Investigational New Drugs

, Volume 37, Issue 5, pp 1044–1051 | Cite as

The antitumor efficacy of monomeric disintegrin obtustatin in S-180 sarcoma mouse model

  • Narine GhazaryanEmail author
  • Naira Movsisyan
  • Joana Catarina Macedo
  • Sara Vaz
  • Naira Ayvazyan
  • Luis Pardo
  • Elsa Logarinho


Obtustatin, isolated from the Levantine Viper snake venom (Macrovipera lebetina obtusa -MLO), is the shortest known monomeric disintegrin shown to specifically inhibit the binding of the α1β1 integrin to collagen IV. Its oncostatic effect is due to the inhibition of angiogenesis, likely through α1β1 integrin inhibition in endothelial cells. To explore the therapeutic potential of obtustatin, we studied its effect in S-180 sarcoma-bearing mice model in vivo as well as in human dermal microvascular endothelial cells (HMVEC-D) in vitro, and tested anti-angiogenic activity in vivo using the chick embryo chorioallantoic membrane assay (CAM assay). Our in vivo results show that obtustatin inhibits tumour growth by 33%. The expression of vascular endothelial growth factor (VEGF) increased after treatment with obtustatin, but the level of expression of caspase 8 did not change. In addition, our results demonstrate that obtustatin inhibits FGF2-induced angiogenesis in the CAM assay. Our in vitro results show that obtustatin does not exhibit cytotoxic activity in HMVEC-D cells in comparison to in vivo results. Thus, our findings disclose that obtustatin might be a potential candidate for the treatment of sarcoma in vivo with low toxicity.


Obtustatin Sarcoma Angiogenesis VEGF 



This work was made possible by the research grant # molbio 3440 from the Armenian National Science and Education Fund (ANSEF) based in New York.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Informed consent

For this type of study, formal consent is not required.

Supplementary material

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  1. 1.
    Aranda-Souza M, Rossato F, Costa R, Figueira T, Castilho R, Guarniere M, Nunes E, Coelho L, Correia M, Vercesi AA (2014) Lectin from Bothrops leucurus snake venom raises cytosolic calcium levels and promotes B16-F10 melanoma necrotic cell death via mitochondrial permeability transition. Toxicon 2(82):97–103CrossRefGoogle Scholar
  2. 2.
    Benassi MS, Gamberi G, Magagnoli G, Molendini L, Ragazzini P, Merli M, Chiesa F, Balladelli A, Manfrini M, Bertoni F (2001) Metalloproteinase expression and prognosis in soft tissue sarcomas. Ann Oncol 12:75–80CrossRefGoogle Scholar
  3. 3.
    Bielenberg DR, Zetter BR (2015) The contribution of angiogenesis to the process of metastasis. Cancer J 21:267–273CrossRefGoogle Scholar
  4. 4.
    Brown MC, Staniszewska I, Del Valle L, Tuszynski GP, Marcinkiewicz C (2008) Angiostatic activity of obtustatin as alpha1beta1 integrin inhibitor in experimental melanoma growth. Int J Cancer 123:2195–2203CrossRefGoogle Scholar
  5. 5.
    Calvete JJ, Mureno-Murciano MP, Theakston DG, Kisiel DG, Marcinkiewicz C (2003) Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering. Biochem J 372:725–734CrossRefGoogle Scholar
  6. 6.
    Carmeliet P, Jain RK (2011) Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov 10:417–427CrossRefGoogle Scholar
  7. 7.
    Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407:249–257CrossRefGoogle Scholar
  8. 8.
    Chen X, Su Y, Fingleton B, Acuff H, Matrisian LM, Zent R, Pozzi A (2005) Increased plasma MMP9 in integrin a1-null mice enhances lung metastasis of colon carcinoma cells. Int J Cancer 116:52–61CrossRefGoogle Scholar
  9. 9.
    Cormier JN, Pollock RE (2004) Soft tissue sarcomas. CA Cancer J Clin 54:94–109CrossRefGoogle Scholar
  10. 10.
    Dean BJF, Whitwell D (2009) Epidemiology of bone and soft-tissue sarcomas. Orthop Traumatol 23:223–230Google Scholar
  11. 11.
    Ebrahim K, Shirazi F, Mirakabadi A, Vatanpour H (2015) Cobra venom cytotoxins; apoptotic or necrotic agents? Toxicon 108:134–140CrossRefGoogle Scholar
  12. 12.
    Ferrara N (2002) VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer 2:795–803CrossRefGoogle Scholar
  13. 13.
    Fletcher CDM, Uni KK, Mertens F (2002) Eds: World Health Organisation classification of tumours. IARC Press, Pathology and genetics Lyon, 427pGoogle Scholar
  14. 14.
    Folkman J (2002) Role of angiogenesis in tumor growth and metastasis. Semin Oncol 29:15–18CrossRefGoogle Scholar
  15. 15.
    Folkman J (2003) Fundamental concepts of the angiogenic process. Curr Mol Med 3:643–651CrossRefGoogle Scholar
  16. 16.
    Ghazaryan NA, Ghulikyan L, Kishmiryan A, Kirakosyan G, Nazaryan O, Ghevondyan T, Zakaryan N, Ayvazyan NM (2015) Anti-tumor effect investigation of obtustatin and crude Macrovipera lebetina obtusa venom in S-180 sarcoma bearing mice. Eur J Pharmacol 11:340–345CrossRefGoogle Scholar
  17. 17.
    Golubkov V, Hawes D, Markland FS (2003) Anti-angiogenicactivity of contortrostatin, a disintegrin from Agkistrodoncontortrixcontortrix snake venom. Angiogenesis2003 6:213–224CrossRefGoogle Scholar
  18. 18.
    Goswami S (2013) Importance of integrin receptors in the field of pharmaceutical & medical science. Adv Biol Chem 3:224–252CrossRefGoogle Scholar
  19. 19.
    Guimarães DO, Lopes DS, Azevedo FV, Gimenes SN, Silva MA, Achê DC, Gomes MS, Vecchi L, Goulart LR, Yoneyama KA, Rodrigues RS, Rodrigues VM (2017) In vitro antitumor and antiangiogenic effects of Bothropoidin, a metalloproteinase from Bothropspauloensis snake venom. Int J Biol Macromol 97:770–777CrossRefGoogle Scholar
  20. 20.
    Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G, Roberge S, Jain RK, McDonald DM (2000) Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol 156:1363–1380CrossRefGoogle Scholar
  21. 21.
    Hedlund EM, Hosaka K, Zhong Z, Cao R, Cao Y (2009) Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature. Proc Natl Acad Sci U SA 106:17505–17510CrossRefGoogle Scholar
  22. 22.
    Hirota K, Semenza GL (2006) Regulation of angiogenesis by hypoxiainducible factor 1. Crit Rev Oncol Hematol 59:15–26CrossRefGoogle Scholar
  23. 23.
    Ibaragi S, Shimo T, Hassan NMM, Isowa S, Kurio N, Mandai H, Kodama S, Sasaki A (2011) Induction of MMP-13 expression in bone-metastasizing cancer cells by type I collagen through integrin a1b1 and a2b1-p38 MAPK signaling. Anticancer Res 31:1307–1313Google Scholar
  24. 24.
    Kisiel DG, Calvete JJ, Katzhendler J, Fertala A, Lazarovici P, Marcinkiewicz C (2004) Structural determinants of the selectivity of KTS-disintegrins for the alpha1beta1 integrin. FEBS Lett 577:478–482CrossRefGoogle Scholar
  25. 25.
    Liang H, Xiao J, Zhou Z, Wu J, Ge F, Li Z, Zhang H, Sun J, Li F, Liu R, Chen C (2018) Hypoxia induces miR-153 through the IRE1α-XBP1 pathway to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis. Oncogene.
  26. 26.
    Lijnen HR (2008) Angiogenesis and obesity. Cardiovasc Res 78:286–293CrossRefGoogle Scholar
  27. 27.
    Marcinkewicz C (2005) Functional characteristic of snake venom disintegrins: potential therapeutic implication. Curr Pharm Des 11:815–827CrossRefGoogle Scholar
  28. 28.
    Marcinkiewicz C, Rosenthal LA, Mosser DM, Kunicki TJ, Niewiarowicz C (1996) Immunological characterization of erististatin and echistatin binding sites on αIIbβ3 and ανβ3 integrins. Biochem J 317:817–825CrossRefGoogle Scholar
  29. 29.
    Marcinkiewicz C, Weinreb PH, Calvete JJ, Kisiel DG, Mousa SA, Tuszynski GP, Lobb RR (2003) Obtustatin: a potent selective inhibitor of alpha1beta1 integrin in vitro and angiogenesis in vivo. Cancer Res 9:2020–2023Google Scholar
  30. 30.
    Naldini A, Carraro F (2005) Role of inflammatory mediators in angiogenesis. Curr Drug Targets Inflamm Allergy 4:3–8CrossRefGoogle Scholar
  31. 31.
    Pozzi A, Moberg PE, Miles LA, Wagner S, Soloway P, Gardner HA (2000) Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization. Proc Natl AcadSci USA 97:2202–2207CrossRefGoogle Scholar
  32. 32.
    Prinholato da Silva C, Costa T, Paiva R, Cintra A, Menaldo D, Antunes L, Sampaio S (2015) Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines. J Venom Anim Toxins Incl Trop Dis.
  33. 33.
    Pugh CW, Ratcliffe PJ (2003) Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 9:677–684CrossRefGoogle Scholar
  34. 34.
    Sanz L, Ayvazyan N, Calvete JJ (2008) Snake venomics of the Armenian mountain vipers Macrovipera lebetina obtusa and Vipera raddei. J Proteome 71:198–209CrossRefGoogle Scholar
  35. 35.
    Senger DR, Perruzzi CA, Streit M, Koteliansky VE, De Fougerolles AR, Detmar M (2002) Theα1β1 and α2β1 integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis. Am J Pathol 160:195–204CrossRefGoogle Scholar
  36. 36.
    Stupack DG, Cheresh DA (2002) Get a ligand, get a life: integrins, signaling and cell survival. J Cell Sci 115:3729–3738CrossRefGoogle Scholar
  37. 37.
    Vihinen P, Riikonen T, Laine A, Heino J (1996) Integrin alpha 2 beta 1 in tumorigenic human osteosarcoma cell lines regulates cell adhesion, migration, and invasion by interaction with type I collagen. Cell Growth Differ 7:439–447Google Scholar
  38. 38.
    Wang GL, Semenza GL (1993) Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem 268:21513–21518Google Scholar
  39. 39.
    Wang YQ, Su J, Wu F, Lu P, Yuan LF, Yuan WE, Sheng J, Jin T (2012) Biscarbamate cross-linked polyethylenimine derivative with low molecular weight, low cytotoxicity, and high efficiency for gene delivery. Int J Nanomedicine 7:693–704Google Scholar
  40. 40.
    Yoshimura K, Meckel KF, Laird LS, Chia CY, Park JJ, Olino KL, Tsunedomi R, Harada T, Iizuka N, Hazama S (2009) Integrin a2 mediates selective metastasis to the liver. Cancer Res 69:7320–7328CrossRefGoogle Scholar
  41. 41.
    Zhang Z, Ramirez NE, Yankeelov TE, Li Z, Ford LE, Qi Y, Pozzi A, Zutter MM (2008) a2b1 integrin expression in the tumor microenvironment enhances tumor angiogenesis in a tumor cell-specific manner. Blood 111:1980–1988CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Narine Ghazaryan
    • 1
    • 2
    Email author
  • Naira Movsisyan
    • 2
    • 3
  • Joana Catarina Macedo
    • 4
  • Sara Vaz
    • 4
  • Naira Ayvazyan
    • 1
  • Luis Pardo
    • 2
  • Elsa Logarinho
    • 4
  1. 1.Laboratory of Toxinology and Molecular SystematicsL.A. Orbeli Institute of PhysiologyYerevanArmenia
  2. 2.Oncophysiology GroupMax Planck Institute for Experimental MedicineGöttingenGermany
  3. 3.Göttingen Graduate School for Neurosciences, Biophysics, and Molecular BiosciencesGöttingenGermany
  4. 4.Aging and Aneuploidy Laboratory, Instituto de Biologia Molecular e Celular, Instituto de Investigação e Inovação em Saúde – i3SUniversidade do PortoPortoPortugal

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