Antiangiogenic therapy with Nintedanib affects hypoxia, angiogenesis and apoptosis in the ventral prostate of TRAMP animals

  • Raquel Frenedoso da Silva
  • Thais Petrochelli Banzato
  • Letícia Ferreira Alves
  • João Ernesto Carvalho
  • Rajesh Agarwal
  • Valéria Helena Alves CagnonEmail author
Regular Article


The antiangiogenic therapy for prostate cancer with Nintedanib, a potent inhibitor of important growth factor receptors, has been proven to delay tumor progression and arrest tumor growth; thus, the aim herein is to evaluate Nintedanib effects on tumor cells, besides angiogenesis and apoptosis processes, metalloproteinases and hypoxia factor in an animal model. Nintedanib promoted growth inhibition and cell death in a dose-dependent manner, showing no tumor selectivity. Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) were treated with Nintedanib (10 mg/kg/day) in different stages of tumor development and the ventral prostate was examined for protein levels by means of immunohistochemistry and Western blotting and apoptosis evaluation. In vitro antiproliferative activity of Nintedanib was also assessed in nine human tumor cell lines. Early Nintedanib treatment has shown decreased levels of FGF-2, VEGFR-1, MMP-9 and HIF-1α and a significantly increased apoptosis of epithelial cells. Furthermore, late Nintedanib treatment decreased FGF-2, VEGFR-1 and FGFR-3 levels. Importantly, even after treatment discontinuation, treated animals displayed a significant decrease in VEGFR-1 as well as MMP-9. Although Nintedanib treatment in late stages of tumor growth has shown some good results, it is noteworthy that the drug presents the best tissue response when administered in the early stages of disease development. Nintedanib treatment has shown to be a promising approach for prostate cancer therapy, especially in the early stages of the disease, interfering in different carcinogenesis progression pathways.


Angiogenesis Prostate cancer Growth factors Tumor cells Nintedanib 



Not applicable.

Authors’ contribution

Study design, collection, analyses and interpretation of data and writing of manuscript: Raquel Frenedoso da Silva and Valéria Helena Alves Cagnon. Collection and analyses of data: Thais Petrochelli Banzato and Letícia Ferreira Alves. Analyses and interpretation of data: Rajesh Agarwal and João Ernesto Carvalho.


This work was supported by a grant from the São Paulo Research Foundation (FAPESP 2013/26677-7) and in part by the NCI R01 grant CA195708 (to RA).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Ethical approval

The Ethics Committee on Animal Use (CEUA-UNICAMP) approved this study under protocol number 3285-1, carried out in agreement with the Ethical Principles for Animal Research established by the Brazilian College for Animal Experimentation (COBEA).

Supplementary material

441_2019_3091_MOESM1_ESM.jpg (2.7 mb)
Fig. S1 Experimental design: Animals received treatment from 8 to 12 weeks of age and were euthanized at the end of the treatment (TC12 and TN12, respectively) or at 22 weeks of age (TC22(8–12) and TN22(8–12), respectively). Furthermore, groups receiving treatment from 12 to 16 weeks of age were euthanized either at the end of the treatment (TC16 and TN16, respectively) or at 22 weeks of age (TC22(12–16) and TN22(12–16), respectively). Nintedanib was administered in a dose of 10 mg/kg/day, orally. (JPG 2721 kb)


  1. Alves LF, da Silva RF, Cagnon VHA (2018) Nintedanib effects on delaying cancer progression and decreasing COX-2 and IL-17 in the prostate anterior lobe in TRAMP mice. Tissue Cell 50:96–103CrossRefPubMedGoogle Scholar
  2. Awasthi N, Schwarz RE (2015) Profile of nintedanib in the treatment of solid tumors: the evidence to date. Onco Targets Ther 8:3691–3701CrossRefPubMedPubMedCentralGoogle Scholar
  3. Awasthi N, Hinz S, Brekken RA, Schwarz MA, Schwarz RE (2015) Nintedanib, a triple angiokinase inhibitor, enhances cytotoxic therapy response in pancreatic cancer. Cancer Lett 358:59–66CrossRefPubMedGoogle Scholar
  4. Boget S, Cereser C, Parvaz P, Leriche A, Revol A (2001) Fibroblast growth factor receptor 1 (FGFR1) is over-expressed in benign prostatic hyperplasia whereas FGFR2-IIIc and FGFR3 are not. Eur J Endocrinol 145:303–310CrossRefPubMedGoogle Scholar
  5. Castellano G, Malaponte G, Mazzarino MC, Figini M, Marchese F, Gangemi P, Travali S, Stivala F, Canevari S, Libra M (2008) Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression. Clin Cancer Res 14:7470–7480CrossRefPubMedGoogle Scholar
  6. da Silva RF, Nogueira-Pangrazi E, Kido LA, Montico F, Arana S, Kumar D, Raina K, Agarwal R, Cagnon VHA (2017) Nintedanib antiangiogenic inhibitor effectiveness in delaying adenocarcinoma progression in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP). J Biomed Sci 24:31CrossRefPubMedPubMedCentralGoogle Scholar
  7. da Silva RF, Dhar D, Raina K, Kumar D, Kant R, Cagnon VHA, Agarwal C, Agarwal R (2018) Nintedanib inhibits growth of human prostate carcinoma cells by modulating both cell cycle and angiogenesis regulators. Sci Rep 8:9540CrossRefPubMedPubMedCentralGoogle Scholar
  8. Damasceno AA, Carvalho CP, Santos EM, Botelho FV, Araujo FA, Deconte SR, Tomiosso TC, Balbi AP, Zanon RG, Taboga SR, Goes RM, Ribeiro DL (2014) Effects of maternal diabetes on male offspring: high cell proliferation and increased activity of MMP-2 in the ventral prostate. Cell Tissue Res 358:257–269CrossRefPubMedGoogle Scholar
  9. Daniyal M, Siddiqui ZA, Akram M, Asif HM, Sultana S, Khan A (2014) Epidemiology, etiology, diagnosis and treatment of prostate cancer. Asian Pac J Cancer Prev 15:9575–9578CrossRefPubMedGoogle Scholar
  10. Doll JA, Reiher FK, Crawford SE, Pins MR, Campbell SC, Bouck NP (2001) Thrombospondin-1, vascular endothelial growth factor and fibroblast growth factor-2 are key functional regulators of angiogenesis in the prostate. Prostate 49:293–305CrossRefPubMedGoogle Scholar
  11. Feng S, Shao L, Yu W, Gavine P, Ittmann M (2012) Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression. Clin Cancer Res 18:3880–3888CrossRefPubMedPubMedCentralGoogle Scholar
  12. Feng S, Shao L, Castro P, Coleman I, Nelson PS, Smith PD, Davies BR, Ittmann M (2017) Combination treatment of prostate cancer with FGF receptor and AKT kinase inhibitors. Oncotarget 8:6179–6192PubMedGoogle Scholar
  13. Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676CrossRefGoogle Scholar
  14. Giri D, Ropiquet F, Ittmann M (1999) Alterations in expression of basic fibroblast growth factor (FGF) 2 and its receptor FGFR-1 in human prostate cancer. Clin Cancer Res 5:1063–1071PubMedGoogle Scholar
  15. Goncalves BF, de Campos SG, Zanetoni C, Scarano WR, Falleiros LR Jr, Amorim RL, Goes RM, Taboga SR (2013) A new proposed rodent model of chemically induced prostate carcinogenesis: distinct time-course prostate cancer progression in the dorsolateral and ventral lobes. Prostate 73:1202–1213CrossRefPubMedGoogle Scholar
  16. Gowardhan B, Douglas DA, Mathers ME, McKie AB, McCracken SR, Robson CN, Leung HY (2005) Evaluation of the fibroblast growth factor system as a potential target for therapy in human prostate cancer. Br J Cancer 92:320–327CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674CrossRefGoogle Scholar
  18. Hernandez S, de Muga S, Agell L, Juanpere N, Esgueva R, Lorente JA, Mojal S, Serrano S, Lloreta J (2009) FGFR3 mutations in prostate cancer: association with low-grade tumors. Mod Pathol 22:848–856CrossRefPubMedGoogle Scholar
  19. Hilberg F, Roth GJ, Krssak M, Kautschitsch S, Sommergruber W, Tontsch-Grunt U, Garin-Chesa P, Bader G, Zoephel A, Quant J, Heckel A, Rettig WJ (2008) BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy. Cancer Res 68:4774–4782CrossRefGoogle Scholar
  20. Huss WJ, Hanrahan CF, Barrios RJ, Simons JW, Greenberg NM (2001) Angiogenesis and prostate cancer: identification of a molecular progression switch. Cancer Res 61:2736–2743PubMedGoogle Scholar
  21. Huss WJ, Barrios RJ, Foster BA, Greenberg NM (2003) Differential expression of specific FGF ligand and receptor isoforms during angiogenesis associated with prostate cancer progression. Prostate 54:8–16CrossRefPubMedGoogle Scholar
  22. Kudo K, Arao T, Tanaka K, Nagai T, Furuta K, Sakai K, Kaneda H, Matsumoto K, Tamura D, Aomatsu K, De Velasco MA, Fujita Y, Saijo N, Kudo M, Nishio K (2011) Antitumor activity of BIBF 1120, a triple angiokinase inhibitor and use of VEGFR2+pTyr+ peripheral blood leukocytes as a pharmacodynamic biomarker in vivo. Clin Cancer Res 17:1373–1381CrossRefPubMedGoogle Scholar
  23. Labi V, Erlacher M (2015) How cell death shapes cancer. Cell Death Dis 6:e1675CrossRefPubMedPubMedCentralGoogle Scholar
  24. Lo Iacono M, Buttigliero C, Monica V, Bollito E, Garrou D, Cappia S, Rapa I, Vignani F, Bertaglia V, Fiori C, Papotti M, Volante M, Scagliotti GV, Porpiglia F, Tucci M (2016) Retrospective study testing next generation sequencing of selected cancer-associated genes in resected prostate cancer. Oncotarget 7:14394–14404CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lowe SW, Lin AW (2000) Apoptosis in cancer. Carcinogenesis 21:485–495CrossRefPubMedGoogle Scholar
  26. McCormack PL (2015) Nintedanib: first global approval. Drugs 75:129–139CrossRefPubMedGoogle Scholar
  27. Mollereau B, Perez-Garijo A, Bergmann A, Miura M, Gerlitz O, Ryoo HD, Steller H, Morata G (2013) Compensatory proliferation and apoptosis-induced proliferation: a need for clarification. Cell Death Differ 20:181CrossRefPubMedGoogle Scholar
  28. Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A et al (1991) Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83:757–766CrossRefPubMedGoogle Scholar
  29. Montico F, Kido LA, Hetzl AC, Lorencini RM, Candido EM, Cagnon VH (2014) Antiangiogenic therapy effects on age-associated matrix metalloproteinase-9 (MMP-9) and insulin-like growth factor receptor-1 (IGFR-1) responses: a comparative study of prostate disorders in aged and TRAMP mice. Histochem Cell Biol 142:269–284CrossRefPubMedGoogle Scholar
  30. Mukherji D, Temraz S, Wehbe D, Shamseddine A (2013) Angiogenesis and anti-angiogenic therapy in prostate cancer. Crit Rev Oncol Hematol 87:122–131CrossRefPubMedGoogle Scholar
  31. Nogueira Pangrazi E, da Silva RF, Kido LA, Montico F, Cagnon VHA (2018) Nintedanib treatment delays prostate dorsolateral lobe cancer progression in the TRAMP model: contribution to the epithelial-stromal interaction balance. Cell Biol Int 42:153–168CrossRefPubMedGoogle Scholar
  32. Noh EM, Park YJ, Kim JM, Kim MS, Kim HR, Song HK, Hong OY, So HS, Yang SH, Kim JS, Park SH, Youn HJ, You YO, Choi KB, Kwon KB, Lee YR (2015) Fisetin regulates TPA-induced breast cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways. Eur J Pharmacol 764:79–86CrossRefPubMedGoogle Scholar
  33. Pal SK, Vuong W, Zhang W, Deng J, Liu X, Carmichael C, Ruel N, Pinnamaneni M, Twardowski P, Lau C, Yu H, Figlin RA, Agarwal N, Jones JO (2015) Clinical and translational assessment of VEGFR1 as a mediator of the premetastatic niche in high-risk localized prostate cancer. Mol Cancer Ther 14:2896–2900CrossRefPubMedGoogle Scholar
  34. Polnaszek N, Kwabi-Addo B, Peterson LE, Ozen M, Greenberg NM, Ortega S, Basilico C, Ittmann M (2003) Fibroblast growth factor 2 promotes tumor progression in an autochthonous mouse model of prostate cancer. Cancer Res 63:5754–5760PubMedGoogle Scholar
  35. Ruggeri B, Singh J, Gingrich D, Angeles T, Albom M, Yang S, Chang H, Robinson C, Hunter K, Dobrzanski P, Jones-Bolin S, Pritchard S, Aimone L, Klein-Szanto A, Herbert JM, Bono F, Schaeffer P, Casellas P, Bourie B, Pili R, Isaacs J, Ator M, Hudkins R, Vaught J, Mallamo J, Dionne C (2003) CEP-7055: a novel, orally active pan inhibitor of vascular endothelial growth factor receptor tyrosine kinases with potent antiangiogenic activity and antitumor efficacy in preclinical models. Cancer Res 63:5978–5991PubMedGoogle Scholar
  36. Sahadevan K, Darby S, Leung HY, Mathers ME, Robson CN, Gnanapragasam VJ (2007) Selective over-expression of fibroblast growth factor receptors 1 and 4 in clinical prostate cancer. J Pathol 213:82–90CrossRefPubMedGoogle Scholar
  37. Siegel RL, Miller KD, Jemal A (2018) Cancer statistics, 2018. CA Cancer J Clin 68:7–30CrossRefGoogle Scholar
  38. Steinemann G, Jacobsen C, Gerwing M, Hauschild J, von Amsberg G, Hopfner M, Nitzsche B, Honecker F (2016) Activity of nintedanib in germ cell tumors. Anti-Cancer Drugs 27:89–98CrossRefPubMedGoogle Scholar
  39. Weibel ER (1963) Principles and methods for the morphometric study of the lung and other organs. Lab Invest 12:131–155PubMedGoogle Scholar
  40. Xu D, Wang X, Lou Y (2017) Association of endothelin-1 gene single-nucleotide polymorphisms and haplotypes with risk of hormone refractory prostate cancer. Pharmazie 72:103–106PubMedGoogle Scholar
  41. Yamaguchi K, Izaki H, Takahashi M, Fukumori T, Nishitani M, Sutou Y, Uema K, Kawano A, Hamao T, Kanayama HO (2014) Changes in levels of prostate-specific antigen and testosterone following discontinuation of long-term hormone therapy for non-metastatic prostate cancer. J Med Investig 61:35–40CrossRefGoogle Scholar
  42. Zar J (1999) Biostatistical analysis. Prentice Hall, New JerseyGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Raquel Frenedoso da Silva
    • 1
    • 2
  • Thais Petrochelli Banzato
    • 3
  • Letícia Ferreira Alves
    • 1
  • João Ernesto Carvalho
    • 3
    • 4
  • Rajesh Agarwal
    • 2
  • Valéria Helena Alves Cagnon
    • 1
    Email author
  1. 1.Department of Structural and Functional Biology, Institute of BiologyState University of Campinas (UNICAMP)CampinasBrazil
  2. 2.Department of Pharmaceutical Sciences, Skaggs School of PharmacyUniversity of Colorado Anschutz Medical CampusAuroraUSA
  3. 3.Chemical, Biological and Agricultural Pluridisciplinary Research CenterState University of Campinas (UNICAMP)São PauloBrazil
  4. 4.Faculty of Pharmaceutical SciencesState University of Campinas (UNICAMP)São PauloBrazil

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