		FIGURE 4. MG132-induced p53 expression, translocation, phosphorylation, acetylation, and stability. A, Cells were treated with 1 µM MG132 for the indicated time. Whole-cell lysates were prepared and subjected to Western blotting using Ab against p53 and actin. B and C, Cells were treated with 1 µM MG132 or PSI for the indicated time, then nuclear extracts were prepared and subjected to Western blotting using Ab against p53, proliferating cell nuclear Ag (PCNA), phospho-p53 (Ser392), or acetyl-p53 (Lys373/Lys382). D, Cells were treated with 1 µM MG132 for 4 h, then cells were treated with 500 nM cycloheximide (CHX) and harvested at the indicated time periods. Whole-cell lysates were subjected to Western blotting using Ab against p53 or actin. p53 Expression normalized with actin was quantified using ImageQuant and the p53 remaining is indicated (bottom).

 
One common mechanism causing NF-{kappa}B activation is the enhanced degradation of I{kappa}B{alpha} protein which is phosphorylated by IKK. Although proteasome inhibitors had been reported to abolish cytokine-induced NF-{kappa}B activation by preventing I{kappa}B{alpha} degradation (19), whether proteasome inhibitors alone affected NF-{kappa}B activation was examined. Surprisingly, I{kappa}B{alpha} degradation was seen after 8 h of treatment with MG132, and almost complete degradation was seen at 16 h (Fig. 5A). MG132 also induced IKK phosphorylation accompanied with I{kappa}B{alpha} degradation (Fig. 5B). The translocation of p65 and p50 to the nucleus was also seen after 2 h of treatment with MG132 and sustained for 16 h (Fig. 5C).


Figure 5
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  		FIGURE 5. MG132-induced I{kappa}B{alpha} degradation, IKK phosphorylation, and NF-{kappa}B translocation. A and B, NCI-H292 cells were treated with 1 µM MG132 for the indicated time. Whole-cell lysates were prepared and subjected to Western blotting using Ab against I{kappa}B{alpha}, phospho-IKK, IKK{alpha}, or actin. C, Nuclear extracts were prepared and subjected to Western blotting using Ab against p65, p50, or proliferating cell nuclear Ag (PCNA).

 
MG132 induced the bindings of p53 and p65 to the DR5 intron

The in vitro binding of p53 and p65 to the respective p53 and NF-{kappa}B site on the DR5 intron was examined by DAPA. Using a biotinylated probe covering p53 and NF-{kappa}B consensus sequences in the first intron, increased bindings of p53 and p65 to the DR5 intron were seen after MG132 treatment (Fig. 6A). When p53 site was mutated, the binding was abolished (Fig. 6B). The in vivo interaction of p53 or NF-{kappa}B with DR5 intron was further confirmed by ChIP assay. In vivo bindings of p53 or p65 to the DR5 intron were seen after MG132 treatment for 2 h and sustained for 8 h (Fig. 6C, lanes 2–4). In contrast, there is no binding of p50 to DR5 intron upon MG132 stimulation (Fig. 6C), which is consistent with the ineffectiveness of p50 siRNA on DR5 expression (data not shown).


Figure 6
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  		FIGURE 6. MG132-induced p53 and p65 binding to DR5 intron in vitro and in vivo. Cells were treated with 1 µM MG132 for the indicated time, then nuclear extracts were prepared and mixed with biotinylated DR5 intron probe containing p53 and NF-{kappa}B sites (A) or p53 site mutation (B), or without probe (Control) and streptavidin-agarose beads. The p65 or p53 in the complex was detected by Western blotting. Results are representative of three independent experiments. C, NCI-H292 cells were treated with 1 µM MG132 for the indicated time. ChIP assays were performed using anti-p65, anti-p50, or anti-p53 Ab or with rabbit nonimmune IgG (Control). The precipitated DR5 intron region (+173 to +409) was assayed as described in Materials and Methods. The 1% of chromatin was assayed to verify equal loading (Input). Results are representative of three independent experiments.

 
ROS scavengers inhibit proteasome inhibitor-induced DR5 expression as well as p53 expression, translocation, and binding activity

ROS-dependent regulation of apoptotic gene expressions had been reported (20). Because our previous study had demonstrated the ROS production induced by proteasome inhibitors (16), its involvement in the p53 activation and DR5 expression was further examined. As shown in Fig. 7, A and B, MG132- or PSI-1-induced DR5 protein and mRNA expression were attenuated by the ROS scavengers NAC and GSH. The p53 expression and its nuclear translocation were also attenuated by these two scavengers (Fig. 7, C and D). However, p65 or p50 translocation and I{kappa}B{alpha} degradation were not affected by these scavengers (Fig. 7, C and D). Consistently, DAPA and ChIP assays demonstrated the binding of p53 to the intron site was also inhibited by NAC and GSH (Fig. 7, E and F). These results indicate that p53, but not p65, translocation and binding activity was dependent on MG-132-induced ROS generation.


Figure 7
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  		FIGURE 7. Effects of ROS scavengers on MG132-induced DR5 expression, p53 expression, translocation, and binding activity. A, Cells pretreated with 20 mM NAC or GSH for 30 min were incubated with 1 µM MG132 or PSI-1 for 16 h. Whole-cell lysates were prepared and subjected to Western blotting using Ab specific for DR5 or actin. B, Using Ab specific for DR5 mRNA, lysates in A were harvested and RT-PCR was performed to quantify DR5 mRNA. C, Whole-cell lysates were prepared and subjected to Western blotting using Ab specific for p53, I{kappa}B{alpha}, or actin. D, Cells pretreated with 20 mM NAC or GSH for 30 min were incubated with 1 µM MG132 or PSI-1 for 16 h. Nuclear extracts were prepared and subjected to Western blotting using Ab specific for p65, p50, p53, or proliferating cell nuclear Ag (PCNA). E, Nuclear extracts were prepared and mixed with biotinylated DR5 intron probe or without probe (Control) and streptavidin-agarose beads. The p53 in the complex was detected by Western blotting. F, ChIP assays were performed using anti-p53 Ab or with rabbit nonimmune IgG (Control) as described in Materials and Methods. The 1% of chromatin was assayed to verify equal loading (Input). Results are representative of three independent experiments.

 
ROS scavengers inhibit apoptosis induced by proteasome inhibitors plus TRAIL

Because ROS was involved in proteasome inhibitor-induced DR5 expression, its role in the enhancement of TRAIL-induced apoptosis was further examined. MG132 or PSI-1 plus TRAIL-induced sub-G1 accumulation was inhibited by NAC from 46.1% to 3.97% and 44.3% to 5.31%, respectively (Fig. 8A). Similarly, the cleavage of pro-caspase-3 induced by the cotreatment was reversed by NAC (Fig. 8B, lanes 3 and 4 and lanes 5 and 6).


Figure 8
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  		FIGURE 8. Effect of proteasome inhibitors plus TRAIL in the presence of ROS scavenger or in the p53-null H1299 cell line. A, NCI-H292 cells pretreated with 20 mM NAC for 30 min were incubated with 1 µM MG132 or PSI plus 100 ng/ml TRAIL for 24 h. Cells were stained with propidium iodide at apoptosis and analyzed at the sub-G1 fraction by FACS. B, Whole-cell lysates were prepared and subjected to Western blotting using Abs against pro-caspase-3 or actin. C, NCI-H292 or H1299 cells were treated with 1 µM PSI for 24 h, and whole-cell lysates were prepared and subjected to Western blotting using Abs against DR5 or actin. D, H1299 cells were treated with 100 ng/ml TRAIL in either absence or presence of 1 µM PSI for 24 h. Whole-cell lysates were prepared and subjected to Western blotting using Ab against pro-caspase-3 or actin. E, MTT assay was performed as described in Materials and Methods. The cell viability was compared with control cells (basal). Results were expressed as the mean ± SEM of three independent experiments performed in triplicate. *, p < 0.05 as compared with basal.

 
p53-null H1299 cells were resistant to proteasome inhibitor-induced DR5 up-regulation and enhancement of TRAIL-induced apoptosis

We further used p53-null H1299 cells to examine the important role of p53 in proteasome inhibitor-induced DR5 expression and enhancement of TRAIL-induced apoptosis. The expression of p53 as well as PSI-1-induced DR5 expression was only detected in NCI-H292 but not in H1299 cells (Fig. 8C). Pro-caspase-3 was not cleaved by the cotreatment (Fig. 8D), and MTT assay revealed that H1299 cells were resistant to PSI-1 plus TRAIL-induced cell death (Fig. 8E), indicating the essential role of p53 in DR5 up-regulation and enhancement of TRAIL-induced apoptosis induced by proteasome inhibitors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Proteasome inhibitors control the process of apoptosis and cell cycle through blockade of the ubiquitin-dependent pathway. PS-341, also known as bortezomib has been shown to exert significant antitumor activity in refractory and relapsed multiple myeloma by inducing myeloma cell apoptosis and disrupting myeloma-stromal cell interaction (21). In addition, combination of PS-341 with TRAIL offers a promising strategy to overcome the resistance to conventional chemotherapy (22). Thus, it is worthwhile to characterize the precise mechanism of proteasome inhibitors on death receptor-mediated apoptosis. In this study, we demonstrated that proteasome inhibitors enhance TRAIL-induced apoptosis and induce DR5 up-regulation. MG132, PSI-1, and lactacystin were capable to induce DR5 expression transcriptionally in malignant cells derived from different origins. Furthermore, reporter assay revealed the involvement of NF-{kappa}B and p53 in MG132-induced DR5 expression. Moreover, we demonstrated that MG132 enhances bindings of p53 and p65 to DR5 intron region in vitro and in vivo. ROS generation plays a pivotal role in the MG132-induced p53 activation, which leads to DR5 expression and enhances TRAIL-mediated apoptosis.

The molecules beneath DR5, such as Fas-associated death domain and caspase-8, are essential for the assembly of death-inducing signaling complexes, which activate extrinsic apoptotic pathway, and defects in either of these molecules lead to TRAIL resistance. To conquer the TRAIL resistance, up-regulation of DR5 is a practicable and promising strategy (23, 24). In this study, TRAIL-resistant alveolar epithelial cells with low level of DR5 were used. Our results revealed that proteasome inhibitors can enhance TRAIL-induced apoptosis in NCI-H292 cells. Caspase-3 activation and the decrease in cell viability were blocked by DR5-neutralizing Ab. It had been reported that sodium butyrate acts via induction of DR5 to enhance TRAIL-induced apoptosis in HCT116 cells (25). Our results showed that MG132 and sodium butyrate indeed induce DR5 expression and enhance TRAIL-induced apoptosis in HCT116 cells (data not shown). These results indicated that proteasome inhibitors induce DR5 up-regulation and enhance TRAIL-induced apoptosis.

It is well documented that DR5 expression is mainly governed by the cis-elements in its promoter region, such as SP1 and CHOP site. For example, CHOP-dependent DR5 expression has been reported in MG132- or tunicamycin-treated human prostate cancer DU145 cells (11, 26), whereas the putative Sp1 site is involved in bile acid- and sodium butyrate-induced DR5 expression (25, 27). In this study, we showed that MG132 were unable to induce DR5 promoter activity in the absence of the first intron, suggesting the important role of the first intron in MG132-induced DR5 expression. By reporter assay, we demonstrated the participations of p53 and NF-{kappa}B elements on the first intron region in MG132-induced DR5 promoter activity. Our results also showed that MG132-induced DR5 expression was abolished by NF-{kappa}B inhibitor helenalin and p53 siRNA, further strengthening the crucial role of p53 and NF-{kappa}B. In agreement with our finding, DNA damage agents were reported to induce DR5 expression in a p53-dependent manner, and the transactivation depends on the intronic p53 binding site (14). In addition to p53, NF-{kappa}B was demonstrated to be involved in the DR5 induction by conventional anticancer drugs such as etoposide (28).

Diverse stresses activate p53 by posttranslational modifications and nuclear accumulation, contributing to its apoptotic activity in several cancer cells. Among posttranslational modifications, ubiquitination, phosphorylation, and acetylation are mostly studied and found to affect the overall appearance and activity of p53 (18). Phosphorylation of Ser392 stabilizes the formation of p53 tetramer, which is critical for enhancing DNA binding and activating gene transcription (29, 30). Acetylation of p53 enhances the stabilization by interrupting its interaction with Mdm2, which possesses E3 ligase activity to induce p53 ubiquitination and degradation (31). Our results showed that MG132 enhanced p53 expression and its nuclear translocation in a time-dependent manner. These events were correlated with the Ser392 phosphorylation and Lys373/Lys382 acetylation of nuclear p53. Thus, posttranslational modifications of p53, which lead to protein stabilization, may explain the ability of p53 to activate DR5 transcription in response to proteasome inhibitors.

I{kappa}B{alpha} ubiquitination and its subsequent degradation leading to nuclear translocation of NF-{kappa}B and an increase in DNA binding are the central dogma of NF-{kappa}B activation. Although it had been reported that proteasome inhibitors abolish cytokine-induced NF-{kappa}B activation by preventing I{kappa}B{alpha} degradation (19), we found MG132 alone can induce IKK phosphorylation, I{kappa}B{alpha} degradation, and translocation of p65 and p50 to the nucleus after 16 h of treatment. NF-{kappa}B activation is generally thought to be associated with resistance to apoptosis (32). However, accumulating evidence suggests the opposite role of NF-{kappa}B by up-regulation of the expression of some proapoptotic genes. For example, some cancer chemotherapeutic agents, such as taxenes, induce apoptosis in certain types of cancer cells through NF-{kappa}B activation (33). This paradoxical effect is dependent on the stimulus and cell type as well as which NF-{kappa}B family member is activated (34). Although the NF-{kappa}B subunit p52 has been reported to cooperate with p53 to regulate DR5 expression and other genes such as PUMA, Gadd45{alpha}, and Chk1 (35), we showed the increased bindings of p53 and p65, but not p50 to DR5 first intron by MG132. To our knowledge, this report is the first to demonstrate that proteasome inhibitors regulate the bindings of p53 and p65 to the first intron region, which in turn induce DR5 expression.

ROS production plays a pivotal role in enhancing the proapoptotic gene expression (20). It had been reported that proteasome inhibitors induced ROS production through mitochondrial dysfunction and endoplasmic reticulum stress (8). We already found that MG132 elevated intracellular ROS production (16). Although several chemopreventive agents such as sulforaphane and curcumin have been found inducing ROS-dependent DR5 expression to sensitize TRAIL-induced apoptosis (36, 37), it is still unclear whether ROS and its downstream molecule are involved in MG132-induced DR5 expression. In this study, we found the correlation between MG132-elevated DR5 expression and p53 expression, translocation, and binding to the DR5 intron. All these events were abrogated by the ROS scavengers NAC and GSH, indicating that MG132 acts through ROS generation to activate p53, which results in the induction of DR5 expression. We found that MG132-induced I{kappa}B{alpha} degradation and p65 translocation were not affected by ROS scavenger, although NF-{kappa}B activation induced by ROS is well documented (38). The precise mechanism of MG132-induced differential regulation of p53 and p65 remains to be further elucidated. In addition, our data showed NAC reversed the proteasome inhibitor-enhanced TRAIL apoptosis and downstream caspase-3 activation. In agreement with the result that ROS-mediated p53 activation plays a role in proteasome inhibitors-induced DR5 expression, p53-null H1299 cells were resistant to proteasome inhibitor-induced DR5 up-regulation and enhancement of TRAIL-induced apoptosis. Thus, we demonstrated that ROS production serve as a direct link between p53-mediated DR5 expression and TRAIL-induced apoptosis.

In conclusion, proteasome inhibitors MG132, PSI-1 and lactacystin induced DR5 expression in epithelial cancer cells. The induction is regulated at transcriptional level and via the ROS-mediated p53 activation, which enhances binding of p53 to DR5 intron. This coordinated with the recruitment of p65 to the DR5 intron, resulting in the transactivation of DR5 expression. Thus, ROS generation may be critical for proteasome inhibitor-induced DR5 up-regulation and the subsequent activation of TRAIL-mediated apoptotic pathways in TRAIL-resistant human lung cancer cells.


   Acknowledgments
 
We thank Dr. T. Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan) and Dr. Spencer B. Gibson (University of Manitoba, Manitoba, Ontario, Canada) for DR5 plasmid construction.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by Research Grant NSC-96-2320-B002-018 from the National Science Council of Taiwan. Back

2 Address correspondence and reprint requests to Dr. Ching-Chow Chen, Department of Pharmacology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section 1, Taipei 10018, Taiwan. E-mail address: chingchowchen{at}ntu.edu.tw Back

3 Abbreviations used in this paper: ROS, reactive oxygen species; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; IKK, I{kappa}B{alpha} kinase; DAPA, DNA affinity protein-binding assay; NAC, N-acetylcysteine; GSH, glutathione; PSI-1; proteasome inhibitor 1; CHOP, C/EBP homologous protein. Back

Received for publication September 11, 2007. Accepted for publication April 9, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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