Radotinib attenuates TGFβ -mediated pulmonary fibrosis in vitro and in vivo: exploring the potential of drug repurposing

Baek et al. BMC Pharmacology and Toxicology https://doi.org/10.1186/s40360-022-00634-x (2022) 23:93 Open Access RESEARCH Radotinib attenuates TGFβ ‑mediated pulmonary fibrosis in vitro and in vivo: exploring the potential of drug repurposing Suji Baek1†, Seung Hae Kwon2†, Joo Yeong Jeon2, Gong Yeal Lee3, Hyun Soo Ju3, Hyo Jung Yun3, Dae Jin Cho3, Kang Pa Lee1* and Myung Hee Nam2* Abstract Background: Tyrosine kinase (TK) plays a crucial role in the pathogenesis of idiopathic pulmonary fibrosis. Here, we aimed to investigate whether radotinib (Rb) could inhibit pulmonary fibrosis by inhibiting TK in vitro and in vivo. Methods: The antifibrotic effects of Rb in transforming growth factor-β (TGF-β)1-stimulated A549 cells were determined using real-time polymerase chain reaction, western blotting, and immunocytochemistry assays. Rb inhibition of bleomycin-induced lung fibrosis in Sprague Dawley (SD) rats was determined by histopathological andimmunohistochemical analyses. Rb-interfering metabolites were analyzed using LC-MS/MS. Results: Rb concentrations of up to 1000 nM did not affect the viability of A549 cells, but Rb (30 nM) significantly reduced expression of TGF-β1 (10 ng/mL)-induced ECM factors, such as Snail, Twist, and F-actin. Rb also regulated TGF-β1-overexpressed signal cascades, such as fibronectin and α-smooth muscle actin. Furthermore, Rb attenuated the phosphorylation of Smad2 and phosphorylation of kinases, such as, extracellular signal-regulated kinase, and protein kinase B. In the inhibitory test against bleomycin (5 mg/kg)-induced lung fibrosis, the Rb (30 mg/kg/daily)-treated group showed a half-pulmonary fibrosis region compared to the positive control group. In addition, Rb significantly reduced collagen type I and fibronectin expression in the bleomycin-induced fibrotic region of SD rats. Further, the identified metabolite pantothenic acid was not altered by Rb. Conclusion: Taken together, these results indicate that Rb inhibits TGF-β1-induced pulmonary fibrosis both in vitro and in vivo. These findings suggest that Rb may be an effective treatment for pulmonary fibrosis-related disorders and idiopathic pulmonary fibrosis. Keywords: Radotinib, Tyrosine kinase, Lung fibrosis, Extracellular signal-regulated kinase, TGF-β1 † Suji Baek and Seung Hae Kwon are co-first authors. † Suji Baek and Seung Hae Kwon contributed equally to this work. *Correspondence: ; 1 Research and Development Center, UMUST R&D Corporation, 84, Madeul‑ro 13‑gil, Dobong‑gu, 01411 Seoul, Republic of Korea 2 Seoul Center, Korean Basic Science Institute, 02841 Seoul, Republic of Korea Full list of author information is available at the end of the article Background Diffuse parenchymal lung disease (DPLD) is an intractable respiratory disease that is difficult to treat and is characterized by a change in the shape of the lungs and a decrease in oxygen supply to the pulmonary blood vessels [1, 2]. Cases where the cause of DPLD is unknown are classified as idiopathic pulmonary fibrosis (IPF) [3]. IPF, which has the worst prognosis among DPLD, has an incidence of 10 per 100,000 people, and an average survival of only 3 to 5 years [4, 5]. Current knowledge in © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Baek et al. BMC Pharmacology and Toxicology (2022) 23:93 the field suggests that the increased incidence of IPF is related to the long-term effects of COVID-19 infection and severe respiratory syndrome [6]. Fibrosis is caused by the accumulation of extracellular matrix (collagen and fibronectin) in the lung tissue damage during the inflammation healing process, causing lung dysfunction [7]. However, the etiological molecular mechanisms underlying IPF have not been clearly elucidated [8]. Recent studies and clinical trials have suggested that transforming growth factor-β (TGF-β)-1 and tyrosine kinase (TK) signaling are both potential IPF treatments [9, 10]. TKs, which includes platelet-derived growth factor receptor (PDGFR)-α, PDGFR-β, vascular endothelial growth factor receptor (VEGFR), and epidermal growth factor, are membrane receptors that activate intracellular signaling pathways when growth factors bind to their extracellular domains [11, 12]. TK also induces fibrosis by activating Smad, mitogen-activated protein kinase (MAPK), and protein kinase B (AKT) [13]. Therefore, subsignal modulation of TK could be a key strategy in IPF treatment. Nintedanib and pirfenidone, both FDA-approved TK targeting-drugs, are the standard treatment for IPF, which have been reported to reduce lung function decline by 50% [14]. Although these drugs prevent pulmonary fibrosis by inhibiting TGF-β and TNF-α, there is a continuous demand for research for new therapeutic agents for IPF. Radotinib (Rb) is a TK that inhibits breakpoint cluster region protein-v-abelson murine leukemia viral oncogene homolog 1 and PDGFR and has been approved by the Korea Ministry of Food and Drug Safety (republic of Korea) approval and is being used as a treatment for acute leukemia [15]. However, studies on the efficacy of Rb against IPF have not yet been conducted. Therefore, this study not only confirmed the potential of Rb for the prevention and treatment of pulmonary fibrosis, but also confirmed the possibility of drug re-purposing by changing the use of an approved drug. In this study, we explored the antifibrosis effect of Rb in TGF-β1-stimulated A549 cells and in a bleomycin-induced rat model to provide basic data for the development of therapeutic agents through the significant inhibition of IPF. Methods Cell culture of A549 cells A549 cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were cultured in Roswell Park Memorial Institute Medium 1640 media containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 ± 2 °C and 5% CO2. Page 2 of 9 Cell viability assay A549 cells were seeded in 96-well plates (1 × 104 cells/ well) and (...truncated)