Regorafenib exerts an inhibitory effect on the proliferation of human lung fibroblasts by reducing the production of several cytokines

Regorafenib exerts an inhibitory effect on the proliferation of human lung fibroblasts by reducing the production of several cytokines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Regorafenib exerts an inhibitory effect on the proliferation of human lung fibroblasts by reducing the production of several cytokines Natsuki Nakamura, Rie Tabata, Chiharu Tabata This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3929682/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Pulmonary fibrosis is a disease that leads to respiratory failure and death. There has been little progress in therapeutic strategies for pulmonary fibrosis. There have been several reports on the cytokines associated with pulmonary fibrosis, including IL-6 and TGF-β 1 . Angiogenesis is one of the most important phenomena in the pathogenesis of PF. Previously, we reported the preventive effects of thalidomide against pulmonary fibrosis via the inhibition of neovascularization by angiogenic factors such as VEGF. Regorafenib is a multikinase inhibitor, which inhibits tyrosine kinase receptors such as VEGFR1-3 and TIE2. In the clinical setting, regorafenib has been widely used for anti-cancer therapy for metastatic colorectal cancer. In this study, we examined the preventive effects of regorafenib against pulmonary fibrosis. Methods We investigated whether regorafenib had an inhibitory effect on the proliferation, viability, and production of several cytokines in lung fibroblasts. Results We demonstrated an inhibitory effect of regorafenib on the proliferation and viability of lung fibroblasts. Moreover, regorafenib reduced the production of several cytokines associated with the pathogenesis of pulmonary fibrosis, including IL-6, VEGF and TGF- β 1 , and collagen synthesis from lung fibroblasts. Conclusions These data suggest that regorafenib may have potential clinical applications in the prevention of pulmonary fibrosis. regorafenib cell proliferation cell viability cytokines pulmonary fibrosis Figures Figure 1 Figure 2 INTRODUCTION Pulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation of lung fibroblasts and varying degrees of inflammation and fibrosis. It frequently occurs with collagen diseases, radiotherapy to the thoracic region, and drugs, such as anticancer agents or antibiotics. In idiopathic pulmonary fibrosis (IPF), the most common type of interstitial lung disease, the pulmonary function is progressively compromised, resulting in a high mortality rate. Although pirfenidone and nintedanib have been used for IPF, there are currently few effective treatments for IPF, leading to high incidence of death (> 50% 5-year mortality rate) due to eventual respiratory failure [ 1 – 6 ]. There have been previous reports on various cytokines being associated with the pathogenesis of pulmonary fibrosis, including IL-6 and TGF-β 1 [ 7 , 8 ]. Angiogenesis is one of the most important phenomena in several mechanisms of pulmonary fibrosis [ 9 , 10 ]. Previously, we reported the preventive effects of thalidomide against pulmonary fibrosis via inhibition of neovascularization by angiogenic factors such as VEGF [ 11 ]. VEGF plays an important role in angiogenesis, and VEGF-A binds to three tyrosine kinase receptors, namely VEGFR1-3. Regorafenib, a multi-kinase inhibitor, has been widely used as an anti-cancer drug for metastatic colorectal cancer [ 12 ], metastatic gastrointestinal stromal tumors (GIST) [ 13 ], and advanced hepatocellular carcinoma [ 14 ]. Regorafenib blocks several kinases, including VEGFR1-3, TIE2, c-Kit, and PDGFR [ 15 ]. In this study, we focused on the effects of regorafenib on lung fibroblasts to investigate whether regorafenib has preventive effects against pulmonary fibrosis. MATERIALS and METHODS Cell culture WI38VA-13, a human lung fibroblast cell line transformed by SV40, was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Chemical Co., St Louis, MO) supplemented with 10% heat-inactivated fetal calf serum. Cells were cultured with antibiotics in a humidified incubator at 37°C under 5% CO 2 . Regorafenib (MedChemExpress, NJ, USA) was diluted in DMSO and added to the growth medium to obtain a final DMSO solvent concentration < 0.01% (v/v). As a control, cells were treated with the same concentration of DMSO; all cultures in this study contained the same final concentration of DMSO. In preliminary experiments, the final concentration of DMSO had no gross effect on the WI38VA13 cells. Cell proliferation assay The cell proliferation assay was performed as previously described [ 16 , 17 ]. The cells were cultured in 96-well flat-bottomed culture plates for 72 h with or without regorafenib (1, 5, and 10 µ µM). A Cell Counting Kit-8 (Dojindo, Tokyo, Japan) was used to characterize cell growth. Cell viability assay Cells were seeded in 10 cm dishes with or without regorafenib (10 µM) for 96 h, and cell viability was measured by trypan blue staining using an Automated Cell Counter, Countess II FL (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. Measurement of IL-6, TGF- β 1 , VEGF and collagen type 1 The concentrations of human IL-6, TGF- β 1 , VEGF, and collagen type 1 in the culture supernatants with or without regorafenib (10 µM) for 72 h were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Oxford, UK), according to the manufacturer’s instructions. Next, we measured the concentration of human collagen type 1 in the culture supernatants with or without regorafenib (10 µM) for 72 h using an enzyme-linked immunosorbent assay (ELISA) kit (ACEL, Kanagawa, Japan) according to the manufacturer’s instructions. Statistical analysis The results are presented as the mean ± SD. The statistical analysis was performed using the Bonferroni/Dunn multiple comparison test. RESULTS Inhibitory effect of regorafenib on the proliferation and cell viability of lung fibroblasts We first investigated the in vitro effects of regorafenib on growth of human lung fibroblasts. The addition of regorafenib suppressed the proliferation of WI38 VA13 cells in a dose-dependent manner (Fig. 1 A). The maximum inhibitory effect was observed at the concentration of 10 µM regorafenib (63% decrease [p < 0.01]). Lower concentrations (1 µM or 5 µM) of regorafenib also had a minor preventive effect in comparison to 10 µM (17% decrease [p < 0.05] and 48% decrease [p < 0.01], respectively). The final concentration of DMSO (< 0.01% (v/v)) had no gross effect on WI38 VA13 cells (data not shown). We next demonstrated that the concentration of 10 µM regorafenib had an inhibitory effect on the cell viability of WI38 VA13 cells (with DMSO only: 95.0% and with 10 µM regorafenib: 57.3%, respectively) (Fig. 1 B). Effect of regorafenib on the production of IL-6, VEGF, TGF- β 1 and collagen synthesis of lung fibroblasts We performed experiments to study the effects of regorafenib on IL-6, TGF- β 1 and VEGF production in lung fibroblasts. The concentration of IL-6, TGF- β 1 and VEGF in the culture supernatant was decreased by the addition of 10 µM regorafenib (81% decrease [p < 0.01], 48% decrease [p < 0.01] and 74% decrease [p < 0.01], respectively) (Fig. 2 A, 2 B and 2 C), and furthermore, 10 µM regorafenib reduced the production of collagen type 1 (100% decrease [p < 0.01]) (Fig. 2 D). However, regorafenib did not affect the production of Ang-1 (data not shown). DISCUSSION In this study, we examined the effect of regorafenib on pulmonary fibrosis. In our in vitro study, we found that regorafenib inhibited lung fibroblast proliferation in a dose-dependent manner. Next, we demonstrated that regorafenib reduced cell viability. In addition, we showed that regorafenib inhibited the production of several cytokines, such as IL-6, TGF- β 1 and VEGF, and collagen synthesis in lung fibroblasts. Several factors have been reported to be associated with pulmonary fibrosis, including IL-1, IL-6, TNF-α, platelet-derived growth factor, VEGF, and TGF-β 1 [ 18 – 20 ]. We previously showed that both IL-6 and TGF-β 1 played important roles in pulmonary fibrosis [ 16 , 17 ]. Specifically, we reported that human lung fibroblasts proliferated by IL-6 in a dose-dependent manner [ 16 ], and the inhibition of IL-6 production in lung fibroblasts was significantly associated with the prevention of pulmonary fibrosis [ 16 , 17 ]. In addition, Saito et al. showed that IL-6 may play an important role in the pathogenesis of bleomycin–induced lung injury and fibrosis [ 21 ]. Therefore, IL-6 is one of the major cytokines involved in the pathogenesis of pulmonary fibrosis. In this study, we showed that regorafenib inhibited the production of IL-6 in lung fibroblasts. TGF-β 1 is one of a major cytokines associated with fibrosis [ 22 ]. Yoshida et al. reported the overexpression of interleukin 6 and TGF-β 1 in fibrotic lesions [ 23 ]. Previously, we demonstrated that inhibition of TGF-β 1 was associated with the prevention of pulmonary fibrosis [ 17 ]. In the present study, we also showed that regorafenib inhibited TGF-β 1 production in lung fibroblasts. Collagen synthesis plays an essential role in pulmonary development. Lung fibroblasts are the major collagen-producing cells in fibrotic lung lesions. It has been reported that serological biomarkers reflecting type I and III collagen turnover are elevated in patients with progressive disease in comparison to those with stable IPF [ 24 ]. In this study, we demonstrated that regorafenib reduces type I collagen production in lung fibroblasts. The association between pulmonary fibrosis and neovascularization has been demonstrated in the lung tissues of patients with IPF [ 25 ] and in a rat bleomycin-induced pulmonary fibrosis model [ 26 ]. VEGF and Ang-1 are the major regulators of angiogenesis [ 27 – 29 ]. VEGF plays an important role in endothelial cell proliferation, vascular permeability, and angiogenesis in several inflammatory lesions [ 27 ]. The expression of VEGF is upregulated by various stimuli, such as low oxygen tension and several cytokines. Ang-1 is also an important regulator of blood vessel growth, maturation, and function. Ang-1 promotes angiogenesis, induces vascular maturation, and decreases vascular permeability [ 28 , 29 ]. Previously, we reported the preventive effects of thalidomide against pulmonary fibrosis via inhibition of neovascularization by angiogenic factors such as VEGF [ 11 ]. In the present study, regorafenib had no effect on Ang-1 production by lung fibroblasts. However, regorafenib inhibited the production of VEGF in lung fibroblasts. In summary, we found that regorafenib prevents lung fibroblast proliferation and reduces cell viability. We also showed that regorafenib inhibited the production of three major cytokines in fibroblasts associated with pulmonary fibrosis, including IL-6 (pro-inflammatory), TGF-β 1 (fibrotic), and VEGF (angiogenic), suggesting the major mechanisms for the inhibitory effect of regorafenib on the proliferation of lung fibroblasts. Although the precise cellular mechanism of regorafenib in lung fibroblasts has not been fully investigated and needs to be studied further for clinical use, our data may lead to the development of novel strategies for the prevention and treatment of pulmonary fibrosis. Abbreviations IPF idiopathic pulmonary fibrosis Declarations Author Contribution Authors' contributions: C.T .(Chiharu Tabata) and R.T. (Rie Tabata) designed the study. N.N. (Natsuki Nakamura) and C.T. performed the research. N.N. and C.T. collected data. C.T. and R.T .analyzed and interpreted the data. C.T .performed statistical analyses. C.T. and R.T. drafted the manuscript. N.N., C.T., and R.T. have read and approved the final manuscript. Acknowledgements: This work was supported by grants from JSPS KAKENHI (Grant Numbers JP19K08633 and JP23K07664. Declaration of Interest Statement: The authors declare no conflicts of interest in association with the present study. References Coultas DB, Zumwalt RE, Black WC, Sobonya RE. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med. 1994;150:967–972. American Thoracic Society/European Respiratory Society. International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med. 2002;165:277–304. Gross TJ, Hunninghake GW. Idiopathic pulmonary fibrosis. N Engl J Med. 2001;345: 517–525. Mason RJ, Schwarz MI, Hunninghake GW, Musson RA. NHLBI Workshop Summary. Pharmacological therapy for idiopathic pulmonary fibrosis. Past, present, and future. Am J Respir Crit Care Med. 1999;160:1771–1777. Richeldi L, Collard HR, Jones MG. Idiopathic pulmonary fibrosis. Lancet. 2017;389(10082):1941–1952. Lederer DJ, MartinezFJ. Idiopathic pulmonary fibrosis. N Engl J Med. 2018;378:1811–1823. Dawson RE, Jenkins BJ, Saad MI. IL-6 family cytokines in respiratory health and disease. Cytokine. 2021;143:155520. Wolters PJ, Collard HR, Jones KD. Pathogenesis of idiopathic pulmonary fibrosis. Annu Rev Pathol. 2014;9:157–79. TE King Jr, A Pardo, M Selman. Idiopathic pulmonary fibrosis. Lancet. 2011;378:1949–1961. Simler NR, Brenchley PE, Horrocks AW, Greaves SM, Hasleton PS, Egan JJ. Angiogenic cytokines in patients with idiopathic interstitial pneumonia. Thorax. 2004;59(7): 581–585. Tabata C, Tabata R, Kadokawa Y, Hisamori S, Takahashi M, Mishima M, Nakano T, Kubo H. Thalidomide prevents bleomycin-induced pulmonary fibrosis in mice. J Immunol. 2007;179:708–714. Grothey A, Van Cutsem E, Sobrero A, Siena S, Falcone A, Ychou M, Humblet Y, Bouché O, Mineur L, Barone C, Adenis A, Tabernero J, Yoshino T, Lenz HJ, Goldberg RM, Sargent DJ, Cihon F, Cupit L, Wagner A. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): An international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2013;381:303–312. von Mehren M, Joensuu H. Gastrointestinal Stromal Tumors. J Clin Oncol. 2018;36(2):136–143. Forner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet. 2018;31:1301–1314. Wilhelm SM, Dumas J, Adnane L, Lynch M, Carter CA, Schütz G, Thierauch KH, Zopf D. Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int J Cancer. 2011;129:245–255. Tabata C, Kubo H, Tabata R, Wada M, Sakuma K, Ichikawa M, Fujita S, Mio T, Mishima M. All-trans retinoic acid modulates radiation-induced proliferation of lung fibroblasts via IL-6/IL-6R system. Am J Physiol Lung Cell Mol Physiol. 2006;290:597–606. Tabata C, Kadokawa Y, Tabata R, Takahashi M, Okoshi K, Sakai Y, Mishima M, Kubo H. All-trans-Retinoic Acid Prevents Radiation- or Bleomycin-induced Pulmonary Fibrosis. Am J Respir Crit Care Med. 2006;174:1352–1360. Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest. 1997;100:768–776. Kolb M, Margetts PJ, Anthony DC, Pitossi F, Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 2001;107:1529–1536. Miyazaki Y, Araki K, Vesin C, Garcia I, Kapanci Y, Whitsett JA, Piguet PF, Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive pulmonary fibrosis. J Clin Invest. 1995;96:250–259. Saito F, Tasaka S, Inoue K, Miyamoto K, Nakano Y, Ogawa Y, Yamada W, Shiraishi Y, Hasegawa N, Fujishima S, Takano H, Ishizaka A. Role of interleukin-6 in bleomycin-induced lung inflammatory changes in mice. Am J Respir Cell Mol Biol. 2008;38(5):566–71. Broekelmann, TJ, Limper AH, Colby TV, McDonald JA. Transforming growth facter-1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA. 1991;88: 6642–6646. Yoshida M, Sakuma J, Hayashi S, Abe K, Saito I, Harada S, Sakatani M, Yamamoto S, Matsumoto N, Kaneda Y. A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor beta 1, or platelet-derived growth factor B gene. Proc Natl Acad Sci U S A. 1995;10:9570–4. Jenkins RG, Simpson JK, Saini G, Bentley JH, Russell AM, Braybrooke R, Molyneaux PL, McKeever TM, Wells AU, Flynn A, Hubbard RB, Leeming DJ, Marshall RP, Karsdal MA, Lukey PT, Maher TM. Longitudinal change in collagen degradation biomarkers in idiopathic pulmonary fibrosis: an analysis from the prospective, multicentre PROFILE study. Lancet Respir Med. 2015;3(6):462–72. Turner-Warwick M. PRECAPILLARY SYSTEMIC-PULMONARY ANASTOMOSES. Thorax. 1963;18:225–237. Peao, MN, Aguas AP, de Sa CM, Grande NR. Neoformation of blood vessels in association with rat lung fibrosis induced by bleomycin. Anat Rec. 1994;238:57–67. Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003;9:685–693. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000;6:460–463. Abdel-Malak NA, Mofarrahi M, Mayaki D, Khachigian LM, Hussain SN. Early growth response-1 regulates angiopoietin-1-induced endothelial cell proliferation, migration, and differentiation. Arterioscler Thromb Vasc Biol. 2009;29: 209–216. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3929682","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271482989,"identity":"96c42962-409b-4cba-abf3-e80bd25e1f44","order_by":0,"name":"Natsuki Nakamura","email":"","orcid":"","institution":"Hyogo Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Natsuki","middleName":"","lastName":"Nakamura","suffix":""},{"id":271482990,"identity":"934b84d6-f42e-46c7-af62-cc6d13eb6b2d","order_by":1,"name":"Rie Tabata","email":"","orcid":"","institution":"Saiseikai-Noe Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rie","middleName":"","lastName":"Tabata","suffix":""},{"id":271482991,"identity":"3961de49-986d-4fda-90ac-94e5fbfaf2b5","order_by":2,"name":"Chiharu Tabata","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYHACNhAhZwDhMINJAwIagFoSGIxJ15K4AVkLXsA/v/nZg58/7NK3SyQfYLpRYc3A336AobgAjxaJY2zmhj0Jybk7Z6QlMOecSWeQOAN05gw8WgzYGMwkeBKYczfcyDH/ndt2mIHhBgODMQ9eLezfJP8k1Kcb3MgxYM79d5hBnrAWHjNpnoTDCRAtDYcZDAhpkTiWU24sk3bccMOZZ0C/HEvnMTyT2IDXL/zNx7c9fGNTLW9wPPkAc06NtZzc8cPHjPGFGAIIJIApoJMY24yJ0sHAfwDOZH5MnJZRMApGwSgYIQAAkVdGWG3IaK8AAAAASUVORK5CYII=","orcid":"","institution":"Hyogo Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chiharu","middleName":"","lastName":"Tabata","suffix":""}],"badges":[],"createdAt":"2024-02-05 03:14:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3929682/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3929682/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50862746,"identity":"d3c1b366-80c2-41a9-a0dd-be96b8d36319","added_by":"auto","created_at":"2024-02-08 14:16:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":714154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of regorafenib on cell proliferation and cell viability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)WI38VA-13 cells were cultured with or without regorafenib (1, 5 and 10 mM) for 72 hours and cell proliferation was assayed. (B) Cells were seeded in 10 cm dishes with or without regorafenib (10 mM) for 96 hours, and cell viability was measured. All results are indicated as the mean ± SD of three separate experiments.\u003c/p\u003e","description":"","filename":"IRTabataCRegFig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3929682/v1/284ff111038846595cd35f3a.jpg"},{"id":50862745,"identity":"c4107372-650e-4ed1-9f3b-225ea4537e09","added_by":"auto","created_at":"2024-02-08 14:16:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":810688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of regorafenib on the production of several cytokines and collagen synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWI38 VA13 cells were cultured in the presence or absence of regorafenib (10 mM) for 72 h, and the supernatants of the cells were collected and examined for IL-6 (A), TGF- b\u003csub\u003e1\u003c/sub\u003e (B), and VEGF (C) concentrations by an ELISA. Next, we measured the concentration of human collagen type 1 (D) in the culture supernatants with or without regorafenib (10 mM) for 72 h using an ELISA. All results are presented as the mean ± SD of three separate experiments.\u0026nbsp;\u003c/p\u003e","description":"","filename":"IRTabataCRegFig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3929682/v1/39f836bad7f0df628f52bb16.jpg"},{"id":51243239,"identity":"e86ec6a0-d6eb-4e5a-acd7-0fb69e940cea","added_by":"auto","created_at":"2024-02-16 18:29:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":376314,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3929682/v1/3becd7a1-2acd-45e4-9add-d235be4c97c7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regorafenib exerts an inhibitory effect on the proliferation of human lung fibroblasts by reducing the production of several cytokines","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation of lung fibroblasts and varying degrees of inflammation and fibrosis. It frequently occurs with collagen diseases, radiotherapy to the thoracic region, and drugs, such as anticancer agents or antibiotics. In idiopathic pulmonary fibrosis (IPF), the most common type of interstitial lung disease, the pulmonary function is progressively compromised, resulting in a high mortality rate. Although pirfenidone and nintedanib have been used for IPF, there are currently few effective treatments for IPF, leading to high incidence of death (\u0026gt;\u0026thinsp;50% 5-year mortality rate) due to eventual respiratory failure [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere have been previous reports on various cytokines being associated with the pathogenesis of pulmonary fibrosis, including IL-6 and TGF-β\u003csub\u003e1\u003c/sub\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Angiogenesis is one of the most important phenomena in several mechanisms of pulmonary fibrosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previously, we reported the preventive effects of thalidomide against pulmonary fibrosis via inhibition of neovascularization by angiogenic factors such as VEGF [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. VEGF plays an important role in angiogenesis, and VEGF-A binds to three tyrosine kinase receptors, namely VEGFR1-3.\u003c/p\u003e \u003cp\u003eRegorafenib, a multi-kinase inhibitor, has been widely used as an anti-cancer drug for metastatic colorectal cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], metastatic gastrointestinal stromal tumors (GIST) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and advanced hepatocellular carcinoma [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Regorafenib blocks several kinases, including VEGFR1-3, TIE2, c-Kit, and PDGFR [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this study, we focused on the effects of regorafenib on lung fibroblasts to investigate whether regorafenib has preventive effects against pulmonary fibrosis.\u003c/p\u003e"},{"header":"MATERIALS and METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eWI38VA-13, a human lung fibroblast cell line transformed by SV40, was cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Sigma Chemical Co., St Louis, MO) supplemented with 10% heat-inactivated fetal calf serum. Cells were cultured with antibiotics in a humidified incubator at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. Regorafenib (MedChemExpress, NJ, USA) was diluted in DMSO and added to the growth medium to obtain a final DMSO solvent concentration\u0026thinsp;\u0026lt;\u0026thinsp;0.01% (v/v). As a control, cells were treated with the same concentration of DMSO; all cultures in this study contained the same final concentration of DMSO. In preliminary experiments, the final concentration of DMSO had no gross effect on the WI38VA13 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eThe cell proliferation assay was performed as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The cells were cultured in 96-well flat-bottomed culture plates for 72 h with or without regorafenib (1, 5, and 10 \u0026micro; \u0026micro;M). A Cell Counting Kit-8 (Dojindo, Tokyo, Japan) was used to characterize cell growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 10 cm dishes with or without regorafenib (10 \u0026micro;M) for 96 h, and cell viability was measured by trypan blue staining using an Automated Cell Counter, Countess II FL (Thermo Fisher Scientific, MA, USA), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of IL-6, TGF- β\u003csub\u003e1\u003c/sub\u003e, VEGF and collagen type 1\u003c/h2\u003e \u003cp\u003eThe concentrations of human IL-6, TGF- β\u003csub\u003e1\u003c/sub\u003e, VEGF, and collagen type 1 in the culture supernatants with or without regorafenib (10 \u0026micro;M) for 72 h were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R\u0026amp;D Systems, Oxford, UK), according to the manufacturer\u0026rsquo;s instructions. Next, we measured the concentration of human collagen type 1 in the culture supernatants with or without regorafenib (10 \u0026micro;M) for 72 h using an enzyme-linked immunosorbent assay (ELISA) kit (ACEL, Kanagawa, Japan) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical analysis was performed using the Bonferroni/Dunn multiple comparison test.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eInhibitory effect of regorafenib on the proliferation and cell viability of lung fibroblasts\u003c/h2\u003e \u003cp\u003eWe first investigated the \u003cem\u003ein vitro\u003c/em\u003e effects of regorafenib on growth of human lung fibroblasts. The addition of regorafenib suppressed the proliferation of WI38 VA13 cells in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The maximum inhibitory effect was observed at the concentration of 10 \u0026micro;M regorafenib (63% decrease [p\u0026thinsp;\u0026lt;\u0026thinsp;0.01]). Lower concentrations (1 \u0026micro;M or 5 \u0026micro;M) of regorafenib also had a minor preventive effect in comparison to 10 \u0026micro;M (17% decrease [p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] and 48% decrease [p\u0026thinsp;\u0026lt;\u0026thinsp;0.01], respectively). The final concentration of DMSO (\u0026lt;\u0026thinsp;0.01% (v/v)) had no gross effect on WI38 VA13 cells (data not shown).\u003c/p\u003e \u003cp\u003eWe next demonstrated that the concentration of 10 \u0026micro;M regorafenib had an inhibitory effect on the cell viability of WI38 VA13 cells (with DMSO only: 95.0% and with 10 \u0026micro;M regorafenib: 57.3%, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of regorafenib on the production of IL-6, VEGF, TGF- β\u003c/b\u003e \u003csub\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand collagen synthesis of lung fibroblasts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe performed experiments to study the effects of regorafenib on IL-6, TGF- β\u003csub\u003e1\u003c/sub\u003e and VEGF production in lung fibroblasts. The concentration of IL-6, TGF- β\u003csub\u003e1\u003c/sub\u003e and VEGF in the culture supernatant was decreased by the addition of 10 \u0026micro;M regorafenib (81% decrease [p\u0026thinsp;\u0026lt;\u0026thinsp;0.01], 48% decrease [p\u0026thinsp;\u0026lt;\u0026thinsp;0.01] and 74% decrease [p\u0026thinsp;\u0026lt;\u0026thinsp;0.01], respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and furthermore, 10 \u0026micro;M regorafenib reduced the production of collagen type 1 (100% decrease [p\u0026thinsp;\u0026lt;\u0026thinsp;0.01]) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, regorafenib did not affect the production of Ang-1 (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we examined the effect of regorafenib on pulmonary fibrosis. In our \u003cem\u003ein vitro\u003c/em\u003e study, we found that regorafenib inhibited lung fibroblast proliferation in a dose-dependent manner. Next, we demonstrated that regorafenib reduced cell viability. In addition, we showed that regorafenib inhibited the production of several cytokines, such as IL-6, TGF- β\u003csub\u003e1\u003c/sub\u003e and VEGF, and collagen synthesis in lung fibroblasts.\u003c/p\u003e \u003cp\u003eSeveral factors have been reported to be associated with pulmonary fibrosis, including IL-1, IL-6, TNF-α, platelet-derived growth factor, VEGF, and TGF-β\u003csub\u003e1\u003c/sub\u003e [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We previously showed that both IL-6 and TGF-β\u003csub\u003e1\u003c/sub\u003e played important roles in pulmonary fibrosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Specifically, we reported that human lung fibroblasts proliferated by IL-6 in a dose-dependent manner [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and the inhibition of IL-6 production in lung fibroblasts was significantly associated with the prevention of pulmonary fibrosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, Saito et al. showed that IL-6 may play an important role in the pathogenesis of bleomycin\u0026ndash;induced lung injury and fibrosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, IL-6 is one of the major cytokines involved in the pathogenesis of pulmonary fibrosis. In this study, we showed that regorafenib inhibited the production of IL-6 in lung fibroblasts.\u003c/p\u003e \u003cp\u003eTGF-β\u003csub\u003e1\u003c/sub\u003e is one of a major cytokines associated with fibrosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Yoshida et al. reported the overexpression of interleukin 6 and TGF-β\u003csub\u003e1\u003c/sub\u003e in fibrotic lesions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Previously, we demonstrated that inhibition of TGF-β\u003csub\u003e1\u003c/sub\u003e was associated with the prevention of pulmonary fibrosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the present study, we also showed that regorafenib inhibited TGF-β\u003csub\u003e1\u003c/sub\u003e production in lung fibroblasts.\u003c/p\u003e \u003cp\u003eCollagen synthesis plays an essential role in pulmonary development. Lung fibroblasts are the major collagen-producing cells in fibrotic lung lesions. It has been reported that serological biomarkers reflecting type I and III collagen turnover are elevated in patients with progressive disease in comparison to those with stable IPF [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, we demonstrated that regorafenib reduces type I collagen production in lung fibroblasts.\u003c/p\u003e \u003cp\u003eThe association between pulmonary fibrosis and neovascularization has been demonstrated in the lung tissues of patients with IPF [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and in a rat bleomycin-induced pulmonary fibrosis model [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. VEGF and Ang-1 are the major regulators of angiogenesis [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. VEGF plays an important role in endothelial cell proliferation, vascular permeability, and angiogenesis in several inflammatory lesions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The expression of VEGF is upregulated by various stimuli, such as low oxygen tension and several cytokines. Ang-1 is also an important regulator of blood vessel growth, maturation, and function. Ang-1 promotes angiogenesis, induces vascular maturation, and decreases vascular permeability [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePreviously, we reported the preventive effects of thalidomide against pulmonary fibrosis via inhibition of neovascularization by angiogenic factors such as VEGF [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In the present study, regorafenib had no effect on Ang-1 production by lung fibroblasts. However, regorafenib inhibited the production of VEGF in lung fibroblasts.\u003c/p\u003e \u003cp\u003eIn summary, we found that regorafenib prevents lung fibroblast proliferation and reduces cell viability. We also showed that regorafenib inhibited the production of three major cytokines in fibroblasts associated with pulmonary fibrosis, including IL-6 (pro-inflammatory), TGF-β\u003csub\u003e1\u003c/sub\u003e (fibrotic), and VEGF (angiogenic), suggesting the major mechanisms for the inhibitory effect of regorafenib on the proliferation of lung fibroblasts.\u003c/p\u003e \u003cp\u003eAlthough the precise cellular mechanism of regorafenib in lung fibroblasts has not been fully investigated and needs to be studied further for clinical use, our data may lead to the development of novel strategies for the prevention and treatment of pulmonary fibrosis.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eidiopathic pulmonary fibrosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthors' contributions: C.T .(Chiharu Tabata) and R.T. (Rie Tabata) designed the study. N.N. (Natsuki Nakamura) and C.T. performed the research. N.N. and C.T. collected data. C.T. and R.T .analyzed and interpreted the data. C.T .performed statistical analyses. C.T. and R.T. drafted the manuscript. N.N., C.T., and R.T. have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from JSPS KAKENHI (Grant Numbers JP19K08633 and JP23K07664.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest in association with the present study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCoultas DB, Zumwalt RE, Black WC, Sobonya RE. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med. 1994;150:967\u0026ndash;972.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmerican Thoracic Society/European Respiratory Society. International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med. 2002;165:277\u0026ndash;304.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGross TJ, Hunninghake GW. Idiopathic pulmonary fibrosis. N Engl J Med. 2001;345: 517\u0026ndash;525.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMason RJ, Schwarz MI, Hunninghake GW, Musson RA. NHLBI Workshop Summary. Pharmacological therapy for idiopathic pulmonary fibrosis. Past, present, and future. Am J Respir Crit Care Med. 1999;160:1771\u0026ndash;1777.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicheldi L, Collard HR, Jones MG. Idiopathic pulmonary fibrosis. Lancet. 2017;389(10082):1941\u0026ndash;1952.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLederer DJ, MartinezFJ. Idiopathic pulmonary fibrosis. N Engl J Med. 2018;378:1811\u0026ndash;1823.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDawson RE, Jenkins BJ, Saad MI. IL-6 family cytokines in respiratory health and disease. Cytokine. 2021;143:155520.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolters PJ, Collard HR, Jones KD. Pathogenesis of idiopathic pulmonary fibrosis. Annu Rev Pathol. 2014;9:157\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTE King Jr, A Pardo, M Selman. Idiopathic pulmonary fibrosis. Lancet. 2011;378:1949\u0026ndash;1961.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimler NR, Brenchley PE, Horrocks AW, Greaves SM, Hasleton PS, Egan JJ. Angiogenic cytokines in patients with idiopathic interstitial pneumonia. Thorax. 2004;59(7): 581\u0026ndash;585.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabata C, Tabata R, Kadokawa Y, Hisamori S, Takahashi M, Mishima M, Nakano T, Kubo H. Thalidomide prevents bleomycin-induced pulmonary fibrosis in mice. J Immunol. 2007;179:708\u0026ndash;714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrothey A, Van Cutsem E, Sobrero A, Siena S, Falcone A, Ychou M, Humblet Y, Bouch\u0026eacute; O, Mineur L, Barone C, Adenis A, Tabernero J, Yoshino T, Lenz HJ, Goldberg RM, Sargent DJ, Cihon F, Cupit L, Wagner A. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): An international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2013;381:303\u0026ndash;312.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evon Mehren M, Joensuu H. Gastrointestinal Stromal Tumors. J Clin Oncol. 2018;36(2):136\u0026ndash;143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet. 2018;31:1301\u0026ndash;1314.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilhelm SM, Dumas J, Adnane L, Lynch M, Carter CA, Sch\u0026uuml;tz G, Thierauch KH, Zopf D. Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int J Cancer. 2011;129:245\u0026ndash;255.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabata C, Kubo H, Tabata R, Wada M, Sakuma K, Ichikawa M, Fujita S, Mio T, Mishima M. All-trans retinoic acid modulates radiation-induced proliferation of lung fibroblasts via IL-6/IL-6R system. Am J Physiol Lung Cell Mol Physiol. 2006;290:597\u0026ndash;606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabata C, Kadokawa Y, Tabata R, Takahashi M, Okoshi K, Sakai Y, Mishima M, Kubo H. All-trans-Retinoic Acid Prevents Radiation- or Bleomycin-induced Pulmonary Fibrosis. Am J Respir Crit Care Med. 2006;174:1352\u0026ndash;1360.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest. 1997;100:768\u0026ndash;776.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolb M, Margetts PJ, Anthony DC, Pitossi F, Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 2001;107:1529\u0026ndash;1536.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyazaki Y, Araki K, Vesin C, Garcia I, Kapanci Y, Whitsett JA, Piguet PF, Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive pulmonary fibrosis. J Clin Invest. 1995;96:250\u0026ndash;259.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaito F, Tasaka S, Inoue K, Miyamoto K, Nakano Y, Ogawa Y, Yamada W, Shiraishi Y, Hasegawa N, Fujishima S, Takano H, Ishizaka A. Role of interleukin-6 in bleomycin-induced lung inflammatory changes in mice. Am J Respir Cell Mol Biol. 2008;38(5):566\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBroekelmann, TJ, Limper AH, Colby TV, McDonald JA. Transforming growth facter-1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA. 1991;88: 6642\u0026ndash;6646.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida M, Sakuma J, Hayashi S, Abe K, Saito I, Harada S, Sakatani M, Yamamoto S, Matsumoto N, Kaneda Y. A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor beta 1, or platelet-derived growth factor B gene. Proc Natl Acad Sci U S A. 1995;10:9570\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJenkins RG, Simpson JK, Saini G, Bentley JH, Russell AM, Braybrooke R, Molyneaux PL, McKeever TM, Wells AU, Flynn A, Hubbard RB, Leeming DJ, Marshall RP, Karsdal MA, Lukey PT, Maher TM. Longitudinal change in collagen degradation biomarkers in idiopathic pulmonary fibrosis: an analysis from the prospective, multicentre PROFILE study. Lancet Respir Med. 2015;3(6):462\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurner-Warwick M. PRECAPILLARY SYSTEMIC-PULMONARY ANASTOMOSES. Thorax. 1963;18:225\u0026ndash;237.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeao, MN, Aguas AP, de Sa CM, Grande NR. Neoformation of blood vessels in association with rat lung fibrosis induced by bleomycin. Anat Rec. 1994;238:57\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain RK. Molecular regulation of vessel maturation. Nat Med. 2003;9:685\u0026ndash;693.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000;6:460\u0026ndash;463.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdel-Malak NA, Mofarrahi M, Mayaki D, Khachigian LM, Hussain SN. Early growth response-1 regulates angiopoietin-1-induced endothelial cell proliferation, migration, and differentiation. Arterioscler Thromb Vasc Biol. 2009;29: 209\u0026ndash;216.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"regorafenib, cell proliferation, cell viability, cytokines, pulmonary fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-3929682/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3929682/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePulmonary fibrosis is a disease that leads to respiratory failure and death. There has been little progress in therapeutic strategies for pulmonary fibrosis. There have been several reports on the cytokines associated with pulmonary fibrosis, including IL-6 and TGF-β\u003csub\u003e1\u003c/sub\u003e. Angiogenesis is one of the most important phenomena in the pathogenesis of PF. Previously, we reported the preventive effects of thalidomide against pulmonary fibrosis via the inhibition of neovascularization by angiogenic factors such as VEGF. Regorafenib is a multikinase inhibitor, which inhibits tyrosine kinase receptors such as VEGFR1-3 and TIE2. In the clinical setting, regorafenib has been widely used for anti-cancer therapy for metastatic colorectal cancer. In this study, we examined the preventive effects of regorafenib against pulmonary fibrosis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe investigated whether regorafenib had an inhibitory effect on the proliferation, viability, and production of several cytokines in lung fibroblasts.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe demonstrated an inhibitory effect of regorafenib on the proliferation and viability of lung fibroblasts. Moreover, regorafenib reduced the production of several cytokines associated with the pathogenesis of pulmonary fibrosis, including IL-6, VEGF and TGF- β\u003csub\u003e1\u003c/sub\u003e, and collagen synthesis from lung fibroblasts.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese data suggest that regorafenib may have potential clinical applications in the prevention of pulmonary fibrosis.\u003c/p\u003e","manuscriptTitle":"Regorafenib exerts an inhibitory effect on the proliferation of human lung fibroblasts by reducing the production of several cytokines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-08 14:16:21","doi":"10.21203/rs.3.rs-3929682/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"704f6a94-2176-4bd6-8dfd-f9bdf8d39ddc","owner":[],"postedDate":"February 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-04T09:03:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-08 14:16:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3929682","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3929682","identity":"rs-3929682","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}