Carcinogenesis Carcinogenesis Advance Access published July 10, 2013
The 5-aminosalicylic acid (5-ASA) anti-neoplastic effect in the intestine is mediated by PPARγ
Journal:
Carcinogenesis
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Manuscript ID:
Manuscript Type:
Date Submitted by the Author:
Original Manuscript
30-May-2013
Rousseaux, Christel; Intestinal Biotech Development, Project Management El-Jamal, Noura; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine Fumery, Mathurin; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine Dubuquoy, Caroline; Intestinal Biotech Development, Project Management Romano, Olivier; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine Chatelain, Denis; CHU d’Amiens, Service d'Anatomopathologie Langlois, Audrey; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine Bertin, Benjamin; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine Buob, David; Centre de Biologie-Pathologie, CHRU Lille, Service d'Anatomie Pathologique Colombel, Jean-Frederic; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine; hôpital Claude Huriez, CHRU Lille, Service des maladies de l’appareil digestif et de la nutrition Cortot, Antoine; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine; hôpital Claude Huriez, CHRU Lille, Service des maladies de l’appareil digestif et de la nutrition Desreumaux, Pierre; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine; hôpital Claude Huriez, CHRU Lille, Service des maladies de l’appareil digestif et de la nutrition Dubuquoy, Laurent; Inserm 995, Faculté de Médecine; Université Lille Nord de France, Faculté de Médecine
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Complete List of Authors:
CARCIN-2012-01164.R1
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Keywords:
5-aminosalicylic acid, PPARgamma, inflammatory bowel disease, colorectal cancer, aberrant crypt foci
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Carcinogenesis
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The 5-aminosalicylic acid (5-ASA) anti-neoplastic effect in the intestine is mediated by PPARγ
Christel Rousseaux (PhD) 1˚, Noura El-Jamal
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˚, Mathurin Fumery (MD)
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, Caroline
Dubuquoy 1, Olivier Romano (MD) 2,3, Denis Chatelain (MD, PhD) 4, Audrey Langlois (MSc) 2,3
, Benjamin Bertin (PhD) 2,3, David Buob (MD, PhD) 5, Jean Frederic Colombel (MD) 2,3,6,
Antoine Cortot (MD) 2,3,6, Pierre Desreumaux (MD, PhD) 2,3,6, Laurent Dubuquoy (PhD) 2,3.
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˚ Authors have equally contributed to the work;
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1
Intestinal Biotech Development, Lille, France;
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INSERM U995, Lille, France;
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Université Lille Nord de France, F-59000 Lille, France;
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Service d'Anatomopathologie, CHU d’Amiens, Amiens, France;
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Service d'Anatomie Pathologique, Centre de Biologie-Pathologie, CHRU Lille, Lille, France;
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Service des maladies de l’appareil digestif et de la nutrition, hôpital Claude Huriez, CHRU
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Lille, Lille, France;
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CORRESPONDANCE: Laurent Dubuquoy. INSERM U995, F-59037 Lille, France; Phone: (33).3.20.97.42.08; Fax: (33).3.20. 97.42.01; E-mail:
[email protected]
RUNNING HEAD: 5-ASA suppresses colon carcinogenesis via PPARγ
Carcinogenesis
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ABSTRACT Epidemiological evidences suggested that 5-ASA therapy may prevent the development of colorectal cancer in inflammatory bowel disease patients. Our aim is to investigate whether PPARγ mediates the anti-neoplastic effects of 5-ASA. HT-29 and Caco-2 cells were treated by 5-ASA, rosiglitazone (PPARγ ligand), or etoposide (anti-carcinogenic drug). Epithelial cell growth, proliferation and apoptosis were assessed by cell count, Ki-67 staining and TUNEL assay respectively. The anti-neoplastic effect of 5-ASA was evaluated in a xenograft tumor
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model in SCID mice and in azoxymethane (AOM)-induced colon carcinogenesis in A/JOlaHsd mice. The role of PPARγ was examined by administration of PPARγ antagonist, GW9662 and in PPAR knock-down cells. Compared to untreated cells, treatment of HT-29 cells by 5-ASA
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inhibited significantly cell growth and cell proliferation (respectively 60% and 63%) and
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induced apoptosis in 75% of cells. These effects were abolished by co-treatment with GW9662 and blunted in PPAR knock-down cells. Contrarily to etoposide, similar inhibitory
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effects of GW9662 were obtained in HT-29 cells treated with rosiglitazone. In the xenograft model, GW9662 abolished the therapeutic effect of 5-ASA which decreased tumor weight
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and volume by 80% in SCID mice compared to untreated mice. In A/JOlaHsd mice, 5-ASA
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suppressed colon carcinogenesis by decreasing the number of aberrant crypt foci (75%) and aberrant crypts (22%) induced by AOM treatment with an absence of 5-ASA response after GW9662 administration. In conclusion, 5-ASA exerts potent anti-neoplastic effects which are mediated through PPARγ. These data provide new rational for designing more effective and safe anti-neoplastic PPARγ ligands with topical effects.
KEY WORDS: 5-aminosalicylic acid, PPARgamma, inflammatory bowel disease, colorectal cancer, aberrant crypt foci.
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Carcinogenesis
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INTRODUCTION One of the most serious complications of colonic inflammatory bowel disease (IBD) is colorectal cancer (CRC). Two prevention strategies of CRC are available: regular surveillance colonoscopy with random biopsies, and chemoprevention with 5-aminosalicylic acid (5ASA) treatment. Surveillance colonoscopies are limited strategies during which only 20% 50% of colonic neoplasms is detected [1]. Epidemiological studies have shown that the chronic use of 5-ASA in IBD has chemopreventive effects on the development of CRC. A
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meta-analysis has estimated that 5-ASA halved the risk of developing CRC or dysplasia in ulcerative colitis (UC) patients, and showed a positive correlation between the protection and the treatment duration [2]. Also, Eaden et al. in a case control study showed that
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mesalasine reduced the risk of CRC by 81% in UC patients [3]. Furthermore, functional
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studies in rodents have shown potent anti-carcinogenic effects for 5-ASA in models of sporadic and colitis-associated cancer [4-6].
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The mechanisms sustaining the putative anti-neoplastic property of 5-ASA are still
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under investigation. However, several in vitro studies have demonstrated that the anti-
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neoplastic effects of 5-ASA are mediated via inflammatory-dependent and -independent mechanisms including the inhibition of NF-κB [7, 8] the Wnt/β-catenin pathway [9], regulation of DNA replication checkpoints [10, 11], and disruption of TFGβ pathway [12, 13]. Another mechanism sustaining the effect of 5-ASA is the induction and activation of Peroxisome proliferator-activated receptor-γ (PPARγ) [14]. PPARs are nuclear receptors that function as transcription factors regulating the expression of genes involved in cellular differentiation, development, metabolism, and tumorigenesis. In the gut, PPARγ is
Carcinogenesis
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4 significantly expressed in colonic epithelial cells and exhibits anti-inflammatory and anticarcinogenic effects, notably by interacting with the β-catenin pathway [15-17].
Our laboratory has already demonstrated that, PPARγ mediates 5-ASA antiinflammatory effects in the colon epithelium in mice and in human cultures colonic biopsies [15, 18, 19]. The aim of the present study was to test the hypothesis that the anti-neoplastic effects of 5-ASA were mediated via PPARγ. We studied the anti-neoplastic effect of 5-ASA
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first in vitro on the proliferation, growth and apoptosis of HT-29 and Caco-2 colon epithelial cell lines and in vivo in a mouse model of colon cancer cell xenograft and in Azoxymethane (AOM)-induced colon carcinogenesis. The involvement of PPARγ in 5-ASA-induced anti-
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neoplastic effect was assessed by the use of PPARγ antagonist GW9662 and the PPARγ knock down cells (HT-29 ShPPARγ).
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Carcinogenesis
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MATERIALS AND METHODS Chemicals 5-ASA, AOM and GW9662 were purchased at Sigma-Aldrich (St Quentin Fallavier, France). Rosiglitazone was ordered at Spi Bio (Massy, France). Etoposide was purchased at (TCI EUROPE N.V., Belgium). For in vivo studies in A/JOlaHsd mice, ethyl celllose granules (Pentasa, Ferring Switzerland) leading to a 5-ASA ileo-colonic release were used.
Cell lines
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HT-29 (ATCC HTB-38) and Caco-2 (ATCC HTB-39) colon carcinoma cell lines were grown in DMEM supplemented with 10 and 20% fetal calf serum (FCS), respectively,
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antibiotics and 1x essential amino acids for Caco-2 cell line. The construction and validation
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of the cell line HT-29 PPARγ knock down (HT-29 ShPPARγ) and its negative control (HT-29 ShLuc), are described in supplementary materials and methods.
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Assessment of cell growth
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HT-29 and Caco-2 cells were treated either with 5-ASA (30 mM) or rosiglitazone (105
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M) with or without GW9662 (10-6M) for 12, 24, or 36h. Etoposide 50 mM was used as a
positive control. These doses of 5-ASA and rosiglitazone were chosen because in previous publications, they have shown to induce an anti-inflammatory effect on cultured cells [15] and are clinically relevant [20, 21]. Cells were detached with the trypsin/EDTA solution before counting. Results were expressed as the mean number of cells counted blindly in 4 different experiments when a coefficient of variation
