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Mol Cancer Ther. 2006;5:1827-1835
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

15-Deoxy-{Delta}12,14-prostaglandin J2 induces death receptor 5 expression through mRNA stabilization independently of PPAR{gamma} and potentiates TRAIL-induced apoptosis

Susumu Nakata1, Tatsushi Yoshida1, Takumi Shiraishi1,3, Mano Horinaka1, Junji Kouhara1,2, Miki Wakada1 and Toshiyuki Sakai1

1 Department of Molecular-Targeting Cancer Prevention and 2 Division of Digestive Surgery, Department of Surgery, Graduate School of Medical Science and 3 Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan

Requests for reprints: Toshiyuki Sakai, Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Phone: 81-75-251-5339; Fax: 81-75-241-0792. E-mail: tsakai{at}koto.kpu-m.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
15-Deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), the terminal derivative of the PGJ series, is emerging as a potent antineoplastic agent among cyclopentenone prostaglandins derivatives and also known as the endogenous ligand of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). On the other hand, death receptor 5 (DR5) is a specific receptor for tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), which is one of the most promising candidates for new cancer therapeutics. Here, we report that 15d-PGJ2 induces DR5 expression at both mRNA and protein levels, resulting in the synergistic sensitization of TRAIL-induced apoptosis in human neoplastic cells, such as Jurkat human leukemia cells or PC3 human prostate cancer cells. 15d-PGJ2 significantly increased DR5 mRNA stability, whereas it did not activate DR5 promoter activity. Synthetic PPAR{gamma} agonists, such as pioglitazone or rosiglitazone, did not mimic the DR5-inducing effects of 15d-PGJ2, and a potent PPAR{gamma} inhibitor GW9662 failed to block DR5 induction by 15d-PGJ2, suggesting PPAR{gamma}-independent mechanisms. Cotreatment with 15d-PGJ2 and TRAIL enhanced the sequential activation of caspase-8, caspase-10, caspase-9, caspase-3, and Bid. DR5/Fc chimera protein, zVAD-fmk pancaspase inhibitor, and caspase-8 inhibitor efficiently blocked the activation of these apoptotic signal mediators and the induction of apoptotic cell death enhanced by cotreatment with 15d-PGJ2 and TRAIL. Moreover, a double-stranded small interfering RNA targeting DR5 gene, which suppressed DR5 up-regulation by 15d-PGJ2, significantly attenuated apoptosis induced by cotreatment with 15d-PGJ2 and TRAIL. These results suggest that 15d-PGJ2 is a potent sensitizer of TRAIL-mediated cancer therapeutics through DR5 up-regulation. [Mol Cancer Ther 2006;5(7):1827–35]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostaglandins are a family of naturally occurring cyclic 20-carbon fatty acids that are synthesized mainly from arachidonate released from membrane phospholipids by the action of phospholipases (1). Cyclopentenone prostaglandins are potent bioactive molecules that possess anti-inflammatory (2) and antiviral (3) activity. In addition, cyclopentenone prostaglandins have been shown to induce cell growth arrest (4–6) and apoptosis (1) in several neoplastic cell types. In particular, the terminal metabolite of prostaglandin J2, 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), is emerging as the most potent antineoplastic agent of this class of prostaglandins (4). Antineoplastic activity of 15d-PGJ2 has been reported both in vitro and in vivo in a multiplicity of tissues, including the prostate (7), hematopoietic (8), breast (9), colon (10), gastric (11), brain (12). In most types of cancer, 15d-PGJ2 inhibits tumor cell proliferation and induces apoptosis; however, the mechanism for antineoplastic activity has not been fully elucidated.

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear receptor superfamily, functions as a transcription factor mediating ligand-dependent transcriptional regulation. 15d-PGJ2 was shown to be a natural endogenous ligand for PPAR{gamma} and to exert some of its effects by binding to PPAR{gamma} (13, 14). More recent evidence, however, indicates that 15d-PGJ2 can also act independently of PPAR{gamma} activation (1).

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis selectively in cancer cells in vitro and in vivo while sparing most normal cells (15, 16). Therefore, TRAIL is one of the most promising new candidates for cancer therapeutics and is currently undergoing clinical trials.

Death receptor (DR) 5 (also called TRAIL-R2, Apo2, TRICK2, or KILLER) is a member of the tumor necrosis factor receptor family and is a receptor for TRAIL (17, 18). Apo2L/TRAIL triggers apoptosis through binding to the DRs DR4 (19) and/or DR5 (20). These receptors contain a cytoplasmic death domain that recruits adaptor molecules involved in caspase activation (21). The resulting active caspase-8 (21) or caspase-10 (22) cleaves and activates effector caspases, such as caspase-3, caspase-6, and caspase-7. On the other hand, Bid, a proapoptotic Bcl-2 family member, is also cleaved by caspase-8 and then activates the mitochondrial apoptotic signaling pathway (23).

Interestingly, some studies have reported that DR5 is expressed more abundantly in cancer cells than in normal cells (24, 25). Thus, the expression of DR5 may partially contribute to the tumor-selective induction of apoptosis mediated by TRAIL. Recently, a study based on receptor-selective TRAIL variants revealed that DR5 might contribute more than DR4 to TRAIL-induced apoptosis in cancer cells that express both DRs (26). Accordingly, DR5 is considered to be a major DR on most tumor cells and an attractive target for cancer therapeutics.

In this study, we show that 15d-PGJ2 up-regulates DR5 expression at both mRNA and protein levels in human malignant tumor cells. This up-regulation is not mediated through PPAR{gamma} activation by 15d-PGJ2 but through increasing DR5 mRNA stability. DR5 up-regulation results in the synergistic sensitization of soluble recombinant human TRAIL-induced apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
15d-PGJ2 (Cayman Chemical, Ann Arbor, MI) and trichostatin A (Wako, Osaka, Japan) were dissolved in ethanol. GW9662 (2-chloro-5-nitrobenzanilide), rosiglitazone (Alexis Biochemicals, San Diego, CA), pioglitazone (kindly provided by Takeda Chemical Industries, Osaka, Japan), and actinomycin D (Sigma, St. Louis, MO) were dissolved in DMSO. Soluble recombinant human Apo2L/TRAIL was purchased from PeproTech (London, United Kingdom). Human recombinant DR5 (TRAIL-R2)/Fc chimera protein and the caspase inhibitors zVAD-fmk and zIETD-fmk were purchased from R&D Systems (Minneapolis, MN).

Cell Lines and Cell Culture
Jurkat human T-cell acute lymphoblastic leukemia cells and PC3 human prostate cancer cells were maintained in RPMI 1640 with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin and incubated at 37°C in a humidified atmosphere of 5% CO2. Normal human peripheral blood mononuclear cells were isolated using a Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ) density gradient according to the manufacturer's instructions and maintained in X-VIVO 20 (Cambrex, Walkersville, MD) serum-free medium with 0.2% bovine serum albumin (Sigma) at 37°C in a humidified atmosphere of 5% CO2.

Northern Blot Analysis and RNase Protection Assay
Northern blot analysis was done as described previously (27). For the DR5 mRNA stability assay, the relative band intensity was assessed by densitometric analysis of digitalized autographic images using Scion Image software (Scion Corp., Frederick, MD). The RNase protection assay was done using an RNase protection assay III kit (Ambion, Austin, TX) and labeled RNA probes generated with hAPO3d template sets (BD PharMingen, San Diego, CA) according to the manufacturer's instructions.

Western Blot Analysis
Western blot analysis was done as described previously (27). Rabbit polyclonal anti-DR5 antibody (Cayman Chemical), mouse monoclonal anti-Bid, anti-caspase-8, anti-caspase-9, and anti-caspase-10 antibodies (MBL, Nagoya, Japan), and rabbit monoclonal anti-caspase-3 antibody (Cell Signaling, Beverly, MA) were used as the primary antibodies. For subcellular fractionation, we used the Subcellular Proteome Extraction kit (Calbiochem, San Diego, CA) according to the manufacturer's instructions.

Quantification of PPAR{gamma} Activity
Nuclear extracts from Jurkat cells that were treated with pioglitazone and/or GW9662 were prepared by using Nuclear Extract kit (Active Motif Japan, Tokyo, Japan) according to the manufacturer's instructions. We measured PPAR{gamma} DNA-binding activity using Trans AM kit (Active Motif Japan) according to the manufacturer's instructions. Briefly, nuclear extracts (10 µg) were applied to a multiwell plate coated with a oligonucleotide of the consensus-binding sequence for PPAR{gamma}, peroxisome proliferator response element. DNA-bound PPAR{gamma} was detected by a colorimetric assay using an anti-PPAR{gamma} antibody and a secondary antibody conjugated to horseradish peroxidase.

Transfection and Luciferase Assay
As described previously (28), a digested SacI-NcoI fragment from the DR5 promoter region of genomic DNA was subcloned into the SacI-NcoI site of the pGVB2 luciferase assay vector (Toyo Ink, Tokyo, Japan) to produce pDR5PF (pDR5/SacI). pDR5PF and vacant vector plasmids (1.0 µg) were transfected into PC3 cells using LipofectAMINE Plus (Invitrogen, Carlsbad, CA). After 24 hours, the cells were treated with or without 15d-PGJ2 or trichostatin A for 24 hours and then harvested. Luciferase assays were then done as described previously (27). Data were analyzed using Student's t test.

Quantification of Apoptosis
DNA fragmentation was quantified by the percentage of cells with hypodiploid DNA (sub-G1). In brief, cells were fixed with 70% ethanol and treated with RNase A (Sigma), and the nuclei were stained with propidium iodide (Sigma). The DNA content was measured using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA). For all assays, 10,000 cells were counted.

Small Interfering RNAs
The DR5 and LacZ small interfering RNA (siRNA) sequences were described previously (synthesized by Proligo, Kyoto, Japan; ref. 29). DR5 or LacZ siRNAs (25 nmol/L) were transfected into cells using a modified Oligofectamine protocol (Invitrogen) as described previously (30). Twenty-four hours after transfection, cells were treated with 15d-PGJ2 (18 µmol/L) and/or TRAIL (5 ng/mL) for 24 hours and then harvested. Data were analyzed using Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
15d-PGJ2 Increases the Expression of DR5
We first investigated the effects of 15d-PGJ2 on the expressions of DR-related genes using an RNase protection assay. As shown in Fig. 1A , 15d-PGJ2 significantly up-regulated DR5 mRNA in Jurkat human T-cell acute lymphoblastic leukemia cells carrying inactivated p53 with a point mutation. The expressions of DR4, TRAIL, DcR1, and DcR2 did not increase, although the expressions of Fas and receptor-interacting protein slightly decreased. We next carried out Northern blot analysis to confirm the up-regulation of DR5 mRNA not only in Jurkat cells but also in PC3, androgen-independent human prostate cancer cells that are p53-null mutant (Fig. 1B). As shown in Fig. 1C, we showed that 15d-PGJ2 increased DR5 expression at the protein level in both Jurkat and PC3 cells by Western blot analysis. Furthermore, we confirmed that DR5, a membrane receptor protein, was increased by 15d-PGJ2 treatment, especially in the membrane protein fraction using the subcellular fractionation technique (Fig. 1D).


Figure 1
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  		Figure 1. 15d-PGJ2 increases the expression of DR5. A, RNase protection assay. Lane 1, probes not treated with RNases; lane 2, RNase-protected probes following hybridization with yeast tRNA; lanes 3 to 4, total RNA from Jurkat cells treated with or without 15d-PGJ2 (18 µmol/L) for 24 h. The housekeeping genes glyceraldehyde-3-phosphate dehydrogenase and L32 are controls. B, Northern blot analysis. Jurkat or PC3 cells were treated with 15d-PGJ2 at indicated concentrations for 24 h. 18S and 28S are loading controls. C, Western blot analysis. Jurkat or PC3 cells were treated with 15d-PGJ2 at the indicated concentrations for 24 h. Western blot analysis was carried out with anti-DR5 antibody. ß-Actin is a loading control. D, Western blot analysis with subcellular fractionation. Jurkat cells were treated with 15d-PGJ2 (18 µmol/L) for 24 h. Cytosol and membrane protein fractions were separated using a subcellular proteome extraction kit. Coomassie brilliant blue staining of the gel (CBB) is shown to ensure equal loading within each fraction. UT, untreated; ET, treatment with solvent ethanol.

 
15d-PGJ2 Does Not Induce the DR5 Gene through PPAR{gamma} Activation
We hypothesized that 15d-PGJ2 might induce DR5 expression through nuclear receptor PPAR{gamma} activation because 15d-PGJ2 is a natural endogenous ligand for PPAR{gamma}. Previous reports have shown that PPAR{gamma} is abundantly expressed in both Jurkat and PC3 cells (31, 32). However, a PPAR{gamma} agonist, such as pioglitazone nor rosiglitazone, failed to up-regulate DR5 expression in both Jurkat and PC3 cells even at very high concentrations (Fig. 2A-B ). Furthermore, pretreatment by GW9662, an irreversible potent inhibitor of PPAR{gamma}, did not block DR5 induction by 15d-PGJ2 at all (Fig. 2C). We confirmed the potency of these reagents that we used by quantifying PPAR{gamma} DNA-binding activity to peroxisome proliferator response element. Indeed, pioglitazone could increase PPAR{gamma} DNA-binding activity, and GW9662 could block the activation (Fig. 2D). These results strongly suggest that 15d-PGJ2 does not induce the DR5 gene through PPAR{gamma} activation.


Figure 2
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  		Figure 2. PPAR{gamma} is not involved in DR5 induction by 15d-PGJ2. A, Western blot analysis. Jurkat cells were treated with pioglitazone at the indicated concentrations for 24 and 48 h. B, Western blot analysis. PC3 cells were treated with pioglitazone or rosiglitazone for 24 h. C, Jurkat or PC3 cells were pretreated with GW9662 (20 µmol/L) for 1 h and then treated with 15d-PGJ2 (18 µmol/L) for 24 h. D, Jurkat cells were treated with 20 µmol/L pioglitazone and/or 20 µmol/L GW9662 for 24 h. PPAR{gamma} DNA-binding activities were measured as described in Materials and Methods. Columns, mean of triplicate experiments; bars, SD. DM, treatment with solvent DMSO.

 
15d-PGJ2 Increases DR5 mRNA Stability
To investigate the further mechanism underlying DR5 up-regulation by 15d-PGJ2, we next examined the effect of 15d-PGJ2 on DR5 promoter activity. We used the DR5 promoter-luciferase reporter plasmid described previously (28) for the transient luciferase assay. As shown in Fig. 3A , 15d-PGJ2 did not increase DR5 promoter activity, whereas trichostatin A increased as we previously reported (27). This result is also consistent with 15d-PGJ2 not inducing DR5 mRNA through nuclear receptor PPAR{gamma}, a transcription factor. We then investigated the effect of 15d-PGJ2 on DR5 mRNA stability. The relative decay rates of DR5 mRNA were determined by Northern blot analysis in PC3 cells under basal conditions and after they had been exposed to 15d-PGJ2 for 18 hours. As shown in Fig. 3B, in untreated PC3 cells, DR5 mRNA decayed with a half-life of ~5 hours after transcription was blocked with actinomycin D. In contrast, the half-life of DR5 mRNA in 15d-PGJ2-treated PC3 cells was strikingly prolonged, and significant decay of DR5 mRNA could not be observed within 10 hours. These data suggest that the 15d-PGJ2-mediated up-regulation of DR5 mRNA occurs via post-transcriptional control involving the increase of DR5 mRNA stability.


Figure 3
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  		Figure 3. 15d-PGJ2 does not increase DR5 promoter activity but increases DR5 mRNA stability. A, PC3 cells were transiently transfected with the DR5 promoter-luciferase reporter plasmid (pDR5PF) or vacant vector pGVB2. Cells were treated with or without 15d-PGJ2 (18 µmol/L) or trichostatin A (1 µmol/L) for 24 h, harvested, and assayed as described in Materials and Methods. Columns, mean of triplicate experiments; bars, SD. *, P < 0.001. B, DR5 mRNA half-life in untreated or 15d-PGJ2-treated PC3 cells. Untreated or 15d-PGJ2-treated (18 µmol/L 15d-PGJ2 for 18 h) cells were exposed to actinomycin D (4 µg/mL) for 2.5, 5, 7.5, and 10 h before cell harvest for total RNA extraction. The DR5 mRNA decay rates were then determined by Northern blotting. DR5 mRNA levels were normalized to 28S and plotted relative to that at time 0, which was taken as 100%. Points, mean of triplicate experiments; bars, SD. CT, treatment with solvent ethanol.

 
15d-PGJ2 Sensitizes TRAIL-Induced Apoptosis in a Synergistic Fashion
We hypothesized that 15d-PGJ2 might enhance exogenous TRAIL-induced apoptosis due to its ability to increase DR5 expression. As shown in Fig. 4A–B , we investigated the induction of apoptosis by concurrent treatment with 15d-PGJ2 and exogenous recombinant human TRAIL or each alone in both Jurkat and PC3 cells. Statistical analysis using factorial ANOVA showed the significant interactions between the administrations of 15d-PGJ2 and TRAIL in both Jurkat and PC3 cells (P < 0.001). The results indicated that 15d-PGJ2 strongly sensitized exogenous recombinant TRAIL-induced apoptosis in a synergistic fashion. Furthermore, we examined the effect of the combined treatment with 15d-PGJ2 and TRAIL on normal human peripheral blood mononuclear cells (Fig. 4C, left). Whereas ~20% of apoptosis was observed by treatment with 18 µmol/L 15d-PGJ2, any enhancement of apoptosis was not observed when cotreated with 50 ng/mL TRAIL. On the other hand, significant induction of DR5 expression was not observed in normal peripheral blood mononuclear cells that were treated with 15d-PGJ2 in the same conditions with Jurkat cells (Fig. 4C, right).


Figure 4
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  		Figure 4. 15d-PGJ2 sensitizes TRAIL-induced apoptosis in a synergistic fashion. Sub-G1 populations (apoptotic cells) were detected using flow cytometry analysis as described in Materials and Methods. Columns, mean of triplicate experiments; bars, SD. A and B, Jurkat or PC3 cells were treated with 15d-PGJ2 and/or TRAIL at the indicated concentrations for 24 h. C, left, normal peripheral blood mononuclear cells (PBMC) were treated with 18 µmol/L 15d-PGJ2 and/or 50 ng/mL TRAIL for 24 h; right, Western blot analysis. Normal peripheral blood mononuclear cells and Jurkat cells were treated with 15d-PGJ2 at the indicated concentrations for 24 h. Western blot analysis was carried out with anti-DR5 antibody. For all of samples, protein (60 µg) was loaded. ß-Actin is a loading control.

 
Synergistic Apoptosis Induced by Combined Treatment with 15d-PGJ2 and TRAIL Is Mediated by TRAIL-DR Interaction, Activation of Caspases, and Bid Cleavage
To confirm that the enhancement of apoptosis by 15d-PGJ2 and TRAIL is mediated through specific interactions between TRAIL and its receptors, we used human recombinant DR5/Fc chimera protein. As shown in Fig. 5 (top), human recombinant DR5/Fc chimera protein efficiently blocked apoptosis caused by 15d-PGJ2 and TRAIL. This result indicated that the enhancement of apoptosis was mediated not by nonspecific toxicity of TRAIL but through specific interactions between TRAIL and its receptors. We also showed that zVAD-fmk pancaspase inhibitor and caspase-8 inhibitor efficiently interrupted the apoptosis induced by 15d-PGJ2 and TRAIL. These observations were consistent with the characteristic features of TRAIL-induced apoptosis (33). As shown in Fig. 5 (bottom), we did Western blot analysis of apoptotic signal mediators in the same conditions with quantitative assays of apoptosis (Fig. 5, top). DR5 up-regulation was also observed when 15d-PGJ2 was given with TRAIL. It was clearly shown that combined treatment with 15d-PGJ2 and TRAIL significantly enhanced the activation of caspase-8, caspase-10, caspase-9, and caspase-3 and Bid cleavage. Moreover, this Western blot analysis also showed that the activation of these apoptotic signal members was blocked by human recombinant DR5/Fc chimera protein, zVAD-fmk pancaspase inhibitor, and caspase-8 inhibitor. These results indicate that the enhanced apoptosis by 15d-PGJ2 and TRAIL depends on TRAIL-DR interaction and caspase activation.


Figure 5
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  		Figure 5. Combined treatment with 15d-PGJ2 and TRAIL induces synergistic apoptosis coupled with activation of caspase-3, caspase-8, caspase-10, and caspase-9 and Bid cleavage that are blocked by DR5/Fc chimera and caspase inhibitors. Top, PC3 cells were treated with 15d-PGJ2 (18 µmol/L) and/or TRAIL (5 ng/mL) for 24 h with or without various inhibitors. DR5/Fc, treated with DR5/Fc chimera protein (1 µg/mL); zVAD-fmk, treated with zVAD-fmk pancaspase inhibitor (20 µmol/L); caspase-8 inhibitor, treated with zIETD-fmk (20 µmol/L). Sub-G1 populations were detected using flow cytometry analysis as described in Materials and Methods. Columns, mean of triplicate experiments; bars, SD. Bottom, PC3 cells were treated with 15d-PGJ2 and/or TRAIL with or without various inhibitors in the same conditions as in top, and then total cell extracts were harvested. Western blot analysis was carried out with anti-DR5, anti-caspase-3, anti-caspase-8, anti-caspase-10, anti-caspase-9, and anti-Bid antibodies. ß-Actin is a loading control. Active forms of caspases are shown.

 
DR5 Up-Regulation Contributes toward the Enhancement of Apoptosis by Combined Treatment with 15d-PGJ2 and TRAIL
To address whether DR5 up-regulation by 15d-PGJ2 contributes toward the sensitization of TRAIL-induced apoptosis, we used transient transfection of a double-stranded siRNA targeting DR5 gene. The Western blot analysis shown in Fig. 6A showed that the siRNA targeting DR5 gene, which induces degradation of DR5 mRNA, could efficiently block DR5 up-regulation through mRNA stabilization by 15d-PGJ2. This reduction of DR5 expression significantly attenuated the apoptotic response for combined treatment with 15d-PGJ2 and TRAIL (Fig. 6B). This result indicates that DR5 up-regulation, at least in part, plays a role in the sensitization of TRAIL-induced apoptosis by 15d-PGJ2.


Figure 6
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  		Figure 6. DR5 up-regulation contributes toward the enhancement of apoptosis by combined treatment with 15d-PGJ2 and TRAIL. PC3 cells were transiently transfected with double-stranded siRNA targeting DR5 or LacZ or treated with transfection reagent (Oligofectamine) alone (mock). Twenty-four hours after transfection, cells were treated with 15d-PGJ2 (18 µmol/L) for 24 h. A, Western blotting was done as described in Materials and Methods. ß-Actin is a loading control. B, sub-G1 populations were detected using flow cytometry analysis as described in Materials and Methods. Columns, mean of triplicate experiments; bars, SD. *, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TRAIL is an attractive candidate for cancer treatment due to its ability to induce apoptosis selectively in cancer cells. The agonistic anti-DR5 or anti-DR4 monoclonal antibodies are also promising for cancer treatment (24, 34). The initial targets of these strategies are DRs expressed on cancer cells; however, some cancer cell types are resistant to TRAIL-induced apoptosis (35). Previous reports showed that DR5 overexpression using an expression vector in TRAIL-resistant cancer cells restored TRAIL sensitivity (36, 37). DR5 expression was also highly correlated with TRAIL sensitivity in several Jurkat subclones (38). These results raise possibilities of a strategy to induce DR5 expression in cancer cells to enhance the susceptibility toward TRAIL or the anti-DR5 agonistic monoclonal antibody.

In this study, we show that 15d-PGJ2, the most potent antineoplastic agent among cyclopentenone prostaglandins derivatives, markedly up-regulates DR5 at both mRNA and protein levels (Fig. 1). This DR5 induction indeed results in the sensitization of TRAIL-induced apoptosis that is mediated by the activation of caspase-8, caspase-10, caspase-9, caspase-3, and Bid. Moreover, we show that siRNA targeting DR5 attenuates the apoptotic response for cotreatment with 15d-PGJ2 and TRAIL (Fig. 6). Our data show the significance of DR5 up-regulation for the enhancement of susceptibility by 15d-PGJ2 for TRAIL-induced apoptosis in human cancer cells. Interestingly, in normal peripheral blood mononuclear cells, DR5 up-regulation and significant sensitization of TRAIL-induced apoptosis by 15d-PGJ2 were not observed.

On the other hand, thiazolidinediones, potent synthetic PPAR{gamma} stimulants, do not mimic the DR5-inducing effects of 15d-PGJ2 (Fig. 2A–B). In addition, potent irreversible PPAR{gamma} inhibitor, GW9662, does not attenuate DR5 induction by 15d-PGJ2 (Fig. 2C). These observations suggest that DR5 induction by 15d-PGJ2 does not occur through nuclear receptor PPAR{gamma}. Recent studies showed that troglitazone, a compound of thiazolidinediones, enhanced TRAIL-induced apoptosis by reducing the survivin level via cyclin D3 repression and cell cycle arrest in a PPAR{gamma}-independent manner (39), whereas the precise mechanisms responsible for the enhancement of apoptosis by pioglitazone were not elucidated (31, 40). Thus, it is suggested that DR5 up-regulation is a characteristic mechanism responsible for sensitization of TRAIL-induced apoptosis by 15d-PGJ2.

Furthermore, we show that 15d-PGJ2 markedly increases DR5 mRNA stability (Fig. 3B). To our knowledge, no particular gene regulated by 15d-PGJ2 through mRNA stabilization has been reported to date. Therefore, our data provide a novel mechanism by which 15d-PGJ2 regulates gene expression. In addition, few agents capable of inducing DR5 expression through mRNA stabilization have been reported thus far. Only one report has shown that thapsigargin, which causes endoplasmic reticulum stress, induces DR5 expression partially through prolonging the DR5 mRNA half-life (41). However, no precise mechanism underlying the stabilization of DR5 mRNA by these agents has been elucidated yet. To date, AU-rich elements in the 3'-untranslated region of a variety of human short-lived mRNA are important for the regulation of mRNA stability (42, 43). Indeed, the 3'-untranslated region of the human DR5 gene contains AU-rich elements, at least two overlapping copies of the UUAUUUAUU nonamer. Therefore, it is possible to postulate that 15d-PGJ2 might effect AU-rich elements in 3'-untranslated region of the human DR5 gene post-transcriptionally.

The DR5 gene was reported as a p53-regulated gene (44–46). Previous reports showed that some conventional chemotherapeutic drugs, including etoposide or doxorubicin, can induce DR5 expression and enhance TRAIL-induced apoptosis in a p53-dependent manner in certain cancer cell types, such as lung cancer and lymphocytic leukemia cells (46, 47). In this study, we show that 15d-PGJ2 can induce DR5 expression in both PC3, p53-null mutant, and Jurkat cells harboring p53 inactivated with a point mutation. Thus, our results suggest that 15d-PGJ2 induces DR5 expression not through p53-mediated transactivation but through DR5 mRNA stabilization. These results implicate possibilities that 15d-PGJ2 can be expected to induce the DR5 gene in a relatively broader spectrum of cancer cell types than conventional chemotherapeutic drugs, such as etoposide or doxorubicin, because p53 is often inactivated in more than half of malignant cancer cell types.

In summary, we have shown that 15d-PGJ2 is a potent p53-independent sensitizer of TRAIL-induced apoptosis via DR5 up-regulation caused by the enhancement of DR5 mRNA stability in a PPAR{gamma}-independent manner. Our results imply that combined treatment of 15d-PGJ2 with TRAIL is a promising strategy to enhance the susceptibility of cancer cells to TRAIL-mediated cancer therapeutics.


   Acknowledgments
 
We thank Dr. Kyohei Hayashi (Department of Epidemiology for Community Health and Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan) for very helpful advice for statistical analysis.


   Footnotes
 
Grant support: Japanese Ministry of Education, Culture, Sports, Science and Technology.

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.

Received 1/13/06; revised 5/12/06; accepted 5/16/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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