Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-β signaling pathway

// Li Li 1, * , Wenting Huang 1, * , Kunlin Li 1, * , Kejun Zhang 2 , Caiyu Lin 1 , Rui Han 1 , Conghua Lu 1 , Yubo Wang 1 , Hengyi Chen 1 , Fenfen Sun 1 , Yong He 1 1 Department of Respiratory Disease, Daping Hospital, Third Military Medical University, Chongqing, China 2 Department of Clinical Labratory, Daping Hospital, Third Military Medical University, Chongqing, China * These authors have contributed equally to this work Correspondence to: Yong He, e-mail: heyong@dphospital.tmmu.edu.cn Keywords: metformin, EGFR-TKI, pulmonary fibrosis, TGF-β Received: June 13, 2015      Accepted: October 05, 2015      Published: October 20, 2015 ABSTRACT Interstitial lung disease (ILD) is a serious side-effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment. Therefore, it is necessary to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential approaches to attenuate it. Metformin is a well-established and widely prescribed oral hypoglycemic drug, and has gained attention for its potential anticancer effects. Recent reports have also demonstrated its role in inhibiting epithelial-mesenchymal transition and fibrosis. However, it is unknown whether metformin attenuates EGFR-TKI-induced pulmonary fibrosis. The effect of metformin on EGFR-TKI-induced exacerbation of pulmonary fibrosis was examined in vitro and in vivo using MTT, Ki67 incorporation assay, flow cytometry, immunostaining, Western blot analysis, and a bleomycin-induced pulmonary fibrosis rat model. We found that in lung HFL-1 fibroblast cells, TGF-β or conditioned medium from TKI-treated lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells, induced significant fibrosis, as shown by increased expression of Collegen1a1 and α-actin, while metformin inhibited expression of fibrosis markers. Moreover, metformin decreased activation of TGF-β signaling as shown by decreased expression of pSMAD2 and pSMAD3. In vivo , oral administration of gefitinib exacerbated bleomycin-induced pulmonary fibrosis in rats, as demonstrated by HE staining and Masson staining. Significantly, oral co-administration of metformin suppressed exacerbation of bleomycin-induced pulmonary fibrosis by gefitinib. We have shown that metformin attenuates gefitinib-induced exacerbation of TGF-β or bleomycin-induced pulmonary fibrosis. These observations indicate metformin may be combined with EGFR-TKI to treat NSCLC patients.