Introduction

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A substantial body of evidence exists that nonsteroidal anti-inflammatory drugs (NSAIDs) have anticolorectal cancer (CRC) activity (Shiff and Rigas, 1997; Garcia Rodriguez and Huerta-Alvarez, 2001). However, the mechanism(s) of the antineoplastic activity of this class of drugs remains unclear. It is well recognized that the majority of NSAIDs inhibit one or both of the cyclooxygenase (COX) enzymes, COX-1 and COX-2 (Shiff and Rigas, 1999). Because a role for the COX isoforms (particularly COX-2) has been implicated in the early stages of intestinal tumorigenesis and at later stages of colorectal carcinogenesis (Chulada et al., 2000; Gupta and DuBois, 2001), COX inhibition has generally been understood to underlie the anti-CRC activity of NSAIDs (Shiff and Rigas, 1999; Gupta and DuBois, 2001). However, several COX-independent mechanisms of action of NSAIDs have also been described in cultured CRC cells in vitro (Tegeder et al., 2001), and some NSAIDs, which lack COX-inhibitory activity, retain potent preventative properties in rodent colon carcinogenesis models in vivo (Shiff and Rigas, 1997). At present, the relative contributions of COX inhibition and COX-independent mechanisms, to the overall anti-CRC activity of individual NSAIDs, remain unclear (Marx, 2001).

The NSAID indomethacin has potent anti-CRC activity in vitro and in vivo (Tanaka et al., 1989; Hixson et al., 1994; Hirota et al., 1996; Chiu et al., 2000; Smith et al., 2000; Brown et al., 2001; Garcia Rodriguez and Huerta-Alvarez, 2001; Turchanowa et al., 2001). We, and others, have previously reported that indomethacin induces G₁ growth arrest and apoptosis of several human CRC cell lines in a concentration-dependent manner in vitro (Hixson et al., 1994; Smith et al., 2000; Turchanowa et al., 2001). The antiproliferative activity of indomethacin against human CRC cells does not require COX-2 inhibition (Smith et al., 2000), and indomethacin has been demonstrated to retain growth inhibitory and proapoptotic effects on transformed murine embryonic fibroblasts, which lack either COX isoform (Zhang et al., 1999). Therefore, it is likely that COX-independent mechanisms contribute to the anti-CRC activity of indomethacin, at least in vitro.

A candidate target for COX-independent activity of indomethacin is the transcription factor peroxisome proliferator-activated receptor (PPAR)  (Gupta and DuBois, 2002). Activation of PPAR by synthetic ligands, e.g., thiaziolidinediones (TZDs), or putative endogenous ligands, e.g., 15-d-prostaglandin (PG) J₂, lead to growth arrest and differentiation of several cell types, including human CRC cell lines (Brockman et al., 1998; Gupta and DuBois, 2002; Shimada et al., 2002). Importantly, indomethacin (and, to a lesser extent, other NSAIDs, e.g., ibuprofen) can directly bind and activate PPAR in monkey CV-1 cells (Lehmann et al., 1997) and human rheumatoid synovial cells (Yamazaki et al., 2002). Alternatively, COX inhibition by indomethacin could lead to decreased synthesis of cyclopentenone PGs such as 15-d-PGJ₂ and hence attenuation of PPAR activity. The contribution, if any, of these two opposing effects of indomethacin on PPAR in malignant colorectal epithelial cells is not known.

Therefore, we tested the hypothesis that the antiproliferative activity of indomethacin against human CRC cells is explained by a PPAR-dependent mechanism of action.

	Materials and Methods

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Cell Culture. The human sporadic CRC cell lines SW480 and HCT116 (European Collection of Animal Cell Cultures, Porton Down, UK) were cultured in RPMI 1640 medium containing Glutamax-I, supplemented with 10% (v/v) fetal bovine serum, 1000 U/ml penicillin, and 500 U/ml streptomycin (all Invitrogen, Paisley, UK), on tissue culture plastic, as described previously (Smith et al., 2000; Hawcroft et al., 2002).

Drugs, Antibodies, and DNA Plasmids. Indomethacin (Sigma-Aldrich, Poole, Dorset, UK) was prepared as a 100 mM stock solution in dimethyl sulfoxide (DMSO; Sigma Chemical). Troglitazone (a kind gift from Parke-Davis Pharmaceutical Research, Ann Arbor, MI) was prepared as a 10 mM stock solution in DMSO. In experiments testing the effects of these drugs, control cell cultures always contained an equivalent v/v dilution of DMSO to that in cultures that contained the highest concentration of drug.

PPAR1

Reverse Transcription-Polymerase Chain Reaction Analysis of PPAR mRNA Expression. Total RNA was prepared from SW480 and HCT116 cells using RNeasy columns (QIAGEN, Crawley, UK) as per manufacturer's instructions and then reverse-transcribed. Polymerase chain reaction (PCR) amplification for human PPAR was performed as described previously (Brockman et al., 1998), giving an amplicon size of 234 base pairs. PCR products were subjected to electrophoresis on 2% agarose in the presence of 0.5 mg ml¹ ethidium bromide (Sigma Chemical).

Western Blot Analysis. Cell monolayers were lysed using 50 mM Tris-HCl, pH 7.2, 0.137 M NaCl containing 1% (v/v) Brij 96 (all Sigma Chemical) as described previously (Smith et al., 2000; Hawcroft et al., 2002) or passive lysis buffer (Promega). The total protein concentration of lysate supernatants was determined using the DC protein assay (Bio-Rad, Hemel Hempstead, UK). NUPAGE NOVEX 10% bis-Tris 1-mm gels in 1× NU-PAGE 3-(N-morpholino) propanesulfonic acid running buffer (all Invitrogen) were used to resolve 20 µg of total protein samples and a MagicMark Western molecular weight standard (Invitrogen). Proteins were transferred to Hybond P polyvinylidene difluoride membranes (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK) using a XCell II wet blot module with 1× NU-PAGE transfer buffer (Invitrogen). Membranes were blocked with a 5% (w/v) solution of nonfat skimmed milk powder in PBS containing 0.02% (v/v) Tween 20 (PBS/T) for 1 h at 20°C, before incubation with primary antibodies [anti-PPAR, 1:200; anti--actin, 1:1000; or anti-cyclin D1, 1:1000 in PBS/T plus 5% (w/v) nonfat skimmed milk powder] for 1 h at 20°C. Subsequently, three washes with PBS containing 0.05% (v/v) Tween 20 were followed by incubation with secondary antibody (1:5000 in PBS/T plus 5% nonfat skimmed milk powder) for 1 h at 20°C. After three further washes with PBS containing 0.05% (v/v) Tween 20, enhanced chemiluminescence was detected as per manufacturer's instructions (Perbio Science, Tattenhall, UK).

Transient DNA Transfection and Dual Luciferase Reporter Assays. GeneJuice transfection reagent (Novagen, Madison, WI) was added to serum-free RPMI 1640 medium containing Glutamax-I and incubated for 5 min before addition of the appropriate DNA. The GeneJuice-DNA mix was incubated for 30 min at 20°C before addition to 35-mm-well cell cultures (30-50% confluence) in RPMI 1640 medium containing Glutamax-I, supplemented with 2.5% (v/v) fetal bovine serum, 250 U/ml penicillin, and 125 U/ml streptomycin. Medium was removed and fresh medium [RPMI 1640 medium containing Glutamax-I, supplemented with 10% (v/v) fetal bovine serum, 1000 U/ml penicillin, and 500 U/ml streptomycin] containing drug or carrier control was added 24 h later. After a further 24 h, dual luciferase reporter assays (Promega) were performed as described previously (Hawcroft et al., 2002). Experiments were performed in triplicate, and all data are expressed as the mean + S.E.M. Firefly luciferase activity relative to Renilla luciferase activity. Cell lysates were used for subsequent Western blot analysis.

Cell Proliferation Assay. Cells were plated at 5 × 10⁴ cells/well in 24-well plates. At 72 h, cells were transfected with PPARAF-2 (4 µg) or mock transfected as described above. After 24 h, indomethacin (600 µM) or carrier control was added, and the cells were incubated for a further 24 h. Adherent cells were then harvested using 0.25% (w/v) trypsin and 1 mM ethylenediaminetetra-acetic solution (Invitrogen). Cell number and viability were measured using a hemocytometer and exclusion of 0.4% trypan blue (Sigma Chemical) in PBS. All conditions were assayed in triplicate.

Statistical Analysis. Student's independent sample t test was used for pairwise comparisons. One-way analysis of variance (ANOVA), with post hoc Bonferroni tests, was used for multiple comparisons. Statistical significance was assumed if the p value was less than 0.05. All analyses were performed using SPSS (version 11) computer software (SPSS Science, Chicago, IL).

	Results

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HCT116 and SW480 Human CRC Cells Express Functional PPAR. First, we determined whether HCT116 and SW480 human CRC cells expressed functional PPAR. These two cell lines were chosen for these PPAR studies because we had previously demonstrated that they were sensitive to the growth inhibitory and proapoptotic effects of indomethacin (100-600 µM; Smith et al., 2000). HCT116 and SW480 cells contain COX-1 but do not express COX-2 constitutively (Smith et al., 2000), which mirrors the phenotype of intestinal epithelial cells at the earliest stages of colorectal carcinogenesis of relevance to CRC chemoprevention (Chapple et al., 2002). HCT116 and SW480 cell lines both expressed PPAR mRNA and protein (Fig. 1, a and b). The HCT15 human CRC cell line also expresses PPAR protein, but it exists in a functionally inactive state in these cells (Brockman et al., 1998). Therefore, we confirmed the functional status of PPAR in HCT116 and SW480 cells by testing that activation of PPAR by the TZD troglitazone led to increased PPRE-luciferase reporter gene activity in HCT116 and SW480 cells. Troglitazone (10-50 µM) significantly increased PPRE-luciferase reporter gene activity in both cell lines, in a concentration-dependent manner (Fig. 1c). PPRE-luciferase gene trans-activation induced by troglitazone was significantly greater in HCT116 cells than SW480 cells (Fig. 1c).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   SW480 and HCT116 human CRC cells express functional PPAR. a, reverse transcription-PCR for PPAR mRNA using SW480 and HCT116 cell total RNA. b, Western blot analysis of PPAR (48-kDa) and -actin (42-kDa) protein expression in SW480 and HCT116 cell lysates. c, SW480 and HCT116 cells contain functional PPAR. A PPRE3-tk-luciferase reporter (1 µg) was transiently cotransfected with a Renilla-tk-luciferase reporter (0.5 µg) into SW480 or HCT116 cells, which were then treated with the PPAR ligand troglitazone (0-50 µM) for 24 h. Cells were lysed and dual luciferase assays were performed. Data from triplicate experiments are expressed as the mean + S.E.M. of the ratio of PPRE reporter Firefly luciferase activity to control Renilla luciferase activity. *, p < 0.05; **, p < 0.01 compared with control values (ANOVA).

Indomethacin Activates PPAR in HCT116 and SW480 Human CRC Cells. Indomethacin induces G₁ arrest and apoptosis of human CRC cells, including HCT116 and SW480 cells in a concentration-dependent manner, at concentrations greater than 100 µM (Hixson et al., 1994; Smith et al., 2000; Turchanowa et al., 2001). These effects are demonstrable from 24 h onwards (Smith et al., 2000). In our previous studies, maximal effects of indomethacin on proliferation and apoptosis of human CRC cells were observed at 600 µM (Smith et al., 2000). At this concentration of indomethacin, we have also previously observed specific down-regulation of -catenin protein levels (but not of its homolog -catenin) in SW480 and HCT116 cells (Hawcroft et al., 2002). Therefore, we initially tested the ability of this concentration of indomethacin to activate PPAR in these human CRC cell lines. Indomethacin treatment (600 µM) significantly increased PPRE-luciferase activity in both cell lines (Fig. 2a). In a similar manner to troglitazone (Fig. 1c), the fold increase in PPAR activation by indomethacin was greater in HCT116 cells than SW480 cells (Fig. 2a). Therefore, we used HCT116 cells as model human CRC cells in all subsequent experiments. Indomethacin induced PPAR activation in a concentration-dependent manner (Fig. 2b) with maximal activation occurring at a concentration of 300 µM. Importantly, 10 µM indomethacin (a concentration below that required for significant, direct PPAR activation (Lehmann et al., 1997; Jaradat et al., 2001; Yamazaki et al., 2002), but at which profound COX inhibition in human CRC cells occurs (Kokoska et al., 1999) did not significantly alter basal PPAR activity in HCT116 cells, with only a slight increase in PPRE-luciferase activity being apparent (Fig. 2b). This suggests that down-regulation of PPAR activity, via inhibition of COX-1-derived PG PPAR ligand synthesis, does not occur at lower concentrations of indomethacin in HCT116 cells.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Indomethacin activates PPAR in SW480 and HCT116 human CRC cells. a, PPRE3-tk-luciferase reporter (1 µg) was transiently cotransfected with a Renilla-tk-luciferase reporter (0.5 µg) into SW480 or HCT116 cells. Cells were then treated with carrier control () or 600 µM indomethacin () for 24 h. b, PPRE3-tk-luciferase reporter (1 µg) was transiently cotransfected with a Renilla-tk-luciferase reporter (0.5 µg) into HCT116 cells, which were then treated with 30 µM troglitazone (trog) or indomethacin (10-600 µM) for 24 h. In both a and b, cells were lysed and dual luciferase assays were performed 24 h after drug treatment. Data from triplicate experiments are expressed as the mean + S.E.M. of the ratio of PPRE reporter Firefly luciferase activity to control Renilla luciferase activity. *, p < 0.05; **, p < 0.01 compared with control values (a, Student's t test; b, ANOVA).

Antiproliferative Activity of Indomethacin Is Not Dependent on PPAR Activation. We then used mutant dominant-negative human PPAR (PPARAF-2) to antagonize endogenous PPAR activity and test the functional relationship between direct PPAR activation and the antiproliferative effects of indomethacin. First, we confirmed that PPARAF-2 had dominant-negative activity in HCT116 cells. PPARAF-2 expression significantly decreased basal and TZD-induced trans-activation of the PPRE-luciferase reporter gene in HCT116 cells by approximately 80% (Fig. 3). We tested the antiproliferative activity of 600 µM indomethacin on HCT116 cells, because this concentration of indomethacin induces maximal growth arrest and apoptosis of HCT116 cells at 24 h (the most relevant time point for the transiently transfected cells in the current experiments; Smith et al., 2000). Indomethacin treatment induced a 65% decrease in control HCT116 cell number at 24 h (Fig. 4). The presence of the transfection reagent alone did not alter HCT116 cell proliferation (Fig. 4). Expression of PPARAF-2 in HCT116 cells was confirmed by Western blot analysis of the FLAG epitope (Fig. 4). Dominant-negative PPAR expression was associated with an increase in HCT116 cell proliferation (Fig. 4), which approached statistical significance (p = 0.11). However, the antiproliferative effect of indomethacin was not altered in the presence of dominant-negative PPAR (Fig. 4). Indomethacin treatment has been associated with down-regulation of the cell cycle gene cyclin D1 in human CRC cells (Hawcroft et al., 2002), and PPAR activation has been demonstrated to repress cyclin D1 expression in HeLa cells (Wang et al., 2001). Therefore, we also investigated whether decreased cyclin D1 expression associated with indomethacin treatment was dependent on endogenous PPAR activation. In keeping with the lack of effect of dominant-negative PPAR on the antiproliferative effects of indomethacin, PPARAF-2 did not abrogate the decrease in cyclin D1 protein levels associated with indomethacin treatment (Fig. 4).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Dominant-negative PPAR inhibits endogenous PPAR activity in HCT116 human CRC cells. A PPRE3-tk-luciferase reporter (1 µg) and a Renilla-tk-luciferase reporter (0.5 µg) were transiently cotransfected in the absence () or presence (+) of PPARAF-2 (4 µg) into HCT116 cells. Cells were treated with carrier control () or 30 µM troglitazone () for 24 h. Cells were then lysed and dual luciferase assays performed. Data from triplicate experiments are expressed as the mean + S.E.M. of the ratio of PPRE reporter Firefly luciferase activity to control Renilla luciferase activity. *, p < 0.01 compared with respective cells treated with carrier control or troglitazone, in the absence of PPARAF-2 (Student's t test).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Indomethacin decreases HCT116 human CRC cell proliferation and cyclin D1 protein levels despite inhibition of endogenous PPAR activation by dominant-negative PPAR. HCT116 cells were transiently transfected with (4 µg) dominant-negative FLAG epitope-tagged PPAR (PPARAF-2) or mock-transfected and then treated with carrier control () or 600 µM indomethacin () for 24 h. The viable cell number was counted in triplicate wells, and the data are expressed as the mean + S.E.M. cell number. Western blot analysis was performed for cyclin D1 (34 kDa), FLAG-PPARAF-2 (54 kDa), and -actin (42 kDa). *, p = 0.11 compared with the number of carrier control-treated cells in the presence of transfection reagent (Student's t test).

	Discussion

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We have demonstrated that indomethacin can activate PPAR in human CRC cells but that this pharmacological mode of action does not contribute to the antiproliferative activity of indomethacin on human CRC cells in vitro.

Previous data showing direct binding and transcriptional activation of PPAR by indomethacin has been obtained from experiments on cells (CV-1 and COS-1 cells transfected with human PPAR, as well as human rheumatoid synoviocytes) with little relevance to CRC (Lehmann et al., 1997; Jaradat et al., 2001; Adamson et al., 2002; Yamazaki et al., 2002). This study has provided definitive evidence that indomethacin also has similar activity in human CRC cells in vitro, at similar (>100 µM) concentrations to those previously reported to activate PPAR (Lehmann et al., 1997; Jaradat et al., 2001; Adamson et al., 2002; Yamazaki et al., 2002). Importantly, negative regulation of PPAR activity at lower concentrations of indomethacin (10 µM), compatible with significant COX inhibition in human CRC cells (Kokoska et al., 1999) and other cultured cell types (Kirtikara et al., 2001), but minimal direct PPAR activation (Lehmann et al., 1997; Jaradat et al., 2001; Yamazaki et al., 2002), did not occur in HCT116 cells. The possibility that lower concentrations of indomethacin and other NSAIDs may decrease cyclopentenone PG (e.g., 15-d-PGJ₂) synthesis, and hence abrogate PPAR activity, requires further investigation in other human CRC cell lines with different COX-1 and COX-2 expression profiles.

Both troglitazone and indomethacin produced more potent PPAR activation in HCT116 cells than SW480 cells, despite similar levels of PPAR mRNA and protein in these two human CRC cell lines. This suggests a human CRC cell line-specific difference in PPAR function, which has previously been described in HCT15 human CRC cells (Brockman et al., 1998). Differential PPAR activity may be related to the phosphorylation status of PPAR (Adams et al., 1997) or heterozygous "loss-of-function" mutation of PPAR (Sarraf et al., 1999).

There was a biphasic response of the PPRE-luciferase reporter gene to indomethacin with maximal PPAR activation at a concentration of 300 µM. A similar biphasic PPAR activation pattern, with diminished PPRE-reporter gene activity at NSAID concentrations above 300 µM, has previously been described in transfected CV-1 cells treated with indomethacin or ibuprofen (Jaradat et al., 2001). At present, the explanation for this phenomenon is unclear.

It remains controversial what NSAID concentrations are achievable in colorectal mucosa and whether in vitro studies using high NSAID concentrations are relevant to NSAID chemoprevention in vivo (Marx, 2001). Plasma indomethacin levels between 1 and 10 µM are obtained after acute dosing (200 mg) in humans (Hucker et al., 1966). However, indomethacin may undergo enterohepatic circulation, which could lead to intestine luminal drug levels significantly higher than plasma values (Hucker et al., 1966; Kwan et al., 1976). For example, the sulfone metabolite of the related NSAID sulindac can attain colorectal mucosal levels of 50 to 100 µM in humans (R. Pamukcu, personal communication). Therefore, indomethacin concentrations capable of significant, direct PPAR activation could be generated in human colonic mucosa.

We used a dominant-negative approach to test the relevance of PPAR activation to the antineoplastic effects of indomethacin on human CRC cells in vitro. Mutant dominant-negative PPARAF-2 has potent inhibitory activity in human CRC cells and is a potent tool for studying PPAR-mediated gene trans-activation in a host of relevant human cell types. Although dominant-negative PPAR did not affect the antiproliferative activity of indomethacin, the basal proliferation rate of HCT116 cells was increased in cells expressing dominant-negative PPARAF-2. This implies that PPAR signaling may down-regulate the proliferation rate of HCT116 cells and is in keeping with data that demonstrate that TZD-induced PPAR activation leads to growth arrest and apoptosis of human CRC cells (Brockman et al., 1998; Shimada et al., 2002). These data are relevant to the continuing controversy about the role of PPAR during colorectal carcinogenesis and its potential as a chemoprevention/chemotherapy target (Gupta and DuBois, 2002). Girnun and colleagues have recently described a complementary approach to our dominant-negative PPAR strategy in which they tested the effect of Ppar haplo-insufficiency (homozygous deletion of Ppar is embryonic lethal) on colorectal carcinogenesis in mice (Girnun et al., 2002). Ppar^+/ mice had an increased incidence of azoxymethane-induced colonic tumors compared with Ppar^+/+ littermates. Therefore, our data on human CRC cell proliferation are consistent with the tumor suppressor activity of PPAR described in this in vivo model.

Several other effects of indomethacin on human CRC cells have been described previously (Tegeder et al., 2001). These include not only COX inhibition but also alterations in WNT signaling (Dihlmann et al., 2001; Hawcroft et al., 2002), BAX-mediated apoptosis (Zhang et al., 2000), increased p38 mitogen-activated protein kinase signaling (Kim et al., 2001), induction of NSAID-associated gene-1 expression (Baek et al., 2002; Kim et al., 2002), decreased ornithine decarboxylase activity (Turchanowa et al., 2001), and induction of nerve growth factor-induced gene B (Kang et al., 2000). In only some instances, the functional relevance of these findings to the antineoplastic activity of indomethacin in vitro and in vivo has been proven (Zhang et al., 2000; Kim et al., 2002). Our data strongly suggest that PPAR activation is not necessary for the antiproliferative action of indomethacin on human CRC cells in vitro. It will now be important to compare the chemopreventive activity of indomethacin in azoxymethane-treated Ppar^+/ and Ppar^+/+ mice (Girnun et al., 2002) to confirm our findings using an in vivo model.

In summary, we have provided evidence that PPAR activation does not underlie the antiproliferative activity of indomethacin on human CRC cells in vitro. We have also confirmed previous data, based on PPAR activation experiments, that PPAR has tumor suppressor activity in human CRC cells, using a novel dominant-negative PPAR strategy.