Tumor Biology

, Volume 36, Issue 8, pp 6037–6043 | Cite as

Oncolytic adenovirus-mediated short hairpin RNA targeting MYCN gene induces apoptosis by upregulating RKIP in neuroblastoma

Research Article


The amplification of MYCN is a typical characteristic of aggressive neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases. We had previously examined the applicability of the replication-competent oncolytic adenovirus, ZD55-shMYCN, to deliver a short hairpin RNA targeting MYCN gene for p53-null and MYCN-amplified neuroblastoma cell line LA1-55N. Our data have shown that ZD55-shMYCN has an additive tumor growth inhibitory response through shRNA-mediated MYCN knockdown and ZD55-mediated cancer cell lysis. In this regard, ZD55-shMYCN can downregulate MYCN and perform anticancer effects, thereby acquiring significance in the administration of MYCN-amplified and p53-null neuroblastomas. Hence, we further investigated the anticancer properties of ZD55-shMYCN in neuroblastomas. Our data showed that ZD55-shMYCN induced G2/M arrest via decreasing the levels of cyclin D1 and cyclin B1 irrespective of p53 status. ZD55-shMYCN effectively induced apoptosis in neuroblastomas through activation of caspase-3 and enhancing PARP cleavage. Furthermore, ZD55-shMYCN could downregulate phosphoinositide 3-kinase and pAkt and upregulate RKIP levels. Similarly, pro-apoptosis was revealed by the histopathologic examination of paraffin-embedded section of resected tumors of mice xenograft. In vitro and in vivo studies, we elucidate the apoptosis properties and mechanisms of action of ZD55-shMYCN, which provide a promising approach for further clinical development.


Neuroblastoma Oncolytic adenovirus MYCN Short hairpin RNA Apoptosis 


Conflicts of interest



  1. 1.
    Matthay KK. Neuroblastoma: a clinical challenge and biologic puzzle. CA: Cancer J Clin. 1995;45(3):179–92.Google Scholar
  2. 2.
    Castleberry RP. Predicting outcome in neuroblastoma. N Engl J Med. 1999;340(25):1992–3. doi: 10.1056/NEJM199906243402510.CrossRefPubMedGoogle Scholar
  3. 3.
    Cornero A, Acquaviva M, Fardin P, Versteeg R, Schramm A, Eva A, et al. Design of a multi-signature ensemble classifier predicting neuroblastoma patients’ outcome. BMC Bioinf. 2012;13(4):S13. doi: 10.1186/1471-2105-13-S4-S13.CrossRefGoogle Scholar
  4. 4.
    Oh YK, Park TG. siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev. 2009;61(10):850–62. doi: 10.1016/j.addr.2009.04.018.CrossRefPubMedGoogle Scholar
  5. 5.
    Kang JH, Rychahou PG, Ishola TA, Qiao J, Evers BM, Chung DH. MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells. Biochem Biophys Res Commun. 2006;351(1):192–7. doi: 10.1016/j.bbrc.2006.10.020.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Novina CD, Sharp PA. The RNAi revolution. Nature. 2004;430(6996):161–4. doi: 10.1038/430161a.CrossRefPubMedGoogle Scholar
  7. 7.
    Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 2003;327(4):761–6.CrossRefPubMedGoogle Scholar
  8. 8.
    Carette JE, Overmeer RM, Schagen FH, Alemany R, Barski OA, Gerritsen WR, et al. Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells. Cancer Res. 2004;64(8):2663–7.CrossRefPubMedGoogle Scholar
  9. 9.
    Wang QZ, Lv YH, Diao Y, Xu R. The design of vectors for RNAi delivery system. Curr Pharm Des. 2008;14(13):1327–40.CrossRefPubMedGoogle Scholar
  10. 10.
    Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science. 1991;252(5007):854–6.CrossRefPubMedGoogle Scholar
  11. 11.
    Toth K, Doronin K, Tollefson AE, Wold WS. A multitasking oncolytic adenovirus vector. Mol Ther: J Am Soc Gene Ther. 2003;7(4):435–7.CrossRefGoogle Scholar
  12. 12.
    Lin E, Nemunaitis J. Oncolytic viral therapies. Cancer Gene Ther. 2004;11(10):643–64. doi: 10.1038/sj.cgt.7700733.CrossRefPubMedGoogle Scholar
  13. 13.
    Aghi M, Martuza RL. Oncolytic viral therapies—the clinical experience. Oncogene. 2005;24(52):7802–16. doi: 10.1038/sj.onc.1209037.CrossRefPubMedGoogle Scholar
  14. 14.
    Zhang ZL, Zou WG, Luo CX, Li BH, Wang JH, Sun LY, et al. An armed oncolytic adenovirus system, ZD55-gene, demonstrating potent antitumoral efficacy. Cell Res. 2003;13(6):481–9. doi: 10.1038/ Scholar
  15. 15.
    Liu XY. Targeting gene-virotherapy of cancer and its prosperity. Cell Res. 2006;16(11):879–86. doi: 10.1038/ Scholar
  16. 16.
    Li Y, Zhang B, Zhang H, Zhu X, Feng D, Zhang D, et al. Oncolytic adenovirus armed with shRNA targeting MYCN gene inhibits neuroblastoma cell proliferation and in vivo xenograft tumor growth. J Cancer Res Clin Oncol. 2013;139(6):933–41. doi: 10.1007/s00432-013-1406-4.CrossRefPubMedGoogle Scholar
  17. 17.
    van Noesel MM, Versteeg R. Pediatric neuroblastomas: genetic and epigenetic ‘danse macabre’. Gene. 2004;325:1–15.CrossRefPubMedGoogle Scholar
  18. 18.
    Waters AM, Beierle EA. The interaction between FAK, MYCN, p53 and Mdm2 in neuroblastoma. Anti Cancer Agents Med Chem. 2014;14(1):46–51.CrossRefGoogle Scholar
  19. 19.
    Singhal J, Yadav S, Nagaprashantha LD, Vatsyayan R, Singhal SS, Awasthi S. Targeting p53-null neuroblastomas through RLIP76. Cancer Prev Res (Phila). 2011;4(6):879–89. doi: 10.1158/1940-6207.CAPR-11-0025.CrossRefGoogle Scholar
  20. 20.
    Singhal SS, Wickramarachchi D, Yadav S, Singhal J, Leake K, Vatsyayan R, et al. Glutathione-conjugate transport by RLIP76 is required for clathrin-dependent endocytosis and chemical carcinogenesis. Mol Cancer Ther. 2011;10(1):16–28. doi: 10.1158/1535-7163.MCT-10-0699.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Chesler L, Schlieve C, Goldenberg DD, Kenney A, Kim G, McMillan A, et al. Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma. Cancer Res. 2006;66(16):8139–46. doi: 10.1158/0008-5472.CAN-05-2769.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chatterjee D, Bai Y, Wang Z, Beach S, Mott S, Roy R, et al. RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis. J Biol Chem. 2004;279(17):17515–23. doi: 10.1074/jbc.M313816200.CrossRefPubMedGoogle Scholar
  23. 23.
    Dangi-Garimella S, Yun J, Eves EM, Newman M, Erkeland SJ, Hammond SM, et al. Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. EMBO J. 2009;28(4):347–58. doi: 10.1038/emboj.2008.294.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Yeung K, Seitz T, Li S, Janosch P, McFerran B, Kaiser C, et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature. 1999;401(6749):173–7. doi: 10.1038/43686.CrossRefPubMedGoogle Scholar
  25. 25.
    Hellmann J, Rommelspacher H, Muhlbauer E, Wernicke C. Raf kinase inhibitor protein enhances neuronal differentiation in human SH-SY5Y cells. Dev Neurosci. 2010;32(1):33–46. doi: 10.1159/000236595.CrossRefPubMedGoogle Scholar
  26. 26.
    Moreno L, Marshall LV, Pearson AD. At the frontier of progress for paediatric oncology: the neuroblastoma paradigm. Br Med Bull. 2013;108:173–88. doi: 10.1093/bmb/ldt033.CrossRefPubMedGoogle Scholar
  27. 27.
    Levy D, Aerts I, Michon J, Lumbroso-Le Rouic L, Cellier C, Orbach D. Childhood cancer: progress but prognosis still very unequal. Example of retinoblastoma and high-risk neuroblastoma. Bull Cancer. 2014;101(3):250–7. doi: 10.1684/bdc.2014.1904.PubMedGoogle Scholar
  28. 28.
    Charron M. Contemporary approach to diagnosis and treatment of neuroblastoma. Q J Nucl Med Mol Imaging. 2013;57(1):40–52.PubMedGoogle Scholar
  29. 29.
    Stacey DW. Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Curr Opin Cell Biol. 2003;15(2):158–63.CrossRefPubMedGoogle Scholar
  30. 30.
    Guo Y, Stacey DW, Hitomi M. Post-transcriptional regulation of cyclin D1 expression during G2 phase. Oncogene. 2002;21(49):7545–56. doi: 10.1038/sj.onc.1205907.CrossRefPubMedGoogle Scholar
  31. 31.
    Gabrielli BG, Sarcevic B, Sinnamon J, Walker G, Castellano M, Wang XQ, et al. A cyclin D-Cdk4 activity required for G2 phase cell cycle progression is inhibited in ultraviolet radiation-induced G2 phase delay. J Biol Chem. 1999;274(20):13961–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Chen Q, Zhang X, Jiang Q, Clarke PR, Zhang C. Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis. Cell Res. 2008;18(2):268–80. doi: 10.1038/cr.2008.11.CrossRefPubMedGoogle Scholar
  33. 33.
    Innocente SA, Abrahamson JL, Cogswell JP, Lee JM. p53 regulates a G2 checkpoint through cyclin B1. Proc Natl Acad Sci U S A. 1999;96(5):2147–52.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Park M, Chae HD, Yun J, Jung M, Kim YS, Kim SH, et al. Constitutive activation of cyclin B1-associated cdc2 kinase overrides p53-mediated G2-M arrest. Cancer Res. 2000;60(3):542–5.PubMedGoogle Scholar
  35. 35.
    Budihardjo I, Oliver H, Lutter M, Luo X, Wang X. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol. 1999;15:269–90. doi: 10.1146/annurev.cellbio.15.1.269.CrossRefPubMedGoogle Scholar
  36. 36.
    Sairanen T, Szepesi R, Karjalainen-Lindsberg ML, Saksi J, Paetau A, Lindsberg PJ. Neuronal caspase-3 and PARP-1 correlate differentially with apoptosis and necrosis in ischemic human stroke. Acta Neuropathol. 2009;118(4):541–52. doi: 10.1007/s00401-009-0559-3.CrossRefPubMedGoogle Scholar
  37. 37.
    Yeung K, Janosch P, McFerran B, Rose DW, Mischak H, Sedivy JM, et al. Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein. Mol Cell Biol. 2000;20(9):3079–85.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Trakul N, Rosner MR. Modulation of the MAP kinase signaling cascade by Raf kinase inhibitory protein. Cell Res. 2005;15(1):19–23. doi: 10.1038/ Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  1. 1.Department of Pediatric SurgeryXuzhou Children’s HospitalXuzhouChina
  2. 2.Laboratory of Biological Cancer TherapyXuzhou Medical CollegeXuzhouChina

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