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Original Research

Simvastatin Increases the Ability of Roflumilast N-oxide to Inhibit Cigarette Smoke-Induced Epithelial to Mesenchymal Transition in Well-differentiated Human Bronchial Epithelial Cells in vitro

, , , , , , & show all
Pages 327-338 | Published online: 10 Sep 2014
 

Abstract

Background: Cigarette smoking contributes to epithelial-mesenchymal transition (EMT) in COPD small bronchi as part of the lung remodeling process. We recently observed that roflumilast N-oxide (RNO), the active metabolite of the PDE4 inhibitor roflumilast, prevents cigarette smoke-induced EMT in differentiated human bronchial epithelial cells. Further, statins were shown to protect renal and alveolar epithelial cells from EMT. Objectives: To analyze how RNO and simvastatin (SIM) interact on CSE-induced EMT in well-differentiated human bronchial epithelial cells (WD-HBEC) from small bronchi in vitro. Methods: WD-HBEC were stimulated with CSE (2.5%). The mesenchymal markers vimentin, collagen type I and α-SMA, the epithelial markers E-cadherin and ZO-1, as well as β-catenin were quantified by real time quantitative PCR or Western blotting. Intracellular reactive oxygen species (ROS) were measured using the H2DCF-DA probe. GTP-Rac1 and pAkt were evaluated by Western blotting. Results: The combination of RNO at 2 nM and SIM at 100 nM was (over) additive to reverse CSE-induced EMT. CSE-induced EMT was partially mediated by the generation of ROS and the activation of the PI3K/Akt/β-catenin pathway. Both RNO at 2 nM and SIM at 100 nM partially abrogated this pathway, and its combination almost abolished ROS/ PI3K/Akt/β-catenin signaling and therefore EMT. Conclusions: The PDE4 inhibitor roflumilast N-oxide acts (over)additively with simvastatin to prevent CSE-induced EMT in WD-HBEC in vitro.

Acknowledgements

Both authors Milara and Peiró contributed equally to this work.

Declaration of Interest Statement

JC received a research grant from Nycomed. HT and JA are full-time employees of Takeda. The other authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

This work was supported by grants SAF2011-26443 (JC), FIS CP11/00293(JM), CIBERES (CB06/06/0027), ADE10/00020 (Spanish government), ACIF/2010/114 (TP) and research grants from Regional Government (Prometeo II/2013/014 (JC, JM), ‘Generalitat Valenciana’. Support from the CENIT programme (Spanish Government) was obtained.

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