Inflammatory Bowel Disease (IBD) patients, such as Crohn’s disease or ulcerative colitis suffer from chronic and relapsing intestinal inflammation that favours the development of colitis associated cancer (CAC). This inflammation is initiated by aberrant activations of the innate immune responses associated to intestinal barrier defects. The conventional medical therapies consist to decrease the inflammatory response, which also decrease the risk of colon carcinoma but lead to severe side-effects. Recently, a number of animal studies have demonstrated that innate immune responses are attenuated by stimulation of the efferent arm of vagus nerve (VN) through its neurotransmitter acetylcholine (ACh), that acts on resident macrophages α7 nicotinic receptor (α7 nAChR). ACh also acts as a signalling molecule in epithetlial cells through cholinergic receptors such as nAChR or muscarinic (mAChR) receptors. In the current study, we aimed to extend these findings to CAC prevention by treating human adenocarcinoma cell lines through targeting cholinergic receptors with nicotine (which binds nAChR) and ACh (which binds both cholinergic receptors). Using HT-29 and Caco-2 cell lines, we demonstrated that ACh-induced activation of mAChR results in cell dissociation together with changes in expression and localization of intestinal tight and adherens junction proteins. ACh-induced modulation of cell adhesion proprieties correlates with the acquisition of invasive potential. By contrast, nicotine-mediated activation of nAChR maintains epithelial cell organisation. ACh-released by VN stimulation (VNS) could effectively preserve epithelium integrity thus limiting inflammatory response and tumor development. However, attention should be paid on the nature of the cholinergic receptor solicited. Indeed, regarding to the protective effects of nAChR signalling on epithelial cells, activation of mAChR would worsen the disease and led to increase inflammation. These data have important repercussions on the therapeutic potential of VNS in IBD and CAC, which may represent “the yin and yang” of the intestinal homeostasis.
Inflammatory Bowel Diseases (IBD), such as Crohn’s disease (CD) or ulcerative colitis (UC), are multifactorial chronic inflammatory diseases of the gastrointestinal tract for which the exact causative mechanism is still unclear. According to a current hypothesis, an increased intestinal permeability due to an epithelial barrier defect, coupled with a dysfunctional immune response participate to the development of chronic intestinal inflammation [
In the long-term, IBD can induce further complication and patients have an increased risk developing other pathologies such as colorectal cancer (CRC). About 20% of CRC cases can be genetically attributed to a family history. Involvement and mechanisms by which environmental factors contribute to the disease are still unclear. Colitis associated cancer (CAC) is a type of colon cancer which is preceded by clinically detectable IBD. CAC develops from dysplasia, which is stimulated by chronic inflammation, rather than polyps. Even though CRC does not always develop after IBD, its high frequency in patients with IBD represents a paradigm for the connection between inflammation and cancer in terms of epidemicology and mechanistic studies in preclinical models (for review [
Vagus nerve (VN) is the main nerve of the parasympathic division of the autonomic nervous system for the thoracic and abdomino-pelvic viscera that controls heart rate, hormone secretion and gastrointestinal motility/secretion [
Inflammation could contribute to carcinogenesis by increasing the level of reactive oxygen species that have a mutagenic effect on DNA (tumor initiation) [
Epithelia form a barrier constituted of specialized cells characterized by structural features including polarized morphology and cell-cell contacts. They lie on a basement membrane, which is organized into a complex structure containing collagen type IV (COIV), various laminin isoforms, and proteoglycans [41-43]. Interactions between cells and this specialized extracellular matrix (ECM) are crucial for essential biological processes such as migration, proliferation, differentiation, and cell survival. The cell-cell or cell-ECM interactions are mediated through various transmembrane receptors, which are linked intracellularly to cytoskeleton components and signal transduction molecules [44,45]. Tight junctions (TJ) are composed of transmembrane proteins (claudins, occludins and junctional adhesion molecules), scaffold proteins like zona occludens (ZOs) that link the actin cytoskeleton, and intracellular regulatory molecules including kinases [
The human colon adenocarcinoma cell lines HT-29 and Caco-2/TC7 were cultured at 37˚C in a 5% CO2 atmosphere in DMEM containing 25 mM glucose (Invitrogen, Cergy Pontoise, France) and supplemented with 10% FCS, 5% penicillin and streptomycin. The medium was changed every day to avoid glucose exhaustion, which leads to differentiation. The differentiation of HT-29 cells was initiated by replacing standard medium by glucose free DMEM (Invitrogen) supplemented with 10% dialyzed foetal calf serum, 5 mM galactose, 15 mM HEPES, selenous acid (10−2 mg/ml), penicillin, and streptomycin [
Polyclonal antibodies directed against M3 mAChR (Cat. N˚. AB41169) provided from Abcam (12964, Abcam, Paris, France). Anti-human E-cadherin (Clone HECD1) monoclonal antibody was obtained from Takara Biochemicals (Cambrex Bio Science, Paris, France). Monoclonal antibody against p120ctn (clone 98) was purchased from BD Biosciences/Transduction Laboratories (Pont de Claix, France). Anti-actin (Cat. N˚. A2066), and anti-β catenin (Cat. N˚. C2206) polyclonal antibodies and a7 nAChR (clone 306) monoclonal antibody were obtained from Sigma Aldrich (L’Isle d’Abeau, France). Polyclonal antibody directed against Src-PTyr418 (AT- 7135) was purchased from MBL Calbiochem (VWR International, Fontenay-sous-Bois, France), monoclonal anti-Src (Clone GD11) was from Millipore (Molsheim, France). Occludine clone (OC-3F10) and ZO-1(clone ZO1-1412) monoclonal antibodies were from Invitrogen (Cergy Pontoise, France). Alexa-conjugated goat antimouse secondary antibody was obtained from Molecular Probes (Eugene, OR). Horse Radish Peroxydase-conjugated goat anti-mouse was from Bio-Rad (Marnes-la-Coquette, France), donkey anti-rabbit antibodies were from Jackson Immunoresearch (Immunotech, Marseille, France).
Total RNA extractions were performed using TrizolTM reagent and 1 µg of total RNA was denaturized and subsequently processed for reverse transcription using MMLV (Invitrogen) according to manufacturer’s instructions and run on thermocycler (Eppendorf). Primer sequences and probes are:
PCR conditions are: 5 min at 92˚C followed by 35 cycles (40 sec at 92˚C, 40 sec at 60˚C and 1 min at 72˚C) and 10 min at 72˚C. PCR were analyzed on 1.5% agarose gel. Quantification was performed using Image J (NIH software). GAPDH were used as housekeeping gene.
Cells were grown to confluence and lysed with a buffer made of 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and supplemented with 1 mM PMSF, 2 mg/mL aprotinin, 10 mg/mL leupeptin, 10 mM pepstatin, 2 mM CaCl2 and MgCl2, for 15 min on ice. Protein concentrations in lysates were determined using the copper reduction/bicinchoninic acid (BCA) assay (Pierce Chemical Co) according to the manufacturer’s instructions. Proteins (30 mg in SDS-b mercaptoethanol sample buffer) were resolved on 10% polyacrylamide gels, transferred into PVDF membranes (Hybond-C super; Amersham), and blocked in 5% bovine serum albumin in 0.1% Tween 20 in TBS for 1 hr at room temperature. After overnight incubation at 4˚C with primary antibodies diluted in the blocking solution, blots were washed in TBS, 0.1% Tween 20 and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (dilution of 1:10000) for 1 hr at room temperature before extensive washes. The blots were revealed by chemiluminescence (Amersham ECL reagents) and quantified with Image J software from NIH. Primary antibodies were used at the following dilutions: antihuman a7 nAChR and M3 mAChR, E-cadherin, p120ctn and b-catenin (1:1000), ZO-1 (1:2000), and anti-actin (1:2000).
Cells were grown on glass coverslips and were treated as described previously [
Tissue culture dishes were coated with LM-332 using the following methods: A431 epidermoid cells were cultured to confluence on various surfaces at 37˚C to allow for the deposit of LM-332, then cells were removed as previously described [51,52]. Briefly, confluent monolayers were sequentially extracted with 1% (v/v) Triton X-100 in PBS, followed by 2M urea in 1M NaCl. All extraction buffers contained protease inhibitors (1mM phenyl-methylsulfonyl fluoride and 2 mM N-ethylmaleimide). Plates were washed in PBS, incubated with 1%BSA, and stored at −20˚C. Human collagen type IV from placenta was obtained from Sigma Aldrich. Coating of plastic Petri dishes (Microtiter plates 96-well, Nunclone; Nunc, Roskilde, Denmark) was performed by overnight incubation with extracellular matrix proteins (10 µg/mL) at 4˚C. Plates were saturated with 3% (w/v) BSA in PBS for 2 hrs at 37˚C to block nonspecific adhesion. HT-29 cells were harvested and pre-treated or not with 100 nM nicotine or ACh for 30 min before to be plated (5 ´ 104 cells/ well) in triplicate in coated 96-well microtiter plates and incubated from 0 to 60 min at 37˚C. Non adherent cells were removed by washing three times with PBS, and cell adhesion was estimated by a colorimetric cell proliferation assay (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay; Promega).
Total HT-29 cells (2.5 ´ 10−5/mL) were placed in the top compartment of a 24-multiwell insert plate (BD Falcon), which was separated from the bottom compartment by BD-Matrigel Matrix membrane, with 0.4-µm pore size. Serum-free RPMI with or without nicotine or ACh (100 nM) were added into the top compartment and 10% FCS into the bottom compartment. After 48 hrs at 37˚C in a 5% CO2 atmosphere, cells that had invaded through the Matrigel were analyzed: cotton swabs were used to remove cells on the upper surface of inserts. After fixation with PFA 4%, migratory cells were stained with Hematoxylin Gill’s formula (Vector Laboratories) and manually counted under the microscope. The mean values of the readings and their SEM were calculated, and statistical differences were analyzed using Student’s t-test for non-paired samples.
Cell were plated in 96-well plates (2000 cells/well) at day 0 and cultured in complete DMEM medium without or with nicotine and ACh (100 nM). Cell proliferation was evaluated from day 1 to 3 using the CellTiter 96 Kit (Promega) according to the manufacturer’s instructions.
Immunoblots shown are representative of at least three independent experiments. All graphs represent the mean value ± SD of protein expression levels measured by densitometric analysis in “Image J” software (NIH). Statistics were performed with unpaired t-test and statistical significance was given by the number of asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).
It has been described that human colon cancer cell lines expressed mainly M3AChR and α4, α5, α7 and β1 nAChR subunits but only α7 subunits form a functional receptor [
nAChR at the extern edge of cells not engaged in cellcell contacts. In presence of cholinergic ligands (nicotine or ACh, 100 nM, 5 hrs) we observed cell dissociation (black spaces appeared between adjacent cells) associated with a modification of the cell shape (more spreading cells) in presence of ACh. Cholinergic treatment induces a hypercholinergic response (desensitization process) which is illustrated by a decrease of cholinergic receptor density at the membrane of epithelial cells with a concomitant increase of cytoplasmic labelling which indicate that they receptors are functional. Then their expression was analyzed at the protein level and according to the status of differentiation of epithelial cell lines (
Disruption of epithelial barrier integrity is identified as one of the pathologic mechanisms in IBD and cancer development. Adequate intestinal TJ protein function determines intestinal barrier integrity. We measured changes in expression and localization of occluding and ZO-1 to determine whether modulation of these TJ proteins correlates with changes observed with cholinergic treatment [58,59]. For this purpose, we use differentiated Caco-2 cells which established “mature” TJ compared to HT-29 cells. Confluent monolayers of Caco-2 cells differentiated for 15 days were harvested and immunoblotted (
sion determined by western blots (
Localization and stability of AJ proteins were controlled by phosphorylation/dephosphorylation events. The kinase Src is one of the kinases involved in this process and it can be activated by cholinergic ligands. We then compared the ability of nicotine and ACh to activate Src by measuring its phosphorylation on tyr418 (
Altogether these data indicate that ACh but not nicotine induces an alteration of epithelial cell integrity characterized by a loss of TJ and AJ proteins subsequently to cell dissociation and protein endocytosis and degradation. This phenomenon could be address by the activation of
the Src kinase.
The AJ’s disassembly correlates with a loss of cell-cell adhesion and an acquisition of migratory potential [
ACh is an autocrine/paracrine factor in various non neuronal cells. Consistent with previous observations, we found that ACh reproducibly induced a 1.7-fold increase in HT-29 cell proliferation at 48 hrs and 72 hrs after seeding (
In this present work, data indicate that ACh-induced mAChR signaling plays a key role in colon cancer cell polarity and adhesion, proliferation and invasion compared to nAChR.
IBD patients suffer from chronic and relapsing intestinal inflammation that favours the development of CAC. This
inflammation is initiated by aberrant activations of the innate immune responses associated to intestinal barrier defects. Recently, a number of animal studies have demonstrated that innate immune responses are attenuated by stimulation of the efferent arm of VN through its neurontransmitter ACh, that acts on resident macrophages a7 nAChR, [15,16]. It appeared that ACh is not only a neurotransmitter but it also acts as a signaling molecule in non-neuronal tissues. In the current study, we aimed to extend these findings to CAC prevention by treating human adenocarcinoma cell lines through targeting cholinergic receptors with nicotine and ACh. nAChRs and mAChRs might affect the inflammatory response in an opposite manner and may represent “the yin and yang” of the intestinal homeostasis [
Emerging evidences indicate that normal and neoplastic non-neuronal cells can produce and release ACh in sufficient quantity to modulate cell function [
All components of the cholinergic system such as synthesis, storage, release, inactivation as well as the expression and function of various cholinergic receptors can be affected in pathophysiological conditions. Some substantial alterations of the non-neuronal cholinergic system have already described in human colon cancers. The cholinergic system for ACh synthesis and degradation is modified in human colon cancer cell lines compared to normal tissues. The activity of AChE was measured in 55 human samples of healthy and malignant colon, sigmoid colon and rectum. It appeared that cancer decreases the average of AChE activity value, such increasing the rate of ACh [
In gastrointestinal tissue, the primary mAChR subunits are M1AChR, M2AChR and M3AChR, but colon epithelial cells mainly express M1AChR and M3AChR [38-40,69]. Frutcht et al. reported that human colon cancer cell lines and colon cancer tissues express M3AChR. Their expression is increased up to 8-fold in cancer compared to normal tissue [
Since the discovery of ubiquitous presence of nAChR in mammalian cells, studies from many laboratories have linked nAChR with various pathological conditions including cancer [
Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells [77-79]. These effects have been attributed to M3AChRinduced secretion of matrix metalloprteinases (MMPs) such as MMP7 that releases EGF ligands or MMP1. Most of M3AChR effects seem to be mediated by transactivation of the EGFR. Co-expression of M3AChR and EGFR in many colon cancer cell lines associated with over expression of these receptors in the majority of colon cancer suggests that the functional interaction between M3AChR and EGFR is important for colon cancer regulation. In human colon cancer cell lines, we found that ACh alters the assembly of intercellular junctions (AJ and TJ), a process associated to epithelial barrier failure. This correlates with the observation that mAChR (in particular M3) increases epithelial cell permeability [
Strong evidences for non-neuronal ACh production are reported in human keratinocytes and small lung cancer cells where this transmitter acts as an autocrine/paracrine growth factor stimulating cell proliferation [91,92]. Similarly, ACh produced and released by H508 colon cancer cells interacts with M3AChR and acts as an autocrine growth factor [
Together, these finding indicate that M3AChR expression plays a strong role in intestinal tumor promotion by modulating key process of carcinogenesis. Using different in vivo models, Raufman et al., showed that M3AChR gene ablation decreases both colon tumor number and size and the degree of dysplasia [
As mAChR, nAChR have been described to modulate epithelial cell-cell contacts, adhesion and motility of respiratory epithelial cells, lung cancer and in a variety of human cancer cell lines [97-99]. Nicotine can induce the transition from a well-differentiated epithelial cell to a highly invasive carcinoma cell involving different signal transduction cascades. Long-term treatment of lung cancer and breast cancer cells is required for these effects. In these models it has been proposed that nicotine, acting via α7 nAChR, stimulates mRNA and protein expression of fibronectin with a concomitant down regulation of junctional protein expression and/or localisation [98,100, 101]. Evidence of nAChR mediated activation of integrin-dependent signaling pathway has also been obtained in colon and gastric cancer [
However in some cases, nicotine and tobacco could exert protective effects. UC patients with a history of smocking usually developed their disease after they had stopped smocking [106-108]. Smoking has been found to reduce development and severity of UC and to worsen the disease in CD patients [106,109,110]. Similarly, nicotine administration ameliorates disease in DSS experimental colitis [
Using a concentration similar to those observed in the blood of smokers (0.01 - 1 mM), we found that nicotine-mediated activation of α7 nAChR exert protective effect on epithelial morphology that can preserve from failure of barrier function. Nicotine increased or maintained the expression of various intercellular proteins such as p120ctn, Z0-1, Occludin. This mechanism may contribute to maintain barrier integrity. McGilligan has described similar results, showing that nicotine decrease Caco-2 permeability by regulating the expression of TJ proteins [
Many mechanisms are responsible for the up-regulation of junction proteins by nicotine. One possibility is the activation of extracellular signal related kinase (ERK) or mitogen activated kinase (MAPK). Nicotine is described to increase the expression of these kinases [118, 119]. Another possibility is a nicotine-mediated decrease of the nuclear factor kappa B (NFkB). This factor has been described to regulate TJ permeability [120,121] and Cox-2 expression [122,123], a CRC tumor promoting factor [
Recently, nAChR has been implicated in regulating the release of the neurotransmitter g-aminobuturic acid (GABA), which may act as a tumor suppressor in particular, in colon carcinoma and lung adenocarcinoma cells [126,127]. The heterotrimeric α4b2 nAChR, which binds ACh and nicotine with higher affinity than α7 nAChR, stimulates the release of GABA. This higher affinity for nicotine is thought to cause the long-term inactivation (or desensitization) of the heteromeric nAChR that has been observed after chronic exposition to nicotine such as smokers. By contrast the sensibility of α7 nAChr remains unchanged, its expression is up-regulated by nicotine and its biological effect is increased [
The effect induced by nicotine may be different in various cell types, differentiated or “totipotent”, and having diverse metabolic properties to convert nicotine in its metabolite such as cotinine which is more toxic. Moreover, these effects may be related to different factors such as: 1) length of exposure (short versus chronic); 2) concentration (high or low); 3) anatomical distribution and expression of receptor subtypes. Finally, the α7 nAChR effects could be modulated by the secreted mammalian LY-6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) that has been recently identified by an endogenous ligand for α7 nAChR which exerts protective effect [
To date different approaches have been done targeting nAChR in cancer therapy in particular tobacco-related carcinogenesis. In this case, the challenge is to develop compound capable of inhibiting α7 nAChR or potentiating α4b2 nAChR to counterbalance the deleterious effects of chronic nicotine (for review [
The authors gratefully acknowledge grant support from Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer, GEFLUC and ESPOIR. B. Ducarouge and M. Pelissier-Rota are the recipient of a fellowship from the Ministère de la Recherche et de l’Enseignement Supérieur. We thank Pierre-Emmanuel Buyse for his technical support.