Applied Biochemistry and Biotechnology

, Volume 187, Issue 4, pp 1539–1550 | Cite as

Investigation of Vipera Anatolica Venom Disintegrin via Intracellular Uptake with Radiolabeling Study and Cell-Based Electrochemical Biosensing Assay

  • Ozge Er
  • Ece Eksin
  • Hale Melis Soylu
  • Bayram Göçmen
  • Ayşe NalbantsoyEmail author
  • Fatma YurtEmail author
  • Arzum ErdemEmail author


Snake venoms are a natural biological source that has potential therapeutic value with various protein compounds. Disintegrins originally were discovered as a family of proteins from snake venoms composed of cysteine rich low molecular weight polypeptides. Disintegrins exhibit specific binding and higher affinity toward integrin with potential inhibition of function. Trans-membrane receptors of the integrin family may involve in many pathological conditions such as inflammation and tumor progression with important processes related to invasion and migration. Since disintegrins have the ability to bind to integrins, they could be used for cancer detection and treatment, and in monitoring of therapy in select cancer types. The main purpose of the study is to investigate disintegrin containing Vipera anatolica (VAT) crude venom potential for radiolabeling and intracellular uptake as well as electrochemical biosensing assay against U87MG human brain glioblastoma cells. For this purpose, VAT crude venom containing U87MG cell-specific disintegrin was investigated in terms of radiolabeling and intracellular uptake as well as electrochemical biosensing assay in comparison with echistatin (ECT) disintegrin in cells. The interaction between VAT crude venom and ECT with HEK293 human non-tumorigenic embryonic kidney cells and glioblastoma U87MG cells was electrochemically investigated using pencil graphite electrodes (PGEs). The interaction of the VAT crude venom and ECT with HEK293 and U87MG cells was detected according to the changes in oxidation signals. Then, VAT crude venom and echistatin were labeled with 131I via iodogen method. Intracellular uptakes of radiolabeled molecules were investigated in U87MG cell line. 131I-VAT can be an agent for imaging of glioblastoma cancer. Further work will focus on the production of large quantities of pure VAT disintegrin with a biotechnological approach to improving imaging agent.


Vipera anatolica crude venom Glioblastoma cancer Cancer imaging Electrochemical biosensing assay 



Vipera anatolica


human brain glioblastoma cells




human non-tumorigenic embryonic kidney cells


pencil graphite electrodes


extracellular matrix




reactive oxygen species


vascular endothelial growth factor


differential pulse voltammetry


thin layer radio chromatography


bicinchoninic acid


Dulbecco’s modified Eagle’s medium F12


fetal bovine serum


3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)


optical density


half maximal inhibition of growth


phosphate buffer solution


radio-immunoprecipitation assay


cellulose-coated plastic


Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Koh, C. Y., & Kini, R. M. (2012). From snake venom toxins to therapeutics - cardiovascular examples. Toxicon, 59, 497–506.CrossRefGoogle Scholar
  2. 2.
    Knight, L. C., Maurer, A. H., & Romano, J. E. (1996). Comparison of iodine-123-disintegrins for imaging thrombi and emboli in a canine model. Journal of nuclear medicine: official publication. Society of Nuclear Medicine, 37(3), 476–482.Google Scholar
  3. 3.
    Calderon, L. A., Sobrinho, J. C., Zaqueo, K. D., de Moura, A. A., Grabner, A. N., Mazzi, M. V., Marcussi, S., Nomizo, A., Fernandes, C. F., Zuliani, J. P., Carvalho, B. M., da Silva, S. L., Stabeli, R. G., Soares, A. M. (2014). Antitumoral activity of snake venom proteins: new trends in cancer therapy. Biomed Res Int, 1–19.Google Scholar
  4. 4.
    Calvete, J. J., Moreno-Murciano, M. P., Theakston, R. D. G., Kisiel, D. G., & Marcinkiewicz, C. (2003). Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering. The Biochemical Journal, 372(3), 725–734.CrossRefGoogle Scholar
  5. 5.
    Takada, Y., Ye, X., & Simon, S. (2007). The integrins. Genome Biology, 8(5), 215.CrossRefGoogle Scholar
  6. 6.
    Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell, 110(6), 673–687.CrossRefGoogle Scholar
  7. 7.
    Kren, A., Baeriswyl, V., Lehembre, F., Wunderlin, C., Strittmatter, K., Antoniadis, H., Fässler, R., Cavallaro, U., & Christofori, G. (2007). Increased tumor cell dissemination and cellular senescence in the absence of beta1-integrin function. The EMBO Journal, 26(12), 2832–2842.CrossRefGoogle Scholar
  8. 8.
    Albelda, S. M. (1993). Role of integrins and other cell adhesion molecules in tumor progression and metastasis. Laboratory Investigation, 68(1), 4–17.Google Scholar
  9. 9.
    Desgrosellier, J. S., & Cheresh, D. A. (2010). Integrins in cancer: biological implications and therapeutic opportunities. Nature Reviews. Cancer, 10(1), 9–22.CrossRefGoogle Scholar
  10. 10.
    Senger, D. R., Claffey, K. P., Benes, J. E., Perruzzi, C. A., Sergiou, A. P., & Detmar, M. (1997). Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins. Proceedings of the National Academy of Sciences of the United States of America, 94(25), 13612–13617.CrossRefGoogle Scholar
  11. 11.
    Senger, D. R., Perruzzi, C. A., Streit, M., Koteliansky, V. E., de Fougerolles, A. R., & Detmar, M. (2002). The α1β1 and α 2β1 integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis. The American Journal of Pathology, 160(1), 195–204.CrossRefGoogle Scholar
  12. 12.
    Lucena, S., Sanchez, E. E., & Perez, J. C. (2011). Anti-metastatic activity of the recombinant disintegrin r-mojastin 1, from the Mohave rattlesnake. Toxicon, 57(5), 794–802.CrossRefGoogle Scholar
  13. 13.
    Wang, J., & Kawde, A. N. (2001). Pencil-based renewable biosensor for label-free electrochemical detection of DNA hybridization. Analytica Chimica Acta, 431(2), 219–224.CrossRefGoogle Scholar
  14. 14.
    Erdreich-Epstein, A., Shimada, H., Groshen, S., Liu, M., Metelitsa, L. S., Kim, K. S., Stins, M. F., Seeger, R. C., & Durden, D. L. (2000). Integrins alpha(v)beta3 and alpha(v)beta5 are expressed by endothelium of high-risk neuroblastoma and their inhibition is associated with increased endogenous ceramide. Cancer Research, 60(3), 712–721.Google Scholar
  15. 15.
    Hemminki, A., Belousova, N., Zinn, K. R., Liu, B., Wang, M., Chaudhuri, T. R., Rogers, B. E., Buchsbaum, D. J., Siegal, G. P., Barnes, M. N., Gomez-Navarro, J., Curiel, D. T., & Alvarez, R. D. (2001). An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imaging of gene expression. Molecular Therapy, 4(3), 223–231.CrossRefGoogle Scholar
  16. 16.
    Alavi, A., Hood, J. D., Frausto, R., Stupack, D. G., & Cheresh, D. A. (2003). Role of Raf in vascular protection from distinct apoptotic stimuli. Science, 301(5629), 94–96.CrossRefGoogle Scholar
  17. 17.
    Huang, T., & Holt, J. (1987). Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex. The Journal of Biological Chemistry, 262(33), 16157–16163.Google Scholar
  18. 18.
    Rivas-Mercado, E. A., & Gara-Ocanas, L. (2017). Disintegrins obtained from snake venom and their pharmacological potential. Medicina Universitaria, 19(74), 32–37.CrossRefGoogle Scholar
  19. 19.
    Gomes, A., Bhattacharjee, P., Mishra, R., Biswas, A. K., Dasgupta, S. C., & Giri, B. (2010). Anticancer potential of animal venoms and toxins. Indian Journal of Experimental Biology, 48(2), 93–103.Google Scholar
  20. 20.
    Vyas, V., Brahmbhatt, K., Bhatt, H., & Parmar, U. (2013). Therapeutic potential of snake venom in cancer therapy: current perspectives. Asian Pacific Journal of Tropical Biomedicine, 3(2), 156–162.CrossRefGoogle Scholar
  21. 21.
    Liu, Z., Wang, F., & Chen, X. (2008). Integrin αvβ3-targeted cancer therapy. Drug Development Research, 69(6), 329–339.CrossRefGoogle Scholar
  22. 22.
    Kumar, C. C. (2003). Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Current Drug Targets, 4(2), 123–131.CrossRefGoogle Scholar
  23. 23.
    Zhou, X. D., Jin, Y., Chen, R. Q., Lu, Q. M., Wu, J. B., Wang, W. Y., & Xiong, Y. L. (2004). Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom. Toxicon, 43(1), 69–75.CrossRefGoogle Scholar
  24. 24.
    Raab-Westphal, S., Marshall, J. F., & Goodman, S. L. (2017). Integrins as therapeutic targets: successes and cancers. Cancers, 9(9), 110.CrossRefGoogle Scholar
  25. 25.
    Goçmen, B., Heiss, P., Petras, D., Nalbantsoy, A., & Süssmuth, R. D. (2015). Mass spectrometry guided venom profiling and bioactivity screening of the Anatolian Meadow Viper, Vipera anatolica. Toxicon, 107(Pt B), 163–174.CrossRefGoogle Scholar
  26. 26.
    Erdem, A., & Ozsoz, M. (2002). Electrochemical DNA biosensors based on DNA-drug interactions. Electroanalysis, 14(14), 965–974.CrossRefGoogle Scholar
  27. 27.
    Top, M., Er, O., Congur, G., Erdem, A., & Lambrecht, F. Y. (2016). Intracellular uptake study of radiolabeled anticancer drug and impedimetric detection of its interaction with DNA. Talanta, 160, 157–163.CrossRefGoogle Scholar
  28. 28.
    Fathi, F., Rahbarghazi, R., & Rashidi, M. (2017). Label-free biosensors in the field of stem cell biology. Biosensors & Bioelectronics, 101, 188–198.CrossRefGoogle Scholar
  29. 29.
    Vanegas, D. C., Gomes, C. L., Cavallaro, N. D., Giraldo-Escobar, D., & McLamore, E. S. (2017). Emerging biorecognition and transduction schemes for rapid detection of pathogenic bacteria in food. Comprehensive Reviews in Food Science and Food Safety, 16(6), 1188–1205.CrossRefGoogle Scholar
  30. 30.
    Felix, F. S., & Angnes, L. (2017). Electrochemical immunosensors – a powerful tool for analytical applications. Biosensors and Bioelectronics, 102, 470–478.CrossRefGoogle Scholar
  31. 31.
    Malekzad, H., Jouyban, A., Hasanzadeh, M., Shadjou, N., & Guardia, M. (2017). Ensuring food safety using aptamer based assays: electroanalytical approach. TrAC Trends in Analytical Chemistry, 94, 77–94.CrossRefGoogle Scholar
  32. 32.
    Yilmaz, N., Eksin, E., Karacicek, B., Erac, Y., & Erdem, A. (2017). Electrochemical detection of interaction between capsaicin and nucleic acids in comparison to agarose gel electrophoresis. Analytical Biochemistry, 535, 56–62.CrossRefGoogle Scholar
  33. 33.
    Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1–2), 55–63.CrossRefGoogle Scholar
  34. 34.
    Yalcın, H. T., Ozen, M. O., Gocmen, B., & Nalbantsoy, A. (2014). Effect of Ottoman viper (Montivipera xanthina (Gray, 1849)) venom on various cancer cells and on microorganisms. Cytotechnology, 66(1), 87–94.CrossRefGoogle Scholar
  35. 35.
    Karadeniz, H., Armagan, G., Erdem, A., Turunç, E., Çalıskan, A., Kanit, L., & Yalçın, A. (2009). The comparison of electrochemical assay and agarose gel electrophoresis for the determination of DNA damage induced by kainic acid. Electroanalysis, 21, 2468–2476.Google Scholar
  36. 36.
    Ersöz, O. A., Soylu, H. M., Er, O., Ocakoglu, K., Lambrecht, F. Y., & Yilmaz, O. (2015). Synthesis, radiolabeling, and bioevaluation of Bis (trifluoromethanesulfonyl) imide. Cancer Biotherapy & Radiopharmaceuticals, 30, 395–399.CrossRefGoogle Scholar
  37. 37.
    Avşar, G., Sari, F. A., Yuzer, A. C., Soylu, H. M., Er, O., Ince, M., & Lambrecht, F. Y. (2016). Intracellular uptake and fluorescence imaging potential in tumor cell of zinc phthalocyanine. International Journal of Pharmaceutics, 505(1-2), 369–375.CrossRefGoogle Scholar
  38. 38.
    DeLand, F. H., & Shih, W. J. (1984). The status of SPECT in tumor diagnosis. Journal of Nuclear Medicine, 25(12), 1375–1379.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Nuclear Applications, Institute of Nuclear SciencesEge UniversityIzmirTurkey
  2. 2.Department of Analytical Chemistry, Faculty of PharmacyEge UniversityIzmirTurkey
  3. 3.The Institute of Natural and Applied Sciences, Biotechnology DepartmentEge UniversityIzmirTurkey
  4. 4.The Institute of Natural and Applied Sciences, Biomedical Technology DepartmentEge UniversityIzmirTurkey
  5. 5.Zoology Section, Department of Biology, Faculty of ScienceEge UniversityIzmirTurkey
  6. 6.Department of Bioengineering, Faculty of EngineeringEge UniversityIzmirTurkey

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