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Cancer Research 67, 3185, April 1, 2007. doi: 10.1158/0008-5472.CAN-06-3431
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Nonsteroidal Anti-inflammatory Drugs Induce Colorectal Cancer Cell Apoptosis by Suppressing 14-3-3{varepsilon}

Jun-Yang Liou1, Dipak Ghelani1,2, Sam Yeh1,2 and Kenneth K. Wu1,2,3

1 The University of Texas Health Science Center; 2 The University of Texas, M. D. Anderson Cancer Center, Houston, Texas; and 3 National Health Research Institutes, Zhunan, Taiwan

Requests for reprints: Kenneth K. Wu, National Health Research Institutes, 35 Keyan Road, Zhunan Township, Miaoli County 350, Taiwan. Phone: 886-37-583042; Fax: 886-37-586402; E-mail: kkgo{at}nhri.org.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine the role of 14-3-3 in colorectal cancer apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs), we evaluated the effects of sulindac on 14-3-3{varepsilon} protein expression in colorectal cancer cells. Sulindac sulfide inhibited 14-3-3{varepsilon} proteins in HT-29 and DLD-1 cells in a time- and concentration-dependent manner. Sulindac sulfone at 600 µmol/L inhibited 14-3-3{varepsilon} protein expression in HT-29. Indomethacin and SC-236, a selective cyclooxygenase-2 (COX-2) inhibitor, exerted a similar effect as sulindac. Sulindac suppressed 14-3-3{varepsilon} promoter activity. As 14-3-3{varepsilon} promoter activation is mediated by peroxisome proliferator–activated receptor {delta} (PPAR{delta}), we determined the correlation between 14-3-3{varepsilon} inhibition and PPAR{delta} suppression by NSAIDs. Sulindac sulfide inhibited PPAR{delta} protein expression and PPAR{delta} transcriptional activity. Overexpression of PPAR{delta} by adenoviral transfer rescued 14-3-3{varepsilon} proteins from elimination by sulindac or indomethacin. NSAID-induced 14-3-3{varepsilon} suppression was associated with reduced cytosolic Bad with elevation of mitochondrial Bad and increase in apoptosis which was rescued by Ad-PPAR{delta} transduction. Stable expression of 14-3-3{varepsilon} in HT-29 significantly protected cells from apoptosis. Our findings shed light on a novel mechanism by which NSAIDs induce colorectal cancer apoptosis via the PPAR{delta}/14-3-3{varepsilon} transcriptional pathway. These results suggest that 14-3-3{varepsilon} is a target for the prevention and therapy of colorectal cancer. [Cancer Res 2007;67(7):3185–91]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human and animal studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) suppress colorectal tumorigenesis (1). Population-based epidemiologic studies have shown that use of aspirin and NSAID reduces risk of colorectal cancer (2, 3). Animal experiments provide evidence for control of colorectal cancer by NSAIDs such as sulindac and selective cyclooxygenase-2 (COX-2) inhibitors (4, 5). Results from clinical trials have documented the efficacy of sulindac and selective COX-2 inhibitors in reducing the size and numbers of colon adenomatous polyps in patients with familial adenomatous polyposis, a precancerous condition (6, 7). The action of NSAIDs on controlling colorectal cancer is attributed to the inhibition of COX-2 (8), which is overexpressed in a majority of patients with colorectal cancer (9, 10). NSAID could also exert its action on colorectal cancer in a COX-2–independent manner (11). It is well documented that NSAIDs, notably sulindac, indomethacin, and selective COX-2 inhibitors exert their actions by inducing colorectal cancer cell apoptosis (12, 13). The mechanism by which these agents induce apoptosis is not entirely clear. It was proposed that NSAID-induced apoptosis might be mediated by down-regulation of peroxisome proliferator–activated receptor-{delta} (PPAR{delta}; ref. 14). Several studies have shown that PPAR{delta} is overexpressed in colorectal cancer and PPAR{delta} is activated by prostacyclin generated by colorectal cancer cells (15). Activation of PPAR{delta} accelerates intestinal adenoma growth whereas genetic disruption of PPAR{delta} decreases the tumorigenicity of human colorectal cancer cells (16, 17). It has been reported that PPAR{delta} plays an important role in cellular resistance to apoptotic insults (18, 19). Activation of PPAR{delta} was reported to be associated with increased expression of phosphoinositide-dependent kinase-1 and activation of Akt (20). Akt is recognized as a key signaling molecule for transmitting survival messages. We have recently reported that PPAR{delta} activation by prostacyclin or L-165041, a synthetic PPAR{delta} ligand, increases the expression of 14-3-3 proteins, especially the 14-3-3{varepsilon} isoform (21). Promoter analysis identifies three PPAR response elements (PPRE) located between –1426 and –1477 of the 14-3-3{varepsilon} promoter region. Deletion of these PPREs abrogates the protective effect of prostacyclin or L-165041 (21). Our previous findings suggest that PPAR{delta}-mediated 14-3-3{varepsilon} up-regulation plays a crucial role in endothelial cell resistance to apoptosis (21). It is unclear whether 14-3-3{varepsilon} is involved in colorectal cancer cell survival. We postulated in this study that NSAIDs induce apoptosis by reducing 14-3-3{varepsilon} via suppressing PPAR{delta}. Reduction in 14-3-3{varepsilon} protein levels results in mitochondrial Bad translocation and colorectal cancer cell apoptosis. The results show that sulindac sulfide, sulindac sulfone, indomethacin, and SC-236 suppressed HT-29 14-3-3{varepsilon} protein and promoter activity, which correlated with the suppression of PPAR{delta} protein expression. The reduction in 14-3-3{varepsilon} levels was accompanied by enhanced mitochondrial Bad translocation and HT-29 apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents. HT-29 and DLD-1 cells were obtained from American Type Culture Collection (Manassas, VA). HT-29 were maintained in DMEM and DLD-1 were maintained in RPMI 1604 supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37°C in a humidified 5% CO2 atmosphere. Cell culture media and antibiotics were purchased from BRL Life Technologies (Grand Island, NY). Sulindac sulfide and sulindac sulfone were obtained from ICN Biomedicals, Inc. (Solon, OH). Indomethacin, SC-236, MG-132, and cell-permeable caspase 3 inhibitor, ac-DVED aldehyde (DEVD-CHO), were from Calbiochem (San Diego, CA). We used 10 µmol/L of MG-132 and 50 µmol/L of DEVD-CHO, which were reported to block proteasome and caspase 3 activities, respectively (22, 23).

Recombinant adenoviral vectors. Replication-defective recombinant adenoviruses were generated by homologous recombination and amplified in 293 cells as described previously (24, 25). The recombinant viruses were purified by CsCl density gradient centrifugation and the virus titers were determined by a plaque assay as previously described (24). Ad-PPAR{delta} was kindly provided by Drs. Kinzler and Vogelstein at Johns Hopkins University. HT-29 cells were transfected with Ad-GFP (green fluorescent protein) or Ad-PPAR{delta} at a multiplicity of infection (moi) of 50 for 48 h before treatment with indomethacin or sulindac sulfide.

Preparation of mitochondrial fraction. HT-29 cells treated with indicated reagents (sulindac sulfide, 160 µmol/L; sulindac sulfone, 300 µmol/L; indomethacin, 800 µmol/L; or SC-236, 40 µmol/L) for 8 h were washed thrice with PBS and harvested by centrifugation. Mitochondrial fractions were prepared by a mitochondria isolation kit (Sigma, St. Louis, MO) as described previously (26). The mitochondrial and cytosolic fractions were purified by two-step gradient centrifugation and stored at –20°C. Mitochondrial Bad protein level was analyzed by Western blotting. Heat shock protein 60 (Hsp60) was used as a mitochondrial marker.

Immunoprecipitation. HT-29 cells treated with the indicated reagents were harvested and lysed in 1x radioimmunoprecipitation assay buffer (Upstate, Charlottesville, VA) with cocktail protease inhibitors (Sigma). Lysate proteins (200 µg) were immunoprecipitated with a human 14-3-3{varepsilon} antibody. The immunoprecipitated complex was pulled down with protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA). After washing five times, the proteins were analyzed by Western blotting using a Bad antibody.

Western blot analysis. Cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors. Protein concentrations were determined by a Bio-Rad (Hercules, CA) protein assay kit. Cell lysate proteins (25 µg) were applied to each lane and analyzed by Western blots as previously described (25). Rabbit polyclonal antibodies against 14-3-3{varepsilon} (diluted at 1:250), and goat polyclonal antibodies against Hsp60 (1:2,000) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibody against PPAR{delta} (1:500) was obtained from Cayman Chemical (Ann Arbor, MI). Rabbit polyclonal antibody against Bad (1:500) was purchased from Cell Signaling (Beverly, MA). Monoclonal antibody against actin (1:5,000) was obtained from Oncogene (Boston, MA). Donkey anti-rabbit or donkey anti-mouse IgG conjugated with horseradish peroxidase were purchased from Santa Cruz Biotechnology. Protein bands were visualized by an enhanced chemiluminescence system (Pierce, Rockford, IL).

Assay of apoptosis. Apoptotic cells were analyzed by Hoechst staining according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Hoechst 33258 is a cell-permeable dye which is sensitive to nuclear DNA damage and is useful in detecting apoptosis of unfixed living cells (27). In brief, HT-29 cells transiently transfected with Ad-PPAR{delta}, Ad-GFP, or stably transfected HT-29 cells were treated with 160 µmol/L of sulindac sulfide for 24 h. After washing, they were incubated with 1 µg/mL of Hoechst 33258 in the dark for 15 min, centrifuged and washed with PBS. The washed cells were examined under fluorescent microscopy (excitation at 352 nm and emission at 461 nm). Apoptotic cells exhibited strong nuclear staining with overt nuclear morphologic changes as previously described (27). The percentage of apoptotic cells was determined by counting at least 200 cells.

Plasmid constructs and luciferase reporter assay. A human 14-3-3{varepsilon} expression construct was amplified by PCR and subcloned into pCDNA 3.1+ vector (Invitrogen) and a 14-3-3{varepsilon} promoter subcloned into pGL3 vector were prepared as previously described (21). PPRE reporter (14) was kindly provided by Drs. Kinzler and Vogelstein at Johns Hopkins University. For the luciferase assay, cells were transfected with the reporter constructs by Effectene transfection kit (Qiagen, Valencia, CA) for 48 h. After treatment with the indicated reagents, cells were lysed and luciferase activity was measured using a kit from Promega (Madison, WI), and the emitted light was determined in a luminometer.

Establishment of 14-3-3{varepsilon} stable cell line. Control vector (pCDNA3.1+) and human 14-3-3{varepsilon} expression vectors which contain a neomycin-resistant cassette were transfected into HT-29 cells by using an Effectene transfection kit for 48 h. The transfected cells were screened by incubation with 500 µg/mL of G418 (Sigma) for 4 weeks. The cell lines which stably expressed 14-3-3{varepsilon} were isolated from individual colonies and maintained in DMEM with 10% fetal bovine serum and 200 µg/mL of G418.

Statistical analysis. ANOVA was used to analyze statistical differences among multiple groups. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Suppression of 14-3-3{varepsilon} by sulindac sulfide and COX-2 inhibitors. To evaluate the effect of sulindac on 14-3-3{varepsilon} protein levels, we treated HT-29 with sulindac sulfide at 0 to 160 µmol/L for 4 to 24 h. 14-3-3{varepsilon} proteins were analyzed by Western blotting. Sulindac sulfide did not have an effect at 4 h of treatment, suppressed 14-3-3{varepsilon} at 8 h only at 160 µmol/L, and exerted a concentration-dependent inhibition of 14-3-3{varepsilon} at 24 h of treatment (Fig. 1A ). Sulindac sulfone had no effect at 300 µmol/L but suppressed 14-3-3{varepsilon} expression at 600 µmol/L (Fig. 1B). Indomethacin, a nonselective COX inhibitor, or SC-236, a selective COX-2 inhibitor, also concentration-dependently inhibited 14-3-3{varepsilon} protein levels (Fig. 1C). Sulindac sulfide suppressed 14-3-3{varepsilon} protein levels in DLD-1 with a pattern similar to that of HT-29 (Fig. 1D).


Figure 1
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  		Figure 1. NSAIDs suppressed 14-3-3{varepsilon} expression. HT-29 cells were treated with (A) sulindac (Sul) sulfide for 4 to 24 h, (B) sulindac sulfone for 24 h, or (C) indomethacin (Indo) or SC-236 for 24 h. D, DLD-1 cells were treated with sulindac sulfide for 24 h. 14-3-3{varepsilon} proteins were determined by Western blotting. Actin was determined as control. Representative of three independent experiments with similar results.

 
Inhibition of 14-3-3{varepsilon} promoter activity by sulindac sulfide. To determine whether sulindac sulfide inhibits 14-3-3{varepsilon} at the transcription level, we transfected HT-29 with a human 14-3-3{varepsilon} promoter construct (–1628/+24) and treated the transfected cells with various concentrations of sulindac sulfide for 24 h. Sulindac sulfide concentration-dependently inhibited 14-3-3{varepsilon} promoter activity (Fig. 2A ). Sulindac-induced 14-3-3{varepsilon} protein suppression was not influenced by MG-132, a proteasome inhibitor, or DEVD-CHO, a caspase 3 inhibitor (Fig. 2B). Taken together, these results suggest that sulindac suppresses 14-3-3{varepsilon} primarily at the transcriptional level.


Figure 2
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  		Figure 2. Sulindac sulfide suppressed 14-3-3{varepsilon} expression through transcriptional inactivation. A, HT-29 cells transfected with a 14-3-3{varepsilon} promoter construct were treated with increasing concentrations of sulindac sulfide for 24 h. Luciferase expression was measured and expressed as relative light units (RLU)/µg protein. Columns, mean of three independent experiments; bars, SD. Statistical difference was analyzed by ANOVA. B, HT-29 cells were pretreated with MG-132 (10 µmol/L) or DEVD-CHO (50 µmol/L) for 30 min followed by sulindac sulfide (160 µmol/L) for 24 h. 14-3-3{varepsilon} proteins were determined by Western blotting. This blot is representative of three experiments with similar results.

 
Restoration of 14-3-3{varepsilon} by adenoviral PPAR{delta} transfer. We have recently discovered that 14-3-3{varepsilon} transcription is mediated by PPAR{delta} (21). We were, therefore, interested in learning whether sulindac-induced 14-3-3{varepsilon} suppression involves PPAR{delta}. HT-29 cells were treated with sulindac sulfide (160 µmol/L) or indomethacin (800 µmol/L) for 24 h and PPAR{delta} proteins were analyzed by Western blots. PPAR{delta} levels became undetectable following treatment with either agent (Fig. 3A ). To ensure that PPAR{delta} is active in HT-29, we transfected the cells with a PPRE construct and treated the cells with sulindac sulfide for 24 h. Sulindac reduced the PPRE activity in a concentration-dependent manner (Fig. 3B). To determine whether PPAR{delta} overexpression rescues 14-3-3{varepsilon} from sulindac or indomethacin suppression, we transduced HT-29 with Ad-PPAR{delta} (50 moi) or Ad-GFP control (50 moi). PPAR{delta} protein levels were increased by Ad-PPAR{delta} transduction (Fig. 4A ), which is accompanied by enhanced PPRE activity (Fig. 4B) and 14-3-3{varepsilon} promoter activity (Fig. 4C). PPAR{delta} proteins in Ad-PPAR{delta}–transduced cells were not altered by sulindac or indomethacin (Fig. 4A), but the PPAR{delta} transcriptional activity (Fig. 4B) and the 14-3-3{varepsilon} promoter activity (Fig. 4C) were partially suppressed. 14-3-3{varepsilon} proteins were increased by Ad-PPAR{delta} overexpression which were suppressed by sulindac sulfide and indomethacin (Fig. 4D).


Figure 3
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  		Figure 3. NSAIDs inhibited PPAR{delta}. A, HT-29 cells treated with sulindac sulfide (160 µmol/L) or indomethacin (800 µmol/L) for 24 h. PPAR{delta} protein was determined by Western blotting. This blot is representative of three experiments with similar results. B, HT-29 cells transfected with a PPRE construct were treated with sulindac sulfide for 24 h. Luciferase expression was measured. Columns, mean of three independent experiments; bars, SD.

 

Figure 4
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  		Figure 4. PPAR{delta} overexpression restored 14-3-3{varepsilon} expression. A, HT-29 cells were transfected with Ad-GFP or Ad-PPAR{delta} for 24 h followed by treatment with sulindac sulfide (160 µmol/L) or indomethacin (800 µmol/L) for an additional 24 h. PPAR{delta} proteins were determined by Western blotting. B to D, HT-29 transfected with Ad-GFP or Ad-PPAR{delta} for 24 h followed by treatment of sulindac sulfide or indomethacin at indicated concentrations for an additional 24 h; PPRE (B) and 14-3-3{varepsilon} (C) promoter activity were determined by luciferase expression. D, 14-3-3{varepsilon} protein was determined by Western blotting. Each blot is representative of three experiments with similar results. NS, statistically nonsignificant; *, P < 0.05; **, P < 0.01.

 
Mitochondrial Bad translocation and cell death by sulindac and COX-2 inhibitors. 14-3-3 proteins bind phosphorylated Bad and sequester it in cytosol. Apoptotic signals induce Bad to release from 14-3-3 and translocate to the mitochondria where it disrupts the protective action of Bcl-XL, thereby promoting apoptosis via the mitochondrial pathway (28, 29). It was reported that up-regulation of 14-3-3{varepsilon} protein levels by PPAR{delta} ligands enhances Bad binding and reduces Bad translocation to mitochondria (21). Because sulindac suppresses 14-3-3{varepsilon} in HT-29, it is predicted that sulindac reduces cytosolic Bad sequestration and reciprocally enhances Bad localization to mitochondria. To confirm this, we did two types of experiments. First, we prepared mitochondrial and cytosolic fractions from HT-29 treated with or without sulindac sulfide or sulfone, indomethacin, or SC-236, and Bad in the mitochondrial fraction was analyzed by Western blotting. As a control, the mitochondria-resident protein Hsp60 was concurrently analyzed. Second, we immunoprecipitated cell lysates of HT-29 treated with or without sulindac sulfide or indomethacin by a 14-3-3{varepsilon} antibody and analyzed Bad in the immunoprecipitate with a Bad antibody. The results show that in the absence of NSAIDs, most Bad were localized to the cytosol, whereas Hsp60 was predominantly detected in the mitochondrial fraction with a trace quantity detected in the cytosolic fraction (Fig. 5A ). Bad was detected at a very low level in mitochondrial fraction (Fig. 5A). Sulindac and indomethacin enhanced mitochondrial Bad (Fig. 5A) and suppressed binding of Bad to 14-3-3{varepsilon} (Fig. 5B). SC-236 enhanced mitochondrial Bad to an extent comparable to sulindac sulfide and indomethacin (Fig. 5A). These results suggest that sulindac, indomethacin and SC-236 induce Bad translocation to mitochondria as a consequence of severe 14-3-3{varepsilon} suppression.


Figure 5
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  		Figure 5. NSAIDs induced Bad translocation to mitochondria and apoptosis. A, HT-29 cells were treated with sulindac sulfone (300 µmol/L), sulindac sulfide (160 µmol/L), indomethacin (800 µmol/L), or SC-236 (40 µmol/L) for 8 h. Mitochondrial and cytosolic fractions were isolated and Bad proteins were determined by Western blotting. Hsp60 was analyzed to validate the homogeneity of mitochondrial fraction. B, HT-29 cells were treated with sulindac sulfide (160 µmol/L) or indomethacin (800 µmol/L) for 8 h. Cell lysates were immunoprecipitated with a 14-3-3{varepsilon} antibody. Bad proteins in the precipitate were analyzed by Western blotting. C, Ad-GFP and Ad-PPAR{delta} transfected HT-29 cells were treated with 160 µmol/L of sulindac sulfide or 800 µmol/L of indomethacin for 24 h. Percentages of apoptotic cells were determined by Hoechst staining. Columns, mean of three independent experiments; bars, SD.

 
Rescue of NSAID-induced apoptosis by Ad-PPAR{delta} and 14-3-3{varepsilon}. Sulindac sulfide and indomethacin induced HT-29 apoptosis as evidenced by an increase in Hoechst-positive cells. Apoptosis was reduced by transduction of cells with Ad-PPAR{delta} (Fig. 5C). To ascertain the role of 14-3-3{varepsilon} in NSAID-induced HT-29 apoptosis, we prepared a HT-29 cell line overexpressing 14-3-3{varepsilon} (HT-2914-3-3{varepsilon}) and a control cell line (HT-29CTR; Fig. 6A ). In HT-29 control cells, sulindac suppressed 14-3-3{varepsilon} proteins (Fig. 6A) accompanied by a large increase in apoptosis (Fig. 6B). Overexpression of 14-3-3{varepsilon} proteins in HT-2914-3-3{varepsilon} cells (Fig. 6A) prevented apoptosis by >50% (Fig. 6B).


Figure 6
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  		Figure 6. 14-3-3{varepsilon} overexpression rescued HT-29 from apoptosis. HT-29 stably transfected with 14-3-3{varepsilon} (HT-2914-3-3{varepsilon}) and control (HT-29CTR) were treated with or without sulindac sulfide (160 µmol/L) for 24 h. A, 14-3-3{varepsilon} proteins were analyzed by Western blotting. B, apoptosis was analyzed by Hoechst staining. Columns, mean of three independent experiments; bars, SD. C, a schematic illustration of multiple biochemical processes via which NSAIDs induce apoptosis. Process 1 refers to a COX-2–dependent pathway in which NSAIDs inhibit COX-2–derived PGI2 and PGE2 productions, and thereby suppress PPAR{delta}-mediated 14-3-3{varepsilon} expression. Processes 2 and 3 are considered COX-2–independent. In process 2, NSAIDs block extracellular signal–regulated kinase (ERK) activation, thereby reducing Bad phosphorylation and enhancing Bad translocation to the mitochondria. In process 3, NSAIDs inhibit Bcl-XL and Bcl-2 protein levels. A common result of these processes is a reduced ratio of Bcl-XL and Bcl-2 to Bad and Bax at mitochondria, and an increased release of proapoptotic factors, notably Smac/DIABLO from mitochondria into the cytosol which contribute to caspase 3 activation and apoptosis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings show for the first time that sulindac and other NSAIDs drastically suppress 14-3-3{varepsilon} expression in colorectal cancer cells, resulting in reduced Bad sequestration in cytosol, increased mitochondrial Bad, and enhanced apoptosis. The 14-3-3 family comprises at least seven members in mammalian cells (30). They share sequence homology and biochemical properties, interact with more than 100 cellular proteins, and have diverse biological activities (31). They bind phosphorylated Bad, thereby sequestering Bad in cytosol and suppressing Bad-induced mitochondrial injury and the consequential apoptosis via the intrinsic (mitochondrial) apoptotic pathway (32). It was reported in endothelial cells that among the 14-3-3 members, 14-3-3{varepsilon} is expressed in abundance, and its cellular level is regulated and is crucial in protecting cells from oxidative damage (21). The results in this study show that 14-3-3{varepsilon} is constitutively expressed in HT-29 and DLD-1 cells and is a target of NSAID-induced apoptosis. Sulindac, indomethacin, and SC-236 suppress 14-3-3{varepsilon} expression in HT-29 accompanied by increased translocation of Bad to mitochondria and apoptosis. Stable overexpression of 14-3-3{varepsilon} alleviates sulindac-induced apoptosis. Taken together, these results strongly suggest that the suppression of 14-3-3{varepsilon} is a major mechanism by which NSAIDs induce colorectal cancer apoptosis.

14-3-3{varepsilon} transcription requires PPAR{delta} and is regulated by the level of PPAR{delta}, as evidenced by increased 14-3-3{varepsilon} proteins in Ad-PPAR{delta}–transduced cells. Our results are consistent with a previous report that sulindac suppresses PPAR{delta} transcription activity (14), and provide new data which attribute reduced PPAR{delta} transcription activity to the suppression of PPAR{delta} protein expression. The results show a good correlation between sulindac- and indomethacin-induced 14-3-3{varepsilon} and PPAR{delta} suppressions. Furthermore, PPAR{delta} overexpression by Ad-PPAR{delta} transduction rescues 14-3-3{varepsilon} from suppressions by sulindac sulfide at 80 µmol/L or indomethacin at 400 µmol/L. These data indicate that sulindac and other NSAIDs suppress PPAR{delta} expression, thereby blocking PPAR{delta}-mediated 14-3-3{varepsilon} transcription. Although Ad-PPAR{delta} abrogates 14-3-3{varepsilon} suppression by sulindac and indomethacin at 80 and 300 µmol/L, respectively, it only partially corrected 14-3-3{varepsilon} levels suppressed by higher concentrations of sulindac (160 µmol/L) or indomethacin (800 µmol/L). Lack of complete correction by Ad-PPAR{delta} may be due to the following reasons. First, activated PPAR{delta} promotes transcription by forming a heterodimer with RXR nuclear receptor (14). Overexpression of PPAR{delta} alone without concurrent RXR overexpression may, therefore, be inadequate to exert maximal transcriptional activation. Second, sulindac and other NSAIDs have diverse COX-2–dependent and COX-2–independent effects on signaling transduction and gene expression. Lack of a complete rescue of 14-3-3{varepsilon} suppression and cell apoptosis may suggest that PPAR{delta} is not the only pathway via which NSAIDs suppress 14-3-3{varepsilon}. Furthermore, differential effects of Ad-PPAR{delta} (50 moi) on rescuing 80 versus 160 µmol/L sulindac-induced 14-3-3{varepsilon} suppression may reflect dose-dependent effects on diverse cellular functions. Lastly, lack of a correlation between the magnitude of PPAR{delta} overexpression and 14-3-3{varepsilon} rescue could also be attributed to the requirement of PPAR{delta} posttranslational modification for its full transactivation activity. Because neither MG-132 nor DEVD-CHO blocks the 14-3-3{varepsilon}–suppressing effect of sulindac, it is unlikely that the reduction in 14-3-3{varepsilon} protein levels are due to degradation by ubiquitin-proteasome or caspase 3.

PPAR{delta} expression is regulated by the ß-catenin-Tcf (T cell factor) transcriptional program (14). The wild-type adenomatous polyposis coli tumor suppressor gene product binds and targets ß-catenin for degradation, thereby suppressing PPAR{delta} expression (14). Adenomatous polyposis coli mutation results in increased ß-catenin, which translocates to the nucleus and complexes with Tcf to form an active transcription factor that binds to specific responsive elements on PPAR{delta} and up-regulates PPAR{delta} expression in familial adenomatous polyposis (14, 33). It is reported that sulindac and celecoxib inhibit ß-catenin expression and/or the transcriptional activity of the ß-catenin/Tcf complex (34, 35). It is, therefore, possible that sulindac, SC-236, and other NSAIDs suppress PPAR{delta} expression through the ß-catenin/Tcf transcription pathway.

Sulindac and other NSAIDs may induce colorectal cancer cell apoptosis by COX-2–dependent and COX-2–independent mechanisms (36–39). COX-2 is overexpressed in colorectal cancer and COX-2-derived prostaglandin I2 (PGI2) has been shown to activate PPAR{delta} in colorectal cancer cells (15). PPAR{delta} activation confers resistance to apoptosis in colorectal cancer as well as a number of non–cancer cells such as endothelial, renal interstitial, cardiomyocytes and keratinocytes (18, 19, 21). Sulindac sulfide and selective COX-2 inhibitors may induce apoptosis by blocking COX-2 derived PGI2 production, thereby suppressing PPAR{delta} activation and the antiapoptosis action of PPAR{delta}. COX-2–derived prostaglandin E2 (PGE2) up-regulates ß-catenin expression (40) and NSAIDs may further suppress ß-catenin–mediated PPAR{delta} expression by blocking PGE2 production. However, a recent report has shown that sulindac, selective COX-2 inhibitors, and a host of classic NSAIDs inhibit ß-catenin by a COX-2–independent mechanism (41). Our results reveal that sulindac sulfone, which does not inhibit COX-2, is capable of inhibiting 14-3-3{varepsilon} expression, suggesting that NSAIDs suppress 14-3-3{varepsilon} by a COX-2–independent mechanism. Regardless of the COX-2 involvement, an important finding is that reduction in 14-3-3{varepsilon} levels by sulindac and other NSAIDs results in a decline in Bad sequestration and an increase in Bad translocation to the mitochondria where Bad binds and inactivates the antiapoptotic Bcl-2 family proteins, notably Bcl-XL and Bcl-2. It was reported that sulindac suppresses Bcl-XL expression, creating an imbalance between Bcl-XL and Bax, and subsequent mitochondria-mediated apoptosis (42). Moreover, sulindac inhibits Bcl-2 expression (43), further enhancing Bad- and Bax-mediated mitochondria damage, Smac/DIABLO release, and apoptosis (44). Taken together, our results and the published data suggest that sulindac and other NSAIDs induce colorectal cancer cell apoptosis by multiple mitochondrial apoptotic processes as proposed in Fig. 6C. First, NSAIDs suppress PPAR{delta}, thereby reducing 14-3-3{varepsilon} level and Bad sequestration. Second, NSAIDs inhibit extracellular signal–regulated kinase, thereby blocking Bad phosphorylation (45), and further reducing Bad sequestration leading to excessive Bad translocation to the mitochondria. Lastly, NSAIDs inhibit Bcl-XL and Bcl-2 expression resulting in a reduction in the ratio of antiapoptotic to proapoptotic Bcl-2 family proteins on the mitochondrial membrane with consequent loss of mitochondrial membrane potential, release of proapoptotic factors especially Smac/DIABLO resulting in caspase activation and apoptosis. These processes are interrelated and mutually reinforcing. As PPAR{delta} or 14-3-3{varepsilon} overexpression rescues apoptosis, the PPAR{delta}/14-3-3{varepsilon} transcriptional pathway represents a major pathway for colorectal cancer cell survival.


   Acknowledgments
 
Grant support: NIH (National Heart, Lung, and Blood Institute, HL-50675).

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.

We thank Susan Mitterling and Nathalie Huang for excellent editorial assistance.

Received 9/15/06. Revised 1/22/07. Accepted 1/29/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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