Cancer Chemotherapy and Pharmacology

, Volume 66, Issue 6, pp 1031–1038 | Cite as

Zonula Occludens-1 alterations and enhanced intestinal permeability in methotrexate-treated rats

  • Kazuma Hamada
  • Yoshihisa Shitara
  • Shuichi Sekine
  • Toshiharu HorieEmail author
Original Article



The molecular mechanisms that underlie the methotrexate (MTX)-mediated disruption of intestinal barrier function have not been fully characterized. Epithelial barrier function is determined in large part by a multiprotein complex located at the most apical part of the lateral membrane, which is referred to as a tight junction (TJ). In the present study, we examined the alteration of zonula occludens-1 (ZO-1), which is a scaffolding protein that plays a pivotal role in the formation of TJs, to identify an additional molecular mechanism for epithelial barrier dysfunction.


Male Wistar rats were administered MTX (15 mg kg−1) orally once daily for 3–5 days. Intestinal mucosal permeability was determined using the in vitro everted intestinal sac technique. Mucosal inflammation was assessed by myeloperoxidase activity and production of reactive oxygen species. Altered expression, tyrosine phosphorylation, and localization of ZO-1 were evaluated by RT–PCR, Western blotting, immunoprecipitation, and immunohistochemistry.


A barrier function study revealed increased intestinal permeability in rats treated with MTX for 4 days, as indicated by enhanced fluorescein isothiocyanate-dextran flux. In addition, mucosal inflammation was linked to enhanced intestinal permeability. Quantitative analysis of ZO-1 expression showed the absence of significant differences in MTX-treated rats, whereas tyrosine dephosphorylation of ZO-1 was observed. Moreover, we also detected an obvious reduction of ZO-1 immunostaining along the apical membrane of intestinal villi.


These results indicate that, in MTX-treated rats, ZO-1 alterations may contribute to disturbance of the TJ barrier, which leads to enhanced intestinal permeability.


Intestinal permeability ZO-1 Methotrexate Tight junction Reactive oxygen species 



This work was supported in part by the Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment), MEXT, Japan.


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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Kazuma Hamada
    • 1
  • Yoshihisa Shitara
    • 1
  • Shuichi Sekine
    • 1
  • Toshiharu Horie
    • 1
    Email author
  1. 1.Department of Biopharmaceutics, Graduate School of Pharmaceutical SciencesChiba UniversityChuo-kuJapan

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