Frontiers of Medicine

, Volume 11, Issue 1, pp 120–128 | Cite as

Cotransfecting norepinephrine transporter and vesicular monoamine transporter 2 genes for increased retention of metaiodobenzylguanidine labeled with iodine 131 in malignant hepatocarcinoma cells

  • Yanlin Zhao
  • Xiao Zhong
  • Xiaohong Ou
  • Huawei Cai
  • Xiaoai Wu
  • Rui Huang
Research Article


Norepinephrine transporter (NET) transfection leads to significant uptake of iodine-131-labeled metaiodobenzylguanidine (131I-MIBG) in non-neuroendocrine tumors. However, the use of 131I-MIBG is limited by its short retention time in target cells. To prolong the retention of 131I-MIBG in target cells, we infected hepatocarcinoma (HepG2) cells with Lentivirus-encoding human NET and vesicular monoamine transporter 2 (VMAT2) genes to obtain NET-expressing, NET-VMAT2-coexpressing, and negative-control cell lines. We evaluated the uptake and efflux of 131I-MIBG both in vitro and in vivo in mice bearing transfected tumors. NET-expressing and NET-VMAT2-coexpressing cells respectively showed 2.24 and 2.22 times higher 131I-MIBG uptake than controls. Two hours after removal of 131I-MIBG-containing medium, 25.4% efflux was observed in NET-VMAT2-coexpressing cells and 38.6% in NET-expressing cells. In vivo experiments were performed in nude mice bearing transfected tumors; results revealed that NET-VMAT2-coexpressing tumors had longer 131I-MIBG retention time than NET-expressing tumors. Meanwhile, NET-VMAT2-coexpressing and NET-expressing tumors displayed 0.54% and 0.19%, respectively, of the injected dose per gram of tissue 24 h after 131I-MIBG administration. Cotransfection of HepG2 cells with NET and VMAT2 resulted in increased 131I-MIBG uptake and retention. However, the degree of increase was insufficient to be therapeutically effective in target cells.


norepinephrine transporter vesicular monoamine transporter 2 131I-MIBG gene therapy lentivirus vector 


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We thank Yuanyou Yang, PhD, for helping in the preparation of 131I-MIBG. This study was funded by the National Natural Science Foundation of China (No. 81271602).


  1. 1.
    Dubois SG, Geier E, Batra V, Yee SW, Neuhaus J, Segal M, Martinez D, Pawel B, Yanik G, Naranjo A, London WB, Kreissman S, Baker D, Attiyeh E, Hogarty MD, Maris JM, Giacomini K, Matthay KK. Evaluation of norepinephrine transporter expression and metaiodobenzylguanidine avidity in neuroblastoma: a report from the Children’s Oncology Group. Int J Mol Imaging 2012; 2012:250834CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Carlin S, Mairs RJ, McCluskey AG, Tweddle DA, Sprigg A, Estlin C, Board J, George RE, Ellershaw C, Pearson AD, Lunec J, Montaldo PG, Ponzoni M, van Eck-Smit BL, Hoefnagel CA, van den Brug MD, Tytgat GA, Caron HN. Development of a real-time polymerase chain reaction assay for prediction of the uptake of meta-[(131)I]iodobenzylguanidine by neuroblastoma tumors. Clin Cancer Res 2003; 9(9): 3338–3344PubMedGoogle Scholar
  3. 3.
    Lode HN, Bruchelt G, Seitz G, Gebhardt S, Gekeler V, Niethammer D, Beck J. Reverse transcriptase-polymerase chain reaction (RTPCR) analysis of monoamine transporters in neuroblastoma cell lines: correlations to meta-iodobenzylguanidine (MIBG) uptake and tyrosine hydroxylase gene expression. Eur J Cancer 1995; 31(4): 586–590CrossRefGoogle Scholar
  4. 4.
    Gonias S, Goldsby R, Matthay KK, Hawkins R, Price D, Huberty J, Damon L, Linker C, Sznewajs A, Shiboski S, Fitzgerald P. Phase II study of high-dose [131I]metaiodobenzylguanidine therapy for patients with metastatic pheochromocytoma and paraganglioma. J Clin Oncol 2009; 27(25): 4162–4168CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kang TI, Brophy P, Hickeson M, Heyman S, Evans AE, Charron M, Maris JM. Targeted radiotherapy with submyeloablative doses of 131I-MIBG is effective for disease palliation in highly refractory neuroblastoma. J Pediatr Hematol Oncol 2003; 25(10): 769–773CrossRefPubMedGoogle Scholar
  6. 6.
    Fitzgerald PA, Goldsby RE, Huberty JP, Price DC, Hawkins RA, Veatch JJ, Dela Cruz F, Jahan TM, Linker CA, Damon L, Matthay KK. Malignant pheochromocytomas and paragangliomas: a phase II study of therapy with high-dose 131I-metaiodobenzylguanidine (131I-MIBG). Ann N Y Acad Sci 2006; 1073(1): 465–490CrossRefPubMedGoogle Scholar
  7. 7.
    Matthay KK, Panina C, Huberty J, Price D, Glidden DV, Tang HR, Hawkins RA, Veatch J, Hasegawa B. Correlation of tumor and whole-body dosimetry with tumor response and toxicity in refractory neuroblastoma treated with (131)I-MIBG. J Nucl Med 2001; 42(11): 1713–1721PubMedGoogle Scholar
  8. 8.
    DuBois SG, Messina J, Maris JM, Huberty J, Glidden DV, Veatch J, Charron M, Hawkins R, Matthay KK. Hematologic toxicity of highdose iodine-131-metaiodobenzylguanidine therapy for advanced neuroblastoma. J Clin Oncol 2004; 22(12): 2452–2460CrossRefPubMedGoogle Scholar
  9. 9.
    Fullerton NE, Boyd M, Ross SC, Pimlott SL, Babich J, Kirk D, Zalutsky MR, Mairs RJ. Comparison of radiohaloanalogues of meta-iodobenzylguanidine (MIBG) for a combined gene- and targeted radiotherapy approach to bladder carcinoma. Med Chem 2005; 1(6): 611–618CrossRefPubMedGoogle Scholar
  10. 10.
    Mairs RJ, Ross SC, McCluskey AG, Boyd M. A transfectant mosaic xenograft model for evaluation of targeted radiotherapy in combination with gene therapy in vivo. J Nucl Med 2007; 48(9): 1519–1526CrossRefPubMedGoogle Scholar
  11. 11.
    Jia ZY, Deng HF, Huang R, Yang YY, Yang XC, Qi ZZ, Ou XH. In vitro and in vivo studies of adenovirus-mediated human norepinephrine transporter gene transduction to hepatocellular carcinoma. Cancer Gene Ther 2011; 18(3): 196–205CrossRefPubMedGoogle Scholar
  12. 12.
    Altmann A, Kissel M, Zitzmann S, Kübler W, Mahmut M, Peschke P, Haberkorn U. Increased MIBG uptake after transfer of the human norepinephrine transporter gene in rat hepatoma. J Nucl Med 2003; 44(6): 973–980PubMedGoogle Scholar
  13. 13.
    Fullerton NE, Mairs RJ, Kirk D, Keith WN, Carruthers R, McCluskey AG, Brown M, Wilson L, Boyd M. Application of targeted radiotherapy/gene therapy to bladder cancer cell lines. Eur Urol 2005; 47(2): 250–256CrossRefPubMedGoogle Scholar
  14. 14.
    Boyd M, Cunningham SH, Brown MM, Mairs RJ, Wheldon TE. Noradrenaline transporter gene transfer for radiation cell kill by 131I meta-iodobenzylguanidine. Gene Ther 1999; 6(6): 1147–1152CrossRefPubMedGoogle Scholar
  15. 15.
    Parsons SM. Transport mechanisms in acetylcholine and monoamine storage. FASEB J 2000; 14(15): 2423–2434CrossRefPubMedGoogle Scholar
  16. 16.
    Kölby L, Bernhardt P, Levin-Jakobsen AM, Johanson V, Wängberg B, Ahlman H, Forssell-Aronsson E, Nilsson O. Uptake of metaiodobenzylguanidine in neuroendocrine tumours is mediated by vesicular monoamine transporters. Br J Cancer 2003; 89(7): 1383–1388CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Temple W, Mendelsohn L, Kim GE, Nekritz E, Gustafson WC, Lin L, Giacomini K, Naranjo A, van Ryn C, Yanik GA, Kreissman SG, Hogarty M, Matthay KK, Du Bois SG. Vesicular monoamine transporter protein expression correlates with clinical features, tumor biology, and MIBG avidity in neuroblastoma: a report from the Children’s Oncology Group. Eur J Nucl Med Mol Imaging 2016; 43(3): 474–481CrossRefPubMedGoogle Scholar
  18. 18.
    Erickson JD, Schafer MK, Bonner TI, Eiden LE, Weihe E. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter. Proc Natl Acad Sci USA 1996; 93(10): 5166–5171CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Liu Y, Schweitzer ES, Nirenberg MJ, Pickel VM, Evans CJ, Edwards RH. Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells. J Cell Biol 1994; 127(5): 1419–1433CrossRefPubMedGoogle Scholar
  20. 20.
    Erickson JD, Eiden LE, Hoffman BJ. Expression cloning of a reserpine-sensitive vesicular monoamine transporter. Proc Natl Acad Sci USA 1992; 89(22): 10993–10997CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Liu Y, Peter D, Roghani A, Schuldiner S, Privé GG, Eisenberg D, Brecha N, Edwards RH. A cDNA that suppresses MPP + toxicity encodes a vesicular amine transporter. Cell 1992; 70(4): 539–551CrossRefPubMedGoogle Scholar
  22. 22.
    Erickson JD, Eiden LE. Functional identification and molecular cloning of a human brain vesicle monoamine transporter. J Neurochem 1993; 61(6): 2314–2317CrossRefPubMedGoogle Scholar
  23. 23.
    Gasnier B, Krejci E, Botton D, Massoulié J, Henry JP. Expression of a bovine vesicular monoamine transporter in COS cells. FEBS Lett 1994; 342(3): 225–229CrossRefPubMedGoogle Scholar
  24. 24.
    Zan LB, Yang YY, Jin JN, Ou XH. Synthesis of 3-trimethylsilylbenzylguanidine as precursor for 125I labelling. Atomic Energy Sci Technol (Yuan Zi Neng Ke Xue Ji Shu) 2007; 41(6): 689–693 (in Chinese)Google Scholar
  25. 25.
    Moroz MA, Serganova I, Zanzonico P, Ageyeva L, Beresten T, Dyomina E, Burnazi E, Finn RD, Doubrovin M, Blasberg RG. Imaging hNET reporter gene expression with 124I-MIBG. J Nucl Med 2007; 48(5): 827–836CrossRefPubMedGoogle Scholar
  26. 26.
    Bomanji J, Levison DA, Flatman WD, Horne T, Bouloux PM, Ross G, Britton KE, Besser GM. Uptake of iodine-123 MIBG by pheochromocytomas, paragangliomas, and neuroblastomas: a histopathological comparison. J Nucl Med 1987; 28(6): 973–978PubMedGoogle Scholar
  27. 27.
    Robson JA, Sidell N. Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid. Neuroscience 1985; 14(4): 1149–1162CrossRefPubMedGoogle Scholar
  28. 28.
    Iavarone A, Lasorella A, Servidei T, Riccardi R, Mastrangelo R. Uptake and storage of m-iodobenzylguanidine are frequent neuronal functions of human neuroblastoma cell lines. Cancer Res 1993; 53(2): 304–309PubMedGoogle Scholar
  29. 29.
    Taupenot L, Harper KL, O’Connor DT. The chromograninsecretogranin family. N Engl J Med 2003; 348(12): 1134–1149CrossRefPubMedGoogle Scholar
  30. 30.
    Stettler H, Beuret N, Prescianotto-Baschong C, Fayard B, Taupenot L, Spiess M. Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granulelike structures in non-endocrine cells. Biochem J 2009; 418(1): 81–91CrossRefPubMedGoogle Scholar
  31. 31.
    Huh YH, Jeon SH, Yoo SH. Chromogranin B-induced secretory granule biogenesis: comparison with the similar role of chromogranin A. J Biol Chem 2003; 278(42): 40581–40589CrossRefPubMedGoogle Scholar
  32. 32.
    Beuret N, Stettler H, Renold A, Rutishauser J, Spiess M. Expression of regulated secretory proteins is sufficient to generate granule-like structures in constitutively secreting cells. J Biol Chem 2004; 279(19): 20242–20249CrossRefPubMedGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Yanlin Zhao
    • 1
  • Xiao Zhong
    • 1
  • Xiaohong Ou
    • 1
  • Huawei Cai
    • 1
  • Xiaoai Wu
    • 1
  • Rui Huang
    • 1
  1. 1.Department of Nuclear Medicine, West China HospitalSichuan UniversityChengduChina

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