Investigational New Drugs

, Volume 35, Issue 3, pp 260–268

A truncated apoptin protein variant selectively kills cancer cells

  • Santiago Ruiz-Martínez
  • Jessica Castro
  • Maria Vilanova
  • Marta Bruix
  • Douglas V. Laurents
  • Marc Ribó
  • Antoni Benito


Apoptin is a nonstructural protein encoded by one of the three open reading frames of the chicken anemia virus genome. It has attracted a great deal of interest due to its ability to induce apoptosis in multiple transformed and malignant mammalian cell lines without affecting primary and non-transformed cells. However, the use of Apoptin as an anticancer drug is restricted by its strong tendency to aggregate. A number of methods to overcome this problem have been proposed, including transduction techniques to deliver the Apoptin gene into tumor cells, but all such methods have certain drawbacks. Here we describe that a truncated variant of Apoptin, lacking residues 1 to 43, is a soluble, non-aggregating protein that maintains most of the biological properties of wild-type Apoptin when transfected into cells. We show that the cytotoxic effect of this variant is also present when it is added exogenously to cancer cells, but not to normal cells. In addition to the interest this protein has attracted as a promising therapeutic strategy, it is also an excellent model to study the structural properties of Apoptin and how they relate to its mechanism of action.


Viral protein Anticancer drug Aggregation Protein engineering Apoptin 

Supplementary material

10637_2017_431_MOESM1_ESM.docx (2.6 mb)
ESM 1(DOCX 2694 kb)


  1. 1.
    Prasetyo AA, Kamahora T, Kuroishi A et al (2009) Replication of chicken anemia virus (CAV) requires apoptin and is complemented by VP3 of human torque Teno virus (TTV). Virology 385:85–92. doi:10.1016/j.virol.2008.10.043 CrossRefPubMedGoogle Scholar
  2. 2.
    Maddika S, Mendoza FJ, Hauff K et al (2006) Cancer-selective therapy of the future: apoptin and its mechanism of action. Cancer Biol Ther 5:10–19. doi:10.4161/cbt.5.1.2400 CrossRefPubMedGoogle Scholar
  3. 3.
    Poon IKH, Oro C, Dias MM et al (2005) A tumor cell-specific nuclear targeting signal within chicken anemia virus VP3/apoptin. J Virol 79:1339–1341. doi:10.1128/JVI.79.2.1339-1341.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Leliveld SR, Zhang Y-H, Rohn JL et al (2003) Apoptin induces tumor-specific apoptosis as a globular multimer. J Biol Chem 278:9042–9051. doi:10.1074/jbc.M210803200 CrossRefPubMedGoogle Scholar
  5. 5.
    Leliveld SR, Dame RT, Mommaas MA et al (2003) Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA. Nucleic Acids Res 31:4805–4813CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Danen-Van Oorschot AAAM, Zhang Y-HH, Leliveld SR et al (2003) Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis. J Biol Chem 278:27729–27736. doi:10.1074/jbc.M303114200 CrossRefPubMedGoogle Scholar
  7. 7.
    Rollano Peñaloza OM, Lewandowska M, Stetefeld J et al (2014) Apoptins: selective anticancer agents. Trends Mol Med 20:519–528. doi:10.1016/j.molmed.2014.07.003 CrossRefPubMedGoogle Scholar
  8. 8.
    Rohn JL (2002) A tumor-specific kinase activity regulates the viral death protein apoptin. J Biol Chem 277:50820–50827. doi:10.1074/jbc.M208557200 CrossRefPubMedGoogle Scholar
  9. 9.
    Poon IKH, Oro C, Dias MM et al (2005) Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumor cells. Cancer Res 65:7059–7064. doi:10.1158/0008-5472.CAN-05-1370 CrossRefPubMedGoogle Scholar
  10. 10.
    Zhou S, Zhang M, Zhang J et al (2012) Mechanisms of apoptin-induced cell death. Med Oncol 29:2985–2991. doi:10.1007/s12032-011-0119-2 CrossRefPubMedGoogle Scholar
  11. 11.
    Leliveld SR, Dame RT, Rohn JL et al (2004) Apoptin’s functional N- and C-termini independently bind DNA. FEBS Lett 557:155–158CrossRefPubMedGoogle Scholar
  12. 12.
    Guelen L, Paterson H, Gäken J et al (2004) TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells. Oncogene 23:1153–1165. doi:10.1038/sj.onc.1207224 CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang M, Guller S, Huang Y (2007) Method to enhance transfection efficiency of cell lines and placental fibroblasts. Placenta 28:779–782. doi:10.1016/j.placenta.2007.01.012 CrossRefPubMedGoogle Scholar
  14. 14.
    Castro J, Ribó M, Puig T et al (2012) A cytotoxic ribonuclease reduces the expression level of P-glycoprotein in multidrug-resistant cell lines. Invest New Drugs 30:880–888. doi:10.1007/s10637-011-9636-2 CrossRefPubMedGoogle Scholar
  15. 15.
    Castro J, Ribó M, Navarro S et al (2011) A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation. BMC Cancer 11:9. doi:10.1186/1471-2407-11-9 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Gras SL, Waddington LJ, Goldie KN (2011) Transmission electron microscopy of amyloid fibrils. Methods Mol Biol 752:197–214. doi:10.1007/978-1-60327-223-0_13 CrossRefPubMedGoogle Scholar
  17. 17.
    Noteborn MH, Todd D, Verschueren CA et al (1994) A single chicken anemia virus protein induces apoptosis. J Virol 68:346–351PubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhang YH, Leliveld SR, Kooistra K et al (2003) Recombinant apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion. Exp Cell Res 289:36–46. doi:10.1016/S0014-4827(03)00188-5 CrossRefPubMedGoogle Scholar
  19. 19.
    Ruiz-Martínez S, Pantoja-Uceda D, Castro J et al (2017) Insights into the mechanism of Apoptin’s exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics. Arch Biochem Biophys 614:53–64. doi:10.1016/ CrossRefPubMedGoogle Scholar
  20. 20.
    Gualfetti PJ, Iwakura M, Lee JC et al (1999) Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein. Biochemistry 38:13367–13378CrossRefPubMedGoogle Scholar
  21. 21.
    Rohn JL, Zhang Y-H, Leliveld SR et al (2005) Relevance of apoptin’s integrity for its functional behavior. J Virol 79:1337–1338. doi:10.1128/JVI.79.2.1337-1338.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Pavlakis N, Vogelzang NJ (2006) Ranpirnase-an antitumour ribonuclease: its potential role in malignant mesothelioma. Expert Opin Biol Ther 6:391–399. doi:10.1517/14712598.6.4.391 CrossRefPubMedGoogle Scholar
  23. 23.
    Zhao J, Gao P, Xiao W et al (2011) A novel human derived cell-penetrating peptide in drug delivery. Mol Biol Rep 38:2649–2656. doi:10.1007/s11033-010-0406-6 CrossRefPubMedGoogle Scholar
  24. 24.
    Sun J, Yan Y, Wang X-T et al (2009) PTD4-apoptin protein therapy inhibits tumor growth in vivo. Int J Cancer 124:2973–2981. doi:10.1002/ijc.24279 CrossRefPubMedGoogle Scholar
  25. 25.
    Zhao M, Hu B, Gu Z et al (2013) Degradable polymeric nanocapsule for efficient intracellular delivery of a high molecular weight tumor-selective protein complex. Nano Today 8:11–20. doi:10.1016/j.nantod.2012.12.003 CrossRefGoogle Scholar
  26. 26.
    Ring J, Seifert J, Jesch F, Brendel W (1977) Anaphylactoid reactions due to non-immune complex serum protein aggregates. Monogr Allergy 12:27–35PubMedGoogle Scholar
  27. 27.
    Benito A, Vilanova M, Ribó M (2008) Intracellular routing of cytotoxic pancreatic-type ribonucleases. Curr Pharm Biotechnol 9:169–179CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Laboratori d’Enginyeria de Proteïnes, Departament de Biologia, Facultat de CiènciesUniversitat de Girona and Institut d’Investigació Biomèdica de Girona Josep Trueta (IdIBGi)GironaSpain
  2. 2.Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones CientíficasMadridSpain

Personalised recommendations