Metformin Attenuates EDFR-TKI-Induced Exacerbation Of Pulmonary Fibrosis Through Inhibition Of TGF-β Signaling Pathway And Emt Reversal

Introduction:Lung cancer is the leading cause of cancer-related death worldwide, with an increasing incidence and mortality rates. Eighty-five per cent of tumours are non-small cell lung cancer(NSCLC). Gefitinib(Iressa) and erlotinib(Tarceva), which are reversible small-molecule epidermal growth factor receptor(EGFR)-tyrosine kinase inhibitors(TKIs), have shown excellent initial clinical responses in NSCLC patients with EGFR-activating mutations, and have been recommended as the standard first-line therapy in these patients. However, despite dramatic therapeutic efficacy, these drugs might induce interstitial lung disease(ILD), which is not common but lethal side-effect that seriously restricts the intial clinical efficacy of these agents. Thus, the potential side effect restricts the therapeutic use of these drugs. Innovative treatment approaches are urgently required to overcome this lethal side effect of EGFR-TKIs.Molecular mechanisms underlying TKI-induced interstitial pneumonia(pulmonary fibrosis) are still not fully understood. A key mediator in the progression of pulmonary fibrosis is transforming growth factor(TGF)-β1. There is high expression of TGF-β1 in tissue samples from both animal and patients with interstitial lung fibrosis, while blocking TGF-β signaling pathway effectively improves pulmonary fibrosis in animal models. Also the progressive pulmonary fibrotic reaction was involved in the epithelial-dependent fibroblast-activated process, known as epithelial–mesenchymal transition(EMT). IL-6 is a downstream molecule of TGF-β, has been demonstrated to contribute to the development or progression of acute interstitial pneumonia. TGF-β/IL-6 was a master promotor of EMT in normal mammalian epithelial cells and has been further proven to initiate and maintain EMT during the organ fibrogenesis and tumor metastasis. Therefore, inhibition of TGF-β signaling pathway may therefore have important theoretical and therapeutic potential in the clinical treatment of pulmonary fibrosis.Metformin, a well-established and widely prescribed oral hypoglycemic drug, has been proven sensitized to EGFR-TKI-resistant human lung cancer cells by overcoming EMT and inhibition of TGF-β/IL-6 signaling pathway in our previously study. Therefore, we conclude that metformin could attneuate EGFR-TKI-induced pulmonary fibrosis.Objectives: Acute interstitial pneumonia is one of lethal side effects of epidermal growth factor receptor(EGFR)-tyrosine kinase inhibitors(TKIs) treatment. Therefore, it is necessary and urgently to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential treatment strategies to attenuate it. Studies have shown that metformin could inhibited the progression of EMT and fibrosis in oher organs. This study aims to investigate the effect of metformin on attenuating EGFR-TKI-induced exacerbation of pulmonary fibrosis in vitro and in vivo by inhibition of TGF-β signaling pathway and EMT reversal.Methods: The effect of metformin on EGFR-TKI-induced pulmonary fibrosis was examined in vitro and in vivo by using MTT, Ki-67 incorporation assay, flow cytometry analysis, immunostaining, immunohistochemistry, Western blot analysis, and bleomycininduced pulmonary fibrosis rat model.Results: In the current study, we found, in human lung fibroblasts HFL-1 cells, TGF-β inserted or conditioned medium from TKI-treated(gefitinib) lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells could induce significant fibrosis, as evidenced by increased expression of Collegen1A1 and α-actin. Importantly, metformin inhibited expression of fibrosis markers. Moreover, metformin decreased TGF-β signaling activation, as evidenced by downregulation of expression of pSmad2 and pSmad3. In vivo, oral administration of gefitinib exacerbated fibrosis in lung tissues from bleomycin-induced pulmonary fibrosis rat, as demonstrated by HE staining and Masson staining. Significantly, oral coadministration of metformin suppressed gefitinib-induced exacerbation of bleomycin-induced pulmonary fibrosis.Conclusions:Taken together, we have demonstrated for the first time that metformin could attenuate lung fibrosis both in HFL-1 cells exacerbated by TGF-β or in bleomycininduced rat, thus providing evidence that metformin may be combined with EGFR-TKI to treat patients with NSCLC.