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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2017, Vol. 11 Issue (1) : 120-128     DOI: 10.1007/s11684-017-0501-3
RESEARCH ARTICLE |
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
Department of Nuclear Medicine, West China Hospital, Sichuan University, Chengdu 610041, China
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Abstract  

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.

Keywords norepinephrine transporter      vesicular monoamine transporter 2      131I-MIBG      gene therapy      lentivirus vector     
Corresponding Authors: Xiaohong Ou   
Just Accepted Date: 09 January 2017   Online First Date: 20 February 2017    Issue Date: 20 March 2017
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0501-3     OR     http://academic.hep.com.cn/fmd/EN/Y2017/V11/I1/120
Fig.1  Left (A) shows the detection of NET protein (60 kDa) using Western blot. Lane 1, positive control; lane 2, HepG2; lane 3, HepG2 co-expressing NET and VMAT2; lane 4, HepG2 co-transfected with NET and negative-control viral; lane 5, HepG2 expressing NET only. Right (B) shows the detection of VMAT2 protein (56 kDa) using Western blot. Lane 1, positive control; lane 2, HepG2; lane 3, HepG2 co-transfected with NET and negative-control viral; lane 4, HepG2 co-expressing NET and VMAT2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase
Fig.2  Dynamic uptake (A) and efflux (B) of MIBG labeled with iodine 131 (131I-MIBG) in HepG2, HepG2+ NET, and HepG2+ NET+ VMAT2 cells (in counts/min (cpm)).
Fig.3  Uptake of MIBG labeled with iodine 131 (131I-MIBG) was inhibited by maprotiline in HepG2+ NET and HepG2+ NET + VMAT2 (in cpm). A significant difference was noted in the accumulation of 131I-MIBG between maprotiline-inhibited and control cells (*P<0.01 for both cell types).
Fig.4  Uptake (in cpm) of MIBG labeled with 131I-MIBG was lower after treatment with tetrabenazine (TBZ) at every measurement in HepG2+ NET+ VMAT2 cells, and the efflux rate was significantly higher than the group that was not treated with tetrabenazine (P<0.001).
Fig.5  Scintigraphic images of tumor bearing-mice subcutaneously transplanted with HepG2+ NET + VMAT2 (left flank) or HepG2+ NET cells (right flank) at 30 min, 2, 4, and 24 h after injection of 131I-MIBG.
Hours after injection
2 4 24
HepG2+ NET + VMAT2 tumors 1.65±0.31 1.16±0.14 0.54±0.06
HepG2+ NET tumor
P value
0.98±0.17
0.02
0.65±0.18
0.01
0.19±0.05
0.001
Tab.1  Activity concentration in tumors at different times after injection of 131I-MIBG
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