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Gene electrotransfer of plasmid AMEP, an integrin-targeted therapy, has antitumor and antiangiogenic action in murine B16 melanoma

Gene Therapy volume 22, pages 578–590 (2015)Cite this article

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Abstract

Gene therapy with Plasmid AMEP (antiangiogenic metargidin peptide) has recently been studied as a potential targeted therapy for melanoma. This plasmid is designed to downregulate α5β1 and αvβ3 integrins. In our study, electroporation was used as a nonviral delivery system. We investigated the antiangiogenic and direct antitumor effectiveness of this gene therapy on low and highly metastatic B16 melanoma variants. In vitro, the antiangiogenic effectiveness as determined by tube formation assay on endothelial cells was predominantly dependent on AMEP expression levels. In vivo, antitumor effectiveness was mediated by the inhibition of proliferation, migration and invasion of melanoma cells and correlated with the expression of integrins on tumor cells after intratumor delivery. In addition, reduced metastatic potential was shown. Intramuscular gene electrotransfer of Plasmid AMEP, for AMEP systemic distribution, had no antitumor effect with this specific preventive treatment protocol, confirming that direct tumor delivery was more effective. This study confirms our previous in vitro data that the expression levels of integrins on melanoma cells could be used as a biomarker for antitumor effectiveness in integrin-targeted therapies, whereas the expression levels of AMEP peptide could be a predictive factor for antiangiogenic effectiveness of Plasmid AMEP in the treatment of melanoma.

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References

  1. Heller LC, Heller R . Electroporation gene therapy preclinical and clinical trials for melanoma. Curr Gene Ther 2010; 10: 312–317.

    Article  CAS  Google Scholar 

  2. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK et al. Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 2008; 26: 5896–5903.

    Article  CAS  Google Scholar 

  3. Cemazar M, Jarm T, Sersa G . Cancer electrogene therapy with interleukin-12. Curr Gene Ther 2010; 10: 300–311.

    Article  CAS  Google Scholar 

  4. Mizejewski GJ . Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med 1999; 222: 124–138.

    Article  CAS  Google Scholar 

  5. Trochon-Joseph V, Martel-Renoir D, Mir LM, Thomaidis A, Opolon P, Connault E et al. Evidence of antiangiogenic and antimetastatic activities of the recombinant disintegrin domain of metargidin. Cancer Res 2004; 64: 2062–2069.

    Article  CAS  Google Scholar 

  6. Daugimont L, Vandermeulen G, Defresne F, Bouzin C, Mir LM, Bouquet C et al. Antitumoral and antimetastatic effect of antiangiogenic plasmids in B16 melanoma: higher efficiency of the recombinant disintegrin domain of ADAM 15. Eur J Pharm Biopharm 2011; 78: 314–319.

    Article  CAS  Google Scholar 

  7. Bosnjak M, Prosen L, Dolinsek T, Blagus T, Markelc B, Cemazar M et al. Biological properties of melanoma and endothelial cells after plasmid AMEP gene electrotransfer depend on integrin quantity on cells. J Membr Biol 2013; 246: 803–819.

    Article  CAS  Google Scholar 

  8. Spanggaard I, Snoj M, Cavalcanti A, Bouquet C, Sersa G, Robert C et al. Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma: safety and efficacy results of a phase I first-in-man study. Hum Gene Ther Clin Dev 2013; 24: 99–107.

    Article  CAS  Google Scholar 

  9. Tesic N, Cemazar M . In vitro targeted gene electrotransfer to endothelial cells with plasmid DNA containing human endothelin-1 promoter. J Membr Biol 2013; 246: 783–791.

    Article  CAS  Google Scholar 

  10. Bosnjak M, Lorente BC, Pogacar Z, Makovsek V, Cemazar M . Different incubation times of cells after gene electrotransfer in fetal bovine serum affect cell viability, but not transfection efficiency. J Membr Biol 2014; 247: 421–428.

    Article  CAS  Google Scholar 

  11. Tevz G, Kranjc S, Cemazar M, Kamensek U, Coer A, Krzan M et al. Controlled systemic release of interleukin-12 after gene electrotransfer to muscle for cancer gene therapy alone or in combination with ionizing radiation in murine sarcomas. J Gene Med 2009; 11: 1125–1137.

    Article  CAS  Google Scholar 

  12. Mir LM, Banoun H, Paoletti C . Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol. Exp Cell Res 1988; 175: 15–25.

    Article  CAS  Google Scholar 

  13. Mir LM, Orlowski S, Belehradek J Jr., Paoletti C . Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. Eur J Cancer 1991; 27: 68–72.

    Article  CAS  Google Scholar 

  14. Sersa G, Cemazar M, Miklavcic D . Antitumor effectiveness of electrochemotherapy with cis-diamminedichloroplatinum(II) in mice. Cancer Res 1995; 55: 3450–3455.

    CAS  PubMed  Google Scholar 

  15. Cemazar M, Miklavcic D, Sersa G . Intrinsic sensitivity of tumor cells to bleomycin as an indicator of tumor response to electrochemotherapy. Jpn J Cancer Res 1998; 89: 328–333.

    Article  CAS  Google Scholar 

  16. Jaroszeski MJ, Dang V, Pottinger C, Hickey J, Gilbert R, Heller R . Toxicity of anticancer agents mediated by electroporation in vitro. Anticancer Drugs 2000; 11: 201–208.

    Article  CAS  Google Scholar 

  17. Sersa G, Miklavcic D . Electrochemotherapy of tumours. J Vis Exp 2008; 15: 1038.

    Google Scholar 

  18. Sedlar A, Dolinsek T, Markelc B, Prosen L, Kranjc S, Bosnjak M et al. Potentiation of electrochemotherapy by intramuscular IL-12 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity. Radiol Oncol 2012; 46: 302–311.

    Article  CAS  Google Scholar 

  19. Teissie J, Escoffre JM, Paganin A, Chabot S, Bellard E, Wasungu L et al. Drug delivery by electropulsation: Recent developments in oncology. Int J Pharm 2012; 423: 3–6.

    Article  CAS  Google Scholar 

  20. Edhemovic I, Brecelj E, Gasljevic G, Marolt Music M, Gorjup V, Mali B et al. Intraoperative electrochemotherapy of colorectal liver metastases. J Surg Oncol 2014; 110: 320–327.

    Article  Google Scholar 

  21. Miklavcic D, Mali B, Kos B, Heller R, Sersa G . Electrochemotherapy: from the drawing board into medical practice. Biomed Eng Online 2014; 13: 29.

    Article  Google Scholar 

  22. Mali B, Miklavcic D, Campana LG, Cemazar M, Sersa G, Snoj M et al. Tumor size and effectiveness of electrochemotherapy. Radiol Oncol 2013; 47: 32–41.

    Article  Google Scholar 

  23. Rols MP, Delteil C, Golzio M, Dumond P, Cros S, Teissie J . In vivo electrically mediated protein and gene transfer in murine melanoma. Nat Biotechnol 1998; 16: 168–171.

    Article  CAS  Google Scholar 

  24. Jaroszeski MJ, Gilbert R, Nicolau C, Heller R . In vivo gene delivery by electroporation. Adv Drug Deliv Rev 1999; 35: 131–137.

    Article  CAS  Google Scholar 

  25. Golzio M, Rols MP, Teissie J . In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression. Methods 2004; 33: 126–135.

    Article  CAS  Google Scholar 

  26. Vidic S, Markelc B, Sersa G, Coer A, Kamensek U, Tevz G et al. MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo. Cancer Gene Ther 2010; 17: 409–419.

    Article  CAS  Google Scholar 

  27. Chabot S, Pelofy S, Paganin-Gioanni A, Teissie J, Golzio M . Electrotransfer of RNAi-based oligonucleotides for oncology. Anticancer Res 2011; 31: 4083–4089.

    CAS  PubMed  Google Scholar 

  28. Dolinsek T, Markelc B, Sersa G, Coer A, Stimac M, Lavrencak J et al. Multiple delivery of siRNA against endoglin into murine mammary adenocarcinoma prevents angiogenesis and delays tumor growth. PLoS One 2013; 8: e58723.

    Article  CAS  Google Scholar 

  29. Kamensek U, Sersa G, Cemazar M . Evaluation of p21 promoter for interleukin 12 radiation induced transcriptional targeting in a mouse tumor model. Mol Cancer 2013; 12: 136.

    Article  Google Scholar 

  30. Prosen L, Markelc B, Dolinsek T, Music B, Cemazar M, Sersa G . Mcam silencing with RNA interference using magnetofection has antitumor effect in murine melanoma. Mol Ther Nucleic Acids 2014; 3: e205.

    Article  CAS  Google Scholar 

  31. Crokart N, Danhier F, Daugimont L, Goncalves N, Jordan BF, Gregoire V et al. Potentiation of radiotherapy by a localized antiangiogenic gene therapy. Radiother Oncol 2013; 107: 252–258.

    Article  Google Scholar 

  32. Robinson SD, Hodivala-Dilke KM . The role of beta3-integrins in tumor angiogenesis: context is everything. Curr Opin Cell Biol 2011; 23: 630–637.

    Article  CAS  Google Scholar 

  33. Niemisto A, Dunmire V, Yli-Harja O, Zhang W, Shmulevich I . Robust quantification of in vitro angiogenesis through image analysis. IEEE Trans Med Imaging 2005; 24: 549–553.

    Article  Google Scholar 

  34. Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402–408.

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge the financial support from the state budget by the Slovenian Research Agency (programs no. P3-0003 and P1-0140 and projects no. J3-4211, J3-4259). The research was conducted in the scope of LEA EBAM (French-Slovenian European Associated Laboratory: Pulsed Electric Fields Applications in Biology and Medicine) and is a result of networking efforts within COST TD1104 Action. We thank Mira Lavric, Ursa Lampreht and Natasa Tesic (Institute of Oncology Ljubljana, Ljubljana, Slovenia) and Miha Butinar (Jozef Stefan Institute) for all the valuable work they contributed to this research.

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Authors and Affiliations

  1. Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, Ljubljana, Slovenia

    M Bosnjak, T Dolinsek, M Cemazar, S Kranjc, T Blagus, B Markelc, M Stimac, U Kamensek & G Sersa

  2. Faculty of Health Sciences, University of Primorska, Polje 42, Izola, Slovenia

    M Cemazar

  3. Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Jamova cesta 39, Ljubljana, Slovenia

    J Zavrsnik & B Turk

  4. Frank Reidy Research Center for Bioelectrics, Old Dominion University, 4211 Monarch Way, Norfolk, Virginia, USA

    L Heller

  5. ONXEO, Boulevard du Général Martial Valin 49, Paris, France

    C Bouquet

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  1. M Bosnjak
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  2. T Dolinsek
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Corresponding author

Correspondence to G Sersa.

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Competing interests

CB is a stockholder and an employee at ONXEO. The remaining authors declare no conflict of interest.

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Bosnjak, M., Dolinsek, T., Cemazar, M. et al. Gene electrotransfer of plasmid AMEP, an integrin-targeted therapy, has antitumor and antiangiogenic action in murine B16 melanoma. Gene Ther 22, 578–590 (2015). https://doi.org/10.1038/gt.2015.26

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  • DOI: https://doi.org/10.1038/gt.2015.26

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