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Carcinogenesis Advance Access originally published online on June 9, 2008
Carcinogenesis 2008 29(7):1407-1414; doi:10.1093/carcin/bgn118
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

PPAR{gamma} is involved in mesalazine-mediated induction of apoptosis and inhibition of cell growth in colon cancer cells

Markus Schwab*, Veerle Reynders, Stefan Loitsch, Yogesh M. Shastri, Dieter Steinhilber1, Oliver Schröder and Jürgen Stein

First Department of Medicine–ZAFES, Johann Wolfgang Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
1 Institute of Pharmaceutical Chemistry–ZAFES, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany

* To whom correspondence should be addressed. Division of Gastroenterology, First Department of Medicine–ZAFES, Johann Wolfgang Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany. Tel: +49 69 6301 5324; Fax: +49 69 6301 6246; Email: m.schwab{at}med.uni-frankfurt.de


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Purpose: Mesalazine has been identified as a candidate chemopreventive agent in colon cancer prophylaxis because of its pro-apoptotic and anti-proliferative effects. However, the precise mechanisms of action are not entirely understood. The aim of our study was to investigate the involvement of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in mesalazine's anticarcinogenic actions in colorectal cancer cells. Experimental design: The effects of mesalazine on cell cycle distribution, cell count, proliferation and caspase-mediated apoptosis were examined in Caco-2, HT-29 and HCT-116 cells used as wild-type, dominant-negative PPAR{gamma} mutant and empty vector cultures. We focused on caspase-3 activity, cleavage of poly(ADP-ribose) polymerase (PARP), caspase-8 and caspase-9, as well as on expression of survivin, X-linked inhibitor of apoptosis (Xiap), phosphatase and tensin homolog deleted from chromosome ten (PTEN) and c-Myc. Techniques employed included transfection assays, immunoblotting, flow cytometry analysis, colorimetric and fluorometric assays. Results: Mesalazine caused a time- and dose-dependent decrease in both cell growth and proliferation. Growth inhibition was accompanied by a G1/G0 arrest, a significant increase in PTEN, caspase-3 activity, cleavage of PARP and caspase-8, whereas the expressions of Xiap, survivin and c-Myc were decreased simultaneously. Cleavage of caspase-9 was not observed. Moreover, PPAR{gamma} expression and activity were elevated. The growth-inhibitory effect of mesalazine was partially reduced in dominant-negative PPAR{gamma} mutant cells, whereas the expression of c-Myc was not affected. Mesalazine-mediated increased caspase-3 activity, the expression of PTEN, cleavage of PARP and caspase-8 as well as reduced levels of survivin and Xiap were completely abolished in the PPAR{gamma} mutant cell lines. Conclusion: This study clearly demonstrates that mesalazine-mediated pro-apoptotic and anti-proliferative actions are regulated via PPAR{gamma}-dependent and -independent pathways in colonocytes.

Abbreviations: 5-ASA, 5-aminosalicylate; CRC, colorectal cancer; G418, Geneticin 418 sulphate; IAP, inhibitor of apoptosis protein; IBD, inflammatory bowel disease; NSAID, non-steroidal anti-inflammatory drug; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PTEN, phosphatase and tension homolog deleted from chromosome ten; UC, ulcerative colitis; Xiap, X-linked inhibitor of apoptosis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Colorectal cancer (CRC) is one of the most fearsome complications of inflammatory bowel diseases (IBDs) mainly ulcerative colitis (UC) but also Crohn's colitis (1,2). CRC risk increases significantly with extent and duration of the disease (1). Main strategy for CRC prevention in chronic UC is currently based on identification of neoplasia by surveillance colonoscopy, but there is great interest in the possibility of primary chemoprevention (3). A series of epidemiological investigations and preliminary clinical trials have affirmatively suggested that regular intake of oral mesalazine [5-aminosalicylate (5-ASA)] may have anti-neoplastic and potentially prophylactic properties reducing the occurrence of CRC in patients with IBD, in particular UC (4,5). Furthermore, chromosomal and microsatellite instability as well as dysplasia was found to be reduced after long-term mesalazine therapy (6–8).

The molecular mechanisms responsible for mesalazine's chemopreventive effects are not entirely understood. The widespread mechanisms leading to its anticarcinogenic actions include the inhibition of inflammatory cascades such as the cyclooxygenase pathway (cyclooxygenase-1 and cyclooxygenase-2), which regulates cell proliferation through formation of prostaglandins (9). Moreover, recent studies suggest that 5-ASA may reduce the cancer risk by mechanisms other than simply controlling inflammation, such as increasing apoptosis, decreasing cellular proliferation and by activating the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (10,11).

In a rodent CRC model, mesalazine inhibits tumour growth and reduces the number of aberrant crypt foci, whereas in patients with sporadic polyps or cancer of the large bowel, mesalazine induces apoptosis and decreases proliferation in the colorectal mucosa (12). In addition, induction of apoptosis through activation of caspase-3 in colon cancer cells has also been indicated (13). However, the precise pathway of activating the caspase cascade is still unknown.

Rousseaux et al. demonstrated that PPAR{gamma} is a target of mesalazine (11,14). The drug increased PPAR{gamma} expression, promoted its translocation from the cytoplasm to the nucleus and induced a modification of its conformation permitting the recruitment of coactivators and the activation of a PPAR{gamma} response element-driven gene (11,14,15). PPAR{gamma} is a transcription factor belonging to the nuclear hormone receptor superfamily (11). The receptor is highly expressed in the colonic epithelium and can not only be activated by natural ligands such as eicosanoids and certain polyunsaturated fatty acids but also by synthetic ligands such as thiazolidinediones and several non-steroidal anti-inflammatory drugs (NSAIDs) (16–18). In the gastrointestinal tract, PPAR{gamma} is known to regulate cellular proliferation, differentiation and induce apoptosis, and may therefore play a regulatory role in carcinogenesis (16,19,20). Several reports demonstrate that PPAR{gamma} ligands such as the thiazolidinediones are potent suppressors of colon tumour formation in rodent models of sporadic colon cancer (21–23). In addition, the growth of cultured human colon tumour cells and of transplanted tumours in nude mice is inhibited by activators of PPAR{gamma}, including troglitazone, rosiglitazone and 15-deoxy-delta12,14-prostaglandin J2 (20,22). Moreover, terminal differentiation, inhibition of cell growth leading to G0/G1 cell cycle arrest and induction of apoptosis via the caspase cascade have also been displayed in colon cancer cell lines treated with PPAR{gamma} agonists (16,24–27).

Based on the recent observations that PPAR{gamma} and its ligands as key players in the control of cell growth and induction of apoptosis, the present study in colonocytes was addressed to elucidate the underlying molecular mechanisms, leading to mesalazine's growth-inhibitory effects including the role of PPAR{gamma}.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell culture
The human CRC cell lines Caco-2, HT-29 and HCT-116 were obtained from the European Collection of Cell Cultures. Cells were cultured in a humidified incubator at 37°C in an atmosphere of 95% air and 5% CO2. Caco-2 cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% foetal calf serum, 1% non-essential amino acids, 1% sodium pyruvate and 1% penicillin–streptomycin. HT-29 and HCT-116 cells were grown in McCoy's 5A Medium, supplemented with 10% foetal calf serum and 1% penicillin–streptomycin. Medium of the dominant-negative PPAR{gamma} mutant and empty vector Caco-2 and HT-29 cells was supplied with 400 µg/ml Geneticin 418 sulphate (G418, Gibco BRL, Eggenstein, Germany). Cells were regularly screened for mycoplasma contamination using the VenorGem Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).

For experiments, cells were seeded in plastic cell culture wells and were cultured in Dulbecco's modified Eagle's medium until 80% confluency was reached. Medium was then removed and replaced by a medium containing mesalazine (10–50 mM). Mesalazine (5-ASA, Sigma chemicals, Deisenhofen, Germany) was dissolved as a 100 mM stock solution in culture medium. The pH of the drug solution was adjusted to 7.0 with NaOH and afterwards the solution was sterile filtrated. All experiments using mesalazine were protected from light. The medium was changed every day. Cells were then harvested at the times indicated in the figure legends.

Transfection assay
The following plasmids were used for transfection: pcDNA3 (Invitrogen, Karlsruhe, Germany), as an empty vector for control transfection, and the plasmid pcDNA3-PPAR{gamma}L468A/E471A, a dominant-negative PPAR{gamma} double mutant, that was kindly provided by V.K.Chatterjee (Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK) (28). These constructs were transfected into sub-confluent Caco-2 and HT-29 cells with Lipofectamine 2000 (Invitrogen) in serum-free conditions. After 6 h, the cells were supplied with fresh medium containing 10% foetal calf serum. Twenty-four hours later, the cells were supplied with medium containing G418 (400 µg/ml) and culture medium supplemented with G418 was replaced twice a week. G418-resistant colonies were collected and used for further analysis.

Cytotoxicity
Cytotoxicity of mesalazine in concentrations used in our experiments was excluded by lactate dehydrogenase release assay using a commercial kit (LDH kit, Roche, Mannheim, Germany).

Cell counts
Cells were suspended and cultured on 96-well dishes at a density of 104 per well (0.28 cm2). Twenty-four hours after plating, cells were incubated for 24–72 h with mesalazine. At given time points following treatment, cell numbers were assessed by crystal violet staining. Medium was removed from the plates and cells were fixed with 5% formaldehyde for 5 min. After washing with phosphate-buffered saline (PBS), cells were stained with 0.5% crystal violet for 10 min, washed again with PBS and unstained with 33% acetic acid. Absorption, which correlates with the cell number, was measured at {lambda}ex = 620 nm.

Assay for cell proliferation
The effect of mesalazine on DNA synthesis of cells was assessed using a cell proliferation ELISA kit (Roche Diagnostics, Tokyo, Japan). This assay is a colorimetric immunoassay for quantification of cell proliferation based on the measurement of bromodeoxyuridine incorporation during DNA synthesis and a non-radioactive alternative to the [3H]-thymidine incorporation assay. Incorporated bromodeoxyuridine was measured colorimetrically.

Caspase-3 activity assay
Caco-2 and HT-29 cells were stimulated with mesalazine at 80% confluency. Fluorometric immunosorbent enzyme assay (Roche) was used according to the manufacturer’s instructions. Protein concentration was analysed and samples were normalized in lysis buffer to equal protein concentrations.

PPAR{gamma} transactivation assay
PPAR{gamma} activity was assayed using an ELISA-based transactivation TransAM® PPAR{gamma} kit (Active Motif, Rixensart, Belgium) following the manufacturer's protocol. The PPAR{gamma} TransAM® kit contains a 96-well plate with immobilized oligonucleotides containing a peroxisome proliferator response element (5'-AACTAGGTCAAAGGTCA-3').

Flow cytometry analysis
Cells were starved for 72 h and then treated with mesalazine (40 mM) up to 48 h. Cells were harvested by trypsinization, fixed with 80% ethanol and kept at –20°C. Cells were then centrifuged for 10 min at 500g and re-suspended in PBS containing 0.25% Triton X-100 for 5 min. After washing with PBS, cells were stained with PBS containing propidium iodide (20 µg/ml; Sigma Chemicals, Deisenhofen, Germany) and RNAse A (2 mg/ml; Sigma Chemicals) at room temperature for 30 min. Subsequently, DNA content was measured by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany) and cell cycle distribution was calculated using Cell Quest Software (Becton Dickinson Technology, Mountain View, CA).

Protein extraction
Caco-2 and HT-29 cells were stimulated with mesalazine at 80% confluency. Cells were washed three times with ice-cold PBS and incubated with cell lysis buffer (Cell Signaling, Beverly, MA) containing multiple protease inhibitors (Complete, Roche) for 20 min at 4°C. Protein extracts were obtained after sonication of cell lysates (2 x 5 s) and centrifugation at 10 000 r.p.m. at 4°C (10 min). Protein content was determined via Bio-Rad colorimetric assay according to the method of Bradford (Bio-Rad Laboratories, Munich, Germany).

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblot analysis
Equal amounts of total protein lysates were separated on a 15% sodium dodecyl sulphate–polyacrylamide gel for poly(ADP-ribose) polymerase (PARP), caspase-8 and -9 and on a 12.5% sodium dodecyl sulphate–polyacrylamide gel for X-linked inhibitor of apoptosis (Xiap), survivin, PPAR{gamma}, PTEN, c-Myc and cytokeratin 20, respectively. Proteins were transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Subsequently, membranes were blocked for 1 h with 5% (wt/vol) non-fat dried milk in Tris-buffered saline containing 0.05% Tween 20. Membranes were then incubated overnight with 1:1000 dilutions of PPAR{gamma}, c-Myc (both from Calbiochem, La Jolla, CA), PTEN, PARP, caspase-8, caspase-9, Xiap and survivin antibody (all from Cell Signaling) or with a 1:500 dilution of human cytokeratin 20-antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in 0.05% Tris-buffered saline containing 0.05% Tween 20 and 5% (wt/vol) non-fat dried milk, respectively. After washing, the blots were incubated for half an hour with corresponding horseradish peroxidase-conjugated antibodies (all from Santa Cruz Biotechnology, dilution 1:2000) in 0.05% Tris-buffered saline containing 0.05% Tween 20 and 5% (wt/vol) non-fat dried milk. The washing steps were repeated, and subsequently enhanced chemoluminescence detection was performed according to the manufacturer's instructions (Enhanced chemoluminescence, Amersham Pharmacia Biotech, Buckinghamshire, UK) on Hyperfilm MP (Amersham International plc, Buckinghamshire, UK). Blots were then reprobed with β-actin antibody (Santa Cruz Biotechnology). For quantitative analysis, bands were detected by scanning densitometry, using a Desaga CabUVIS scanner and Desaga ProVilDoc software (Desaga, Wiesloch, Germany).

Statistics
All statistical analyses were performed using GraphPad Prism 4.01 (Graphpad Software, San Diego, CA). Analysis of variance was performed when more than two groups were compared and, when significant (P < 0.05), multiple comparisons were performed with the Newman–Keuls test. If not otherwise stated, data are expressed as means ± SD from three independent experiments. P value < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Mesalazine reduces cell count, inhibits cell proliferation and affects cell cycle progression of CRC cells
First, we evaluated the effect of mesalazine on cell number and proliferation of Caco-2, HT-29 and HCT-116 cells. Cells were treated with increasing concentrations of mesalazine (10–50 mM) for 24, 48 and 72 h. As shown in Figure 1A, treatment of CRC cells with mesalazine for 48 h significantly reduced the cell count in a dose-dependent manner. Similar effects were obtained for 24 and 72 h and for the inhibition of cell proliferation, measured by the bromodeoxyuridine test (data not shown). To establish whether the reduction in cell growth and proliferation after mesalazine treatment of colon cancer cells was due to changes in cell cycle progression, HT-29 and HCT-116 cells were cultured in presence of the drug (40 mM) for 24 and 48 h, and cell cycle distribution was analysed by flow cytometry (Figure 1B). Both cell lines showed changes in the cell cycle profile within 48 h, with an apparent accumulation of cells in the G0/G1 phase.


Figure 1
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  		Fig. 1. (A) Cell counts of Caco-2, HT-29 and HCT-116 cells after incubation with increasing concentrations of mesalazine (10–50 mM) for 48 h compared with control. n = 8, P value < 0.001 (versus control) was obtained for all concentrations and all cell lines. (B) Cell cycle distribution in mesalazine-treated cells. HT-29 and HCT-116 cells were treated with mesalazine (40 mM) up to 48 h. After staining with propidium iodide, the cellular DNA content was visualized by flow cytometry, and the cell cycle distribution was analysed. In each diagram, distribution of cells (in percentage) in the G0/G1, S and G2/M phase of the cell cycle of three independent experiments is shown.

 
Mesalazine up-regulates the expression and activity of PPAR{gamma}
As depicted in Figure 2A, stimulation of HT-29 cells with mesalazine (30–50 mM) for 48 h led to a concentration-dependent up-regulation of PPAR{gamma} protein expression. To evaluate receptor activity of PPAR{gamma} in HT-29 wild-type, dominant-negative PPAR{gamma} and empty vector HT-29 cells, PPAR{gamma} activation was determined via a transcriptional factor assay (Figure 2B). Treatment of both HT-29 wild-type and empty vector cells with mesalazine (30 mM) for 48 h increased the activity PPAR{gamma}. As expected, PPAR{gamma} activity was not affected in the dominant-negative PPAR{gamma} cell line. Similarly, treatment of Caco-2 wild-type and empty vector cells with mesalazine (30 mM) caused a +1.4-fold increase of cytokeratin 20 expression (versus control, P < 0.01, 48 h), a specific target gene of PPAR{gamma} activity in CRC cells (Figure 2C) (29). In contrast, the induction of cytokeratin 20 could not be observed in dominant-negative PPAR{gamma} mutant Caco-2 cells (Figure 2C).


Figure 2
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  		Fig. 2. (A) Western blot for PPAR{gamma} expression after treatment of HT-29 wild-type cells with mesalazine (30–50 mM) for 48 h. One representative blot of three independent experiments is shown. (B) Effect of mesalazine (30 mM) on PPAR{gamma} transcriptional activity in HT-29 wild-type, PPAR{gamma} dominant-negative and empty vector cells after 48 h of stimulation. ***P < 0.001, n.s., not significant. (C) Cytokeratin 20 expression in Caco-2 wild-type, dominant-negative PPAR{gamma} and empty vector cells after treatment with mesalazine (30 mM) for 48 h. A representative immunoblot of three independent experiments is shown.

 
PPAR{gamma} is partially involved in mesalazine-induced inhibition of cell proliferation
In order to elucidate a potential role for PPAR{gamma} in mesalazine-induced inhibition of cell proliferation and changes in cell cycle distribution, the effect of the drug was studied in dominant-negative PPAR{gamma} colon cancer cells. Mesalazine (50 mM) treatment led to a significant decrease in cell proliferation in both HT-29 wild-type (–50%, P < 0.001) and empty vector cells (–49%, P < 0.001) after 72 h of treatment (Figure 3A). In contrast, in the dominant-negative PPAR{gamma} cell line, the decrease in cell proliferation was partially reversed (–34%, P < 0.001, versus wild-type and empty vector). Similar effects were obtained in the cell line Caco-2 (data no shown). In dominant-negative PPAR{gamma} HT-29 cells, change in cell cycle distribution in response to mesalazine (40 mM) was partially neutralized (data no shown). Nevertheless, statistical significant effects compared with the wild-type were not reached.


Figure 3
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  		Fig. 3. (A) Cell proliferation measurement of HT-29 wild-type, dominant-negative PPAR{gamma} and empty vector cells after incubation with mesalazine (50 mM) for 72 h compared with control. n = 8, ***P < 0.001, n.s., not significant. (B) Western blot for the expression of c-Myc after treatment of HT-29 cells with mesalazine (30 mM) for 4 h. One representative blot of three independent experiments is shown. (C) Western blot for the expression of the tumour suppressor PTEN in HT-29 wild-type and dominant-negative PPAR{gamma} cells in response to mesalazine (40 mM) up to 8 h. One representative blot of three independent experiments is shown.

 
Mesalazine down-regulates the expression of c-Myc in a PPAR{gamma}-independent mechanism
To study further molecular mechanisms responsible for mesalazine's growth-inhibitory effects, expression of the oncoprotein c-Myc after incubation with the drug was determined. c-Myc is known to modulate a broad range of biological activities including cell proliferation and growth (30). Stimulation of HT-29 wild-type, dominant-negative PPAR{gamma} and empty vector (data not shown) cells with mesalazine (40 mM) for 4 h decreased the expression of c-Myc, indicating that PPAR{gamma} does not contribute to the down-regulation of the oncoprotein in response to the drug (Figure 3B).

PPAR{gamma} is involved in mesalazine-mediated up-regulation of the tumour suppressor gene PTEN
The tumour suppressor gene PTEN modulates several cellular functions, including cell survival signalling (31). In particular, it is well established that PTEN decreases cell proliferation through cell cycle arrest in the G0/G1 phase and induces apoptosis via the caspase cascade (32,33). Treatment of HT-29 wild-type and empty vector (data not shown) cells with mesalazine (40 mM) up to 8 h results in a significant increase of PTEN protein expression (for all time points: ~+1.6-fold, P < 0.001) (Figure 3C). In dominant-negative PPAR{gamma} HT-29 cells, the effect of mesalazine on the expression of PTEN was annihilated (Figure 3C).

Mesalazine-induced up-regulation of caspase-3 activity by the caspase-8 signalling pathway occurs via PPAR{gamma}
Challenge of both, Caco-2 wild-type and empty vector cells, with mesalazine (30 mM) resulted in increased cleavage of the nuclear PARP protein after 48 h (+2.2-fold, P < 0.001), a marker for caspase-3 activity (Figure 4A). In contrast, up-regulation of cleaved PARP protein expression in response to mesalazine (30 mM) was found to be attenuated in dominant-negative PPAR{gamma} mutant Caco-2 cells. Analogue effects could be observed in HT-29 wild-type, dominant-negative PPAR{gamma} and empty vector cells after mesalazine treatment (both wild-type and empty vector cells: ~+2.5-fold, P < 0.001; dominant-negative PPAR{gamma} cells: not significant). Direct determination of caspase-3 activity with a fluorometric immunosorbent enzyme assay (Figure 4B) showed a similar pattern of results as compared with the cleavage of PARP (Figure 4A). Incubation with mesalazine resulted in a significant increase in caspase-3 activity after 48 h both in Caco-2 wild-type and empty vector cells (+1.4-fold, P < 0.001). In contrast, up-regulation of caspase-3 activity in response to mesalazine was found to be almost reversed in PPAR{gamma} dominant-negative Caco-2 cells. Comparable results were also obtained in the cell line HT-29 (both wild-type and empty vector cells: ~+1.7-fold, P < 0.001; dominant-negative PPAR{gamma} cells: not significant).


Figure 4
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  		Fig. 4. (A) Effect of mesalazine (30 mM) on cleaved PARP protein expression after 48 h of treatment in Caco-2 cells used as wild-type, dominant-negative PPAR{gamma} mutant and empty vector cultures. One representative western blot of three independent experiments is shown. The band at 89 kDa corresponds to the cleaved PARP protein. Densitometric data are corrected for β-actin levels. ***P < 0.001, n.s., not significant. (B) Caspase-3 activity in Caco-2 wild-type, dominant-negative PPAR{gamma} mutant and empty vector cells measured via fluorometric immunosorbent enzyme assay after stimulation with mesalazine (30 mM) for 48 h. n = 6, ***P < 0.001, n.s., not significant.

 
To further unravel the responsible elements in the caspase pathway leading to increased caspase-3 activity after mesalazine treatment, cleavage of caspase-8 and -9 was examined. Incubation with mesalazine (30 mM) for 48 h led to elevated cleavage of caspase-8 (+1.5-fold, P < 0.01) in Caco-2 wild-type and empty vector cells. In contrast, no cleavage of caspase-9 in response to mesalazine could be observed. In dominant-negative PPAR{gamma} Caco-2 cells, mesalazine-mediated (30 mM) cleavage of caspase-8 was also reversed.

PPAR{gamma} controls the expression of inhibitor of apoptosis proteins after mesalazine treatment
To specify the molecular mechanism of mesalazine-induced apoptosis, changes in protein levels of the inhibitor of apoptosis proteins (IAPs), survivin (Figure 5A) and Xiap (Figure 5B) were examined. Levels of survivin and Xiap were reduced in HT-29 wild-type and empty vector cells in response to mesalazine after 48 h. In contrast, mesalazine-mediated down-regulation of both IAP family members was completely neutralized when incubated in HT-29 dominant-negative PPAR{gamma} mutant cells. A similar pattern of results was obtained in the cell line Caco-2. Incubation with mesalazine resulted in a significant decrease in the expression of survivin (30 mM: -13%, P < 0.05; 50 mM: -15%, P < 0.05) and Xiap (30 mM: -27%, P < 0.001; 50 mM -40%, P < 0.001) in both wild-type and empty vector Caco-2 cells. Again, in the dominant-negative PPAR{gamma} cell line, the decrease in both IAPs was completely annihilated.


Figure 5
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  		Fig. 5. (A) Effect of mesalazine (30 mM) on the expression of survivin in HT-29 wild-type, dominant-negative PPAR{gamma} mutant and empty vector cells after incubation for 48 h. One representative western blot of three independent experiments is shown. Quantitative data are normalized for β-actin levels. ***P < 0.001, n.s., not significant. (B) Effect of mesalazine (30 mM) on the expression of Xiap in HT-29 wild-type, dominant-negative PPAR{gamma} mutant and empty vector cells after incubation for 48 h. One representative western blot of three independent experiments is shown. Densitometric data are corrected for β-actin levels. ***P < 0.001, n.s., not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The increased risk for the development of CRC in patients suffering from UC and Crohn's colitis remains a significant problem in the long-term management of IBD (1). One of the first candidate chemopreventive drugs that has specific relevance in IBD patients seems to be mesalazine, an anti-inflammatory drug which has been used extensively in the treatment of IBD for >50 years (34). Several retrospective studies have suggested that long-term use of 5-ASA in IBD patients may significantly reduce the risk for the development of CRC by decreasing cell proliferation and inducing apoptosis (4,9). However, despite comprehensive clinical and experimental experience with the drug, the mechanisms leading to its pro-apoptotic actions are largely unknown. Unravelling the signalling pathways behind these effects could deliver insights into mesalazine's mode of action in colon cancer prevention.

There is increasing evidence supporting the role of PPAR{gamma} as a regulator of proliferation and a modulator of cell growth (16,23,35). Recent studies demonstrate that activators of PPAR{gamma} suppress the growth response of colon cancer cells and inhibit colon tumorigenesis in animal models (20–23). Thus, inhibition of cell growth is partly due to the induction of apoptosis upon PPAR{gamma} activation. In addition, several reports describe not only an induction of caspase-3 by PPAR{gamma} ligands in a variety of cancer cells (27,36) but also an association between loss of function mutations of PPAR{gamma} and the development of CRC (16,23,37). These observations raised the possibility that somatic mutations of PPAR{gamma} contribute to the carcinogenic process and the receptor functions as a tumour suppressor gene by inhibiting cell growth and inducing apoptosis.

Recent data showed that PPAR{gamma} was the major functional receptor mediating the common mesalazine activities in IBD and have corroborated involvement of the receptor in controlling intestinal inflammation (14). Mesalazine has been identified to act as an agonist of PPAR{gamma} and was shown to up-regulate the expression and activity of the receptor in HT-29 cells (14), which is in accordance to the findings in the present study. Moreover, PPAR{gamma} has also been proposed to mediate the anti-proliferative and pro-apoptotic effects of mesalazine (10). In the present study, we shed light on PPAR{gamma} as a possible key target in mesalazine-mediated anticarcinogenic abilities. For that purpose, mesalazine-induced anti-proliferative effects and pro-apoptotic actions along the caspase signalling cascade were examined in colonocytes transfected with a dominant-negative PPAR{gamma} vector to inhibit wild-type PPAR{gamma} receptor action (28).

Mesalazine seems to act locally within the colonic mucosa, since systemic concentrations are low following both oral and rectal dosing (38,39). Getting oral 5-ASA to its site of action, however, necessitates some form of colonic delivery system since the drug is unstable in gastric acid and is rapidly absorbed from the small intestine (38). Slow release and delayed release coatings and azo bonding to an inert carrier are the usual methods (38). In the colon, 5-ASA is rarely absorbed where it acts from the luminal side and passes the stool. Thus, the therapeutic effect of 5-ASA depends more on the direct contact of the molecule with the colonic epithelium than to tissue concentration in the colon, demonstrating that a high perimucosal concentration of 5-ASA is required for its action (14). It has been demonstrated that stool concentrations in patients conventionally treated with 5-ASA are in the median order of 30 mM, ranging from 10 to 100 mM (14). These concentrations correspond to luminal concentrations of 5-ASA 100 times greater than the concentrations in the colonic mucosa and have been demonstrated in vivo to modulate several cellular functions including inhibition of cell proliferation (13,14,38). Hence, the concentrations used in the present study are clinically and biological relevant and are in accordance with several in vitro models (13,14,40).

In our in vitro model, mesalazine decreased cell proliferation of colonocytes in a time- and dose-dependent manner. These anti-proliferative abilities were partially reversed in the PPAR{gamma} dominant-negative cell lines. However, the inhibition was not complete, indicating that other mechanisms besides PPAR{gamma} are involved in mesalazine's anti-proliferative effects. The anti-proliferative actions of mesalazine on colon carcinoma cell lines are in line with several in vivo and in vitro studies (9,13,40,41). In addition, PPAR{gamma} agonists like rosiglitazone and troglitazone have been shown to decrease proliferation of colon cancer cell lines merely mediated in part by a PPAR{gamma}-dependent mechanism (42,43). Moreover, our data clearly demonstrate that mesalazine induced a G0/G1 arrest in colon cancer cells which is in line with the robust G1 arrest of other NSAIDs, e.g. indomethacin, sulindac and direct PPAR{gamma} agonists, e.g. rosiglitazone (44–46). Our results differ from the observations of former studies illustrating a S or G2 phase arrest in response to mesalazine; however, the authors could not reasonably explain the discrepancy compared with other NSAIDs (13,41).

To unravel further mechanisms contributing to mesalazine's growth-inhibitory abilities in colonocytes, expressions of the oncoprotein c-Myc and of the tumour suppressor gene PTEN were determined. c-Myc is over-expressed in nearly 70% of CRC. Moreover, dysplasia of colonocytes in UC is also associated with increased expression of the oncoprotein (20,30). c-Myc is known to modulate a broad range of biological activities including cellular proliferation and cell growth (20,30). De-regulated c-Myc has been shown to increase apoptosis, genomic instability and to block differentiation (20,30). In colonocytes including HT-29, it was recently shown that mesalazine decreased the expression of c-Myc (30). Although several PPAR{gamma} agonists inhibit the expression of the oncoprotein (20,47), the effects seem to be PPAR{gamma} independent because no peroxisome proliferator response element has been found in its promoter (47). Therefore, it is not surprising that a PPAR{gamma}-independent down-regulation of c-Myc in response to mesalazine was obtained in our in vitro setting which seems to contribute to the modulation of the PPAR{gamma}-independent growth-inhibitory effects caused by the drug.

PTEN is a tumour suppressor gene involved in the regulation of cell survival signalling through the phosphatidylinositol 3-kinase/Akt pathway (48). Phosphatidylinositol 3-kinase/Akt signalling has been shown to be required for an extremely diverse array of cellular activities mainly involved in cell growth, proliferation apoptosis and survival mechanisms (48,49). Activated Akt protects cell from apoptotic death by inactivating compounds of the cell death machinery such as procaspases (48). PTEN exercises its role as a tumour suppressor by antagonizing the phosphatidylinositol 3-kinase/Akt pathway (48). In CRC cells, enforced expression of PTEN, e.g. by NSAIDs, has been demonstrated to decrease cell proliferation through cell cycle arrest in the G0/G1 phase and to activate the caspase cascade (33,49,50). Stimulation of HT-29 wild-type cells with mesalazine in our in vitro model increased the expression of PTEN. In contrast, in the dominant-negative PPAR{gamma} cell line, the effect of mesalazine on the expression of PTEN was reversed. Besides the ability of PPAR{gamma} agonists to up-regulate the expression of PTEN, confirmed by inhibitor and anti-sense experiments, two peroxisome proliferator response elements in the genomic sequence upstream of the tumour suppressor gene have been found (31,51), supporting the findings of our investigations. The PPAR{gamma}-dependent increase of PTEN caused by mesalazine in our experiments not only indicates that the tumour suppressor gene contributes to the growth-inhibitory activities of the drug but also may trigger its pro-apoptotic actions.

In addition to the contribution of PPAR{gamma} in mesalazine-induced growth inhibition, our data provide insight into the molecular mechanisms leading to activation of the caspase cascade, demonstrated directly by increased caspase-3 activity and indirectly by augmented levels of cleaved PARP. Our findings are supported by former studies demonstrating increased cleavage of PARP and caspase-3 activity after mesalazine treatment in HT-29 and colo205 cells, respectively (13,40). Caspase-3 can be activated via two signalling pathways, the extrinsic pathway and the intrinsic trail, initiated by caspase-8 or caspase-9 signalling, respectively (52). The increase in levels of cleaved caspase-8 by mesalazine in the present study indicates that the extrinsic signalling pathway takes part in caspase-3 activation. Both caspase-dependent and caspase-independent mechanisms have been reported for mesalazine's apoptotic processes, indicating that the drug activates multiple cell death signalling pathways, e.g. caspase-9, Bcl-2, intestinal sphingomyelinase and the induction of intracellular peroxides (40,53). Moreover, in our in vitro model, mesalazine significantly decreased the protein levels of survivin and Xiap, two anti-apoptotic proteins known to potently block caspase-3, thereby inhibiting apoptosis (52). Accordingly, a similar down-regulation of IAPs has not only been demonstrated for sulindac but also for PPAR{gamma} ligands e.g. pioglitazone in colorectal carcinoma cells (42,54). Mesalazine-mediated effects on both pro-apoptotic and anti-apoptotic markers were almost reversed in PPAR{gamma} dominant-negative cells. Taken together, these results indicate the involvement of PPAR{gamma} in mesalazine-mediated apoptosis via activating the caspase cascade. The underlying mechanism by which PPAR{gamma} controls the caspase signalling pathways is not fully explained. It may be speculated that the receptor regulates the cascade via modulation of the IAPs by a spermidine/spermidine N1-acetyltransferase-dependent mechanism (55,56). Nevertheless, we suggest that the inhibition of cell growth in response to mesalazine is more complex than if it were based solely on studied molecules and pathways. It is known that activation of PPAR{gamma} can increase terminal differentiation and that cytokeratins are involved in this regulation (29,57,58). Since we measured a PPAR{gamma}-dependent up-regulation of cytokeratin 20 caused by mesalazine in the present study, we suggest that an increase in terminal differentiation might also contribute to the observed inhibition of cell growth.

In conclusion, this study provides evidence for the involvement of PPAR{gamma}-dependent and -independent mechanisms, responsible for mesalazine's pro-apoptotic and anti-proliferative abilities which appear to be triggered at least in part by the modulation of PTEN and c-Myc, respectively (Figure 6). In addition, activation of caspase-8 and down-regulation of Xiap and survivin contribute to elevated caspase-3 activity caused by mesalazine. Revealing this transduction pathway is not only important to understand the fundamental biological processes but also provides new opportunities in the chemoprevention of IBD.


Figure 6
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  		Fig. 6. Possible molecular mechanism of mesalazine-induced apoptosis and cell growth in colorectal carcinoma cells.

 

   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
This work was supported by an unrestricted research grant from the Else Kröner-Fresenius-Foundation, Bad Homburg, Germany. Markus Schwab is supported by the Frankfurt International Research Graduate School for Translational Biomedicine (FIRST).


   Acknowledgments
 
The authors would like to thank Annett Häussler for the excellent technical assistance by performing flow cytometry experiments.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received January 3, 2008; revised May 6, 2008; accepted May 6, 2008.


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