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Methotrexate Modulates Tight Junctions Through NF-κB, MEK, and JNK Pathways

Beutheu Youmba, Stéphanie*; Belmonte, Liliana*; Galas, Ludovic; Boukhettala, Nabile*; Bôle-Feysot, Christine*; Déchelotte, Pierre*; Coëffier, Moïse*

Journal of Pediatric Gastroenterology & Nutrition: April 2012 - Volume 54 - Issue 4 - p 463–470
doi: 10.1097/MPG.0b013e318247240d
Original Articles: Gastroenterology

Objectives: Chemotherapy often induces intestinal mucositis, which is associated with an increase in intestinal permeability; however, underlying mechanisms remain incompletely understood. Thus, we aimed to study the regulation of 3 tight junction (TJ) proteins, claudin-1, occludin, and zonula occludens-1, after anticancer treatment.

Methods: Methotrexate (MTX) was subcutaneously injected for 3 consecutive days in Sprague-Dawley rats to induce intestinal mucositis and was applied on Caco-2 cell monolayers. TJ protein expression and cellular distribution were studied by Western blot and microscopy, respectively. In Caco-2 cells, the paracellular permeability was evaluated by both transepithelial electrical resistance and flux of fluorescein isothiocyanate-dextran marker. Cytokine production and signaling pathways were also assessed.

Results: In MTX-treated rats, the cellular distribution of the 3 TJ proteins was altered and claudin-1 and occludin expression was reduced during the acute phase of mucositis compared with controls. During the recovery phase, these parameters were restored. In vitro, MTX treatment led to an increase in proinflammatory cytokine production at the apical side but did not affect Caco-2 cell apoptosis and necrosis. Increase in paracellular permeability was associated with altered occludin and zonula occludens-1 expression and cellular distribution. All of these alterations were prevented by MEK1 and 2, JNK, and NF-κB inhibitors.

Conclusions: MTX treatment induced an increase in intestinal permeability partially related to alteration of TJs protein expression and cellular distribution that may be mediated by MAPK and NF-κB pathways. These are potential targets to limit the adverse effects of chemotherapy.

*INSERM U1073, Institute for Biomedical Research, IFRMP23, Rouen University, Rouen

PRIMACEN, Cell Imaging Platform of Normandy, IFRMP23, Rouen University, Mont Saint Aignan, France.

Address correspondence and reprint requests to Moïse Coëffier, INSERM U1073, 22 Boulevard Gambetta 76183 Rouen Cedex 1, France (e-mail:

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Received 28 June, 2011

Accepted 4 October, 2011

The study was supported by the University of Rouen and by the Région Haute-Normandie through the GRR IFRMP23/Dynacell program.

The authors report no conflicts of interest.

Copyright 2012 by ESPGHAN and NASPGHAN