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

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

Introduction

As a metabolically stable false analog of norepinephrine, metaiodobenzylguanidine (MIBG) actively accumulates in neuroectodermal tumors expressing the norepinephrine transporter (NET) [ 1– 3]. MIBG labeled with iodine 131 (¹³¹I-MIBG) was used for many years for clinical imaging and targeted radiotherapy in patients with neural crest-derived tumors, such as pheochromocytoma and neuroblastoma [ 4– 8]. Attempts were made to expand this therapeutic strategy to non-neuroendocrine tumors by coupling ¹³¹I-MIBG administration with NET gene transfer into target tumor cells; several recent studies showed promising results [ 9, 10]. Effects of NET gene transfection were investigated in several non-neuroendocrine cells with five-fold (HepG2 cells), 36-fold (rat Morris hepatoma cells), 13-fold (bladder cancer cells), and 15-fold (glioblastoma cells) increases in radiolabeled MIBG accumulation [ 11– 14]. Although efficient ¹³¹I-MIBG uptake was achieved in NET-transfected tumors, the target cells had short ¹³¹I-MIBG retention time [ 12]; therefore, the absorbed dose of radiation in vivo was not expected to be therapeutically effective. For example, NET-expressing hepatoma cell lines accumulated norepinephrine up to 36 times more than the wild-types; however, 38% and 43% of radioactivity was released 1 and 4 h, respectively, after removal of ¹³¹I-MIBG-containing medium [ 12].

Retention time of ¹³¹I-MIBG should be increased to efficiently treat tumors with ¹³¹I-MIBG after NET gene transfer. Amines in cytoplasm are transported into storage vesicles by vesicular monoamine transporters (VMATs), which are specific intracellular membrane-bound transporters in neurons and endocrine cells. A combination of transmembrane protons and electrochemical gradients drive the transport of monoamines in storage vesicles against a large concentration gradient (>10⁵); the electrochemical gradient is generated by vesicular H⁺-adenosine triphosphatase [ 15]. Similar to the observations with monoamine accumulation, MIBG is efficiently absorbed through plasma membranes with NET followed by reaccumulation into storage organelles through VMATs; these processes are crucial in MIBG accumulation in neural crest-derived tumors [ 16]. The expression level of VMAT2 correlates with its affinity for MIBG in neuroendocrine tumors [ 17]. VMATs were also cloned and functionally expressed in different cell types, including several non-neuroendocrine cells, such as CV-1 cells and Chinese hamster ovary cells. Permeabilization of plasma membrane with digitonin demonstrated that monoamine substrates could be directly accumulated in an ATP-dependent fashion by intracellular compartments in Chinese hamster ovary cells stably transfected by VMATs [ 18]. In immunofluorescence studies with transfected Chinese hamster ovary cells, VMAT proteins colocalized with recycling endocytotic vesicles and markers for the Golgi complex; such result is consistent with the presence of V-type adenosine triphosphatase in the endocytotic pathway and in intermediate and distal parts of the Golgi [ 19]. Results further indicated that VMATs contain signals for sorting toward the endocytic pathway and do not rely on association with other synaptic vesicle proteins. Sorting to a population of acidic vesicles thereby allows functional expression of the transporter in non-neuroendocrine cell lines. Therefore, VMAT gene transfection may be a potential strategy for increasing MIBG retention in NET-expressing tumor cells.

Two subtypes of VMATs, namely, VMAT1 and VMAT2, have structural similarities, although they differ in substrate specificity and tissue distribution [ 18, 20– 22]. Pharmacological studies showed that VMAT2 has a three- to five-fold higher apparent affinity for monoamines and catecholamines than VMAT1 [ 18, 23]. In this study, we aimed to cotransfect NET and VMAT2 genes to allow greater ¹³¹I-MIBG aggregation in transfected cells and to achieve longer biological half-life to improve the absorbed dose of radiation of tumors.

Materials and methods

Chemical and biological reagents

The plasmid containing human NET gene was provided by SG Amara (Yale, New Haven, USA). The plasmid containing the VMAT2 gene and lentiviral vectors (GV287 and GV341) were purchased from Genechem Co. (Shanghai, China), with GV287 carrying the green fluorescent protein gene and GV341 carrying the puromycin N-acetyl transferase gene, of which the gene product catalyzes the antibiotic puromycin. Rabbit monoclonal antibody to NET and mouse monoclonal antibody to VMAT2 were purchased from Abcam Inc. (Cambridge, MA, USA). ABclonal Biotech Co. (UK) supplied the goat anti-mouse and goat anti-rabbit immunoglobulin G antibody chemically conjugated to horseradish peroxidase rabbit anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal immunoglobulin G; the ¹³¹I-MIBG used in this work was prepared in accordance with the method of Zan et al. [ 24]. 3-Trimethylsilylbenzylguanidine was synthesized in five steps using 3-bromotoluene as starting material. With this precursor, free-carrier ¹³¹I-MIBG was obtained, and radiochemical purity was more than 98% after purification with high-performance liquid chromatography.

Instruments and equipment

We obtained the AA2250 electronic balance from Denver Instruments (Denver, CO, USA), FJ-2021 γ counter from Xi’an No. 262 Factory (Xi’an, Shaanxi Province, China), and PHILIPS Skylight γ-camera from PHILIPS Medical Systems (Cleveland, USA).

Cell lines

We obtained HepG2 cell lines from the State Key Laboratory of Biotherapy (Sichuan University, Sichuan, China). HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% heat-inactivated fetal calf serum with 100 IU/ml penicillin and 100 µg/ml streptomycin. All cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C.

Construction, amplification, and purification of lentiviral NET and VMAT2 vectors

The human NET and VMAT2 genes were obtained individually through polymerase chain reaction of their original plasmid, after which both were sequenced and compared with the sequences in GenBank (National Center for Biotechnology Information, Bethesda, MD, USA). The right human NET and the right VMAT2 sequences were recombined and inserted in the GV341 and GV287 plasmids, respectively. Recombinant lentiviral vectors encoding the human NET gene (LV-NET) and VMAT2 gene (LV-VMAT2) under the control of Cytomegalovirus promoter were constructed and transfected to 293T cells for packaging, amplification, and purification. The titers of the LV-NET and LV-VMAT2 were measured, wherein both results showed 2.0 × 10⁸ transduction unit/ml (TU/ml). Recombinant lentiviral vector not expressing transgenes was used as negative control in this study.

Construction of stably expressed cell line

To prepare stable cell lines overexpressing human NET and VMAT2, we transfected HepG2 cells with LV-NET and LV-VMAT2. First, HepG2 cells were seeded in 12-well culture plates at a confluence of 70%. LV-NET was then added to the medium at a multiplicity of infection of 100. Transfected cells were selected by culturing with 2.5 µg/ml puromycin. Single colonies of stable transfects were isolated and expanded. We named these colonies “HepG2+ NET cells” and further transfected them with LV-VMAT2 to develop “HepG2 + NET+ VMAT2” cells. Negative control transfects were used as controls. After 72 h of infection, green fluorescent protein was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany). When the cells were confluent, they were trypsinized and resuspended in medium per the protocol. Western blot was used to verify the expression of NET in HepG2+ NET cells and HepG2+ NET+ VMAT2 cells and VMAT2 expression in HepG2+ NET + VMAT2 cells.

Measurement of ¹³¹I-MIBG uptake and release

To evaluate MIBG uptake, HepG2 cells and newly constructed cell lines were seeded in 24-well plates at 5 × 10⁴/ml per well. Eight hours later, the medium was aspirated, and plated cells were incubated with the medium containing 74 kBq of ¹³¹I-MIBG and incubated for 5, 10, 15, 20, 30, 60, and 90 min. Medium was then aspirated, and cells were quickly washed twice with ice-cold phosphate-buffered saline (PBS). Afterward, the cells were lysed by incubation with 95% ethanol, and radioactivity of cell lysate was measured with the γ counter. Three independent experiments were performed in triplicate. Values are expressed as mean values±standard deviation (SD).

For the determination of ¹³¹I-MIBG efflux, HepG2 cells and newly constructed cells were cultured for 30 min in a medium containing 74 kBq of ¹³¹I-MIBG in 24-well plates. After the plates were washed twice with phosphate-buffered saline, the cells were lysed as described above, or fresh nonradioactive medium was added. After 5, 10, 30, 60, and 120 min, radioactivity in cell lysates and the medium was measured with a γ counter. Three independent experiments were performed in triplicate. Values are expressed as mean values±SD.

In vitro competition experiment

A total of 5 × 10⁴/ml per well HepG2 + NET and HepG2 + NET+ VMAT2 cells were seeded in 24-well plates. Eight hours later, the medium was aspirated and plated cells were incubated with a medium containing 74 kBq of ¹³¹I-MIBG and 30 µmol/ml maprotiline (a selective inhibitor of NET) for 20 min. The medium was then aspirated, and cells were quickly washed twice with ice-cold PBS. Cells were lysed by incubation with 95% ethanol, and radioactivity of the cell lysate was measured with a γ counter.

The HepG2+ NET+ VMAT2 cells were seeded in 24-well plates at 5 × 10⁴/ml per well. Eight hours later, the medium was aspirated, and plated cells were incubated with medium containing 74 kBq of ¹³¹I-MIBG and 30 µmol/ml tetrabenazine (a selective inhibitor of VMAT2) and incubated for 35, 40, 45, 50, 55, 60, and 90 min. The medium was aspirated again, and cells were quickly washed twice with ice-cold PBS. Afterward, cells were lysed by incubation with 95% ethanol, and radioactivity of the cell lysate was measured by a γ counter. For these experiments, three independent experiments were performed in triplicate.

¹³¹I-MIBG uptake in tumor tissue

A total of 1.0 × 10⁶ HepG2 + NET+ VMAT2 or HepG2+ NET cells were transplanted subcutaneously into the left (HepG2 + NET+ VMAT2) or right (HepG2+ NET) flanks of young male BALB/c nude mice (purchased from the Laboratory Animal Center of Sichuan University, China) weighing 20–25 g. Two weeks later, mice bearing tumors with minimal diameter of 5 mm were selected. For scintigraphy studies, four nude mice were anesthetized with pentobarbital (50 mg/kg, intraperitoneally) and administered with 18.5 MBq ¹³¹I-MIBG through the vena caudalis. Scintigraphy was performed immediately at 30 min, 2, 4, and 24 h after injection by acquiring 5.0 × 10⁵ counts on a 256 × 256 matrix, at 364 keV peak, and 20% window using a Skylight γ-camera equipped with high-energy and high-resolution parallel-hole collimator. The absolute amount of radioactivity in tumors (percentage of injected dose per gram of wet tissue) was determined in 12 mice at 2, 4, and 24 h after administration of 11 MBq ¹³¹I-MIBG. The mice were sacrificed, and tumor tissue was analyzed using a γ counter. All animal experiments were approved by the Animal Ethics Committee of Sichuan University.

Statistical analyses

One-way analysis of variance was used to test differences in radiopharmaceutical uptake of NET- and VMAT2-expressing cells. Post-hoc analyses used Bonferroni correction for multiple comparisons. Two-sided student’s t-test was used to compare the two groups. All data are expressed as means±SD. P<0.05 was considered statistically significant.

Results

Identification of stable cell strains

We confirmed through sequencing that the product was the preferred gene of NET (GenBank, SLC6A2) and VMAT2 (GenBank, SLC18A2). HepG2 cells were infected with recombinant lentiviral vector LV-NET and stable human NET-expressing cell lines (HepG2 + NET) established by puromycin selection. After transfection of LV-VMAT2, a green fluorescent protein was observed. Western blot confirmed that NET protein was expressed in HepG2 + NET and HepG2+ NET+ VMAT2 cells; VMAT2 protein was also expressed in HepG2 + NET+ VMAT2 cells (Fig. 1).

Uptake and release of ¹³¹I-MIBG in vitro

In the dynamic ¹³¹I-MIBG uptake study, LV-NET-infected cells (HepG2+ NET and HepG2+ NET+ VMAT2 cells) significantly accumulated more ¹³¹I-MIBG than controls (Fig. 2A). The uptake in HepG2+ NET cells peaked at 20 min, whereas uptake of ¹³¹I-MIBG in HepG2+ NET+ VMAT2 cells continued after that time point with the maximal uptake measured at 30 min. After 90 min of incubation, the levels plateaued, implicating steady-state uptake of ¹³¹I-MIBG. HepG2 + NET and HepG2+ NET+ VMAT2 cells showed 2.24 and 2.22 times higher uptake of ¹³¹I-MIBG, respectively, than the control HepG2 line after reaching the peak value. A significant difference in ¹³¹I-MIBG accumulation was observed between NET-transfected and control cells (P<0.001 for both cell types). However, HepG2+ NET + VMAT2 and HepG2 + NET cells (P = 1.000) did not show significant difference in ¹³¹I-MIBG concentration.

To investigate the efflux, uptake of ¹³¹I-MIBG in all cell lines was allowed to proceed for 30 min before replacing the medium with a nonradioactive one. The amount of ¹³¹I-MIBG remaining in HepG2+ NET cells decreased rapidly, whereas radioactivity of HepG2+ NET+ VMAT2 cells decreased steadily with time (Fig. 2B). After 1 h, HepG2+ NET+ VMAT2 cells released about 20.1% cellular radioactivity into the medium, and 25.4% efflux was observed after 2 h, indicating a slower effusion of the radiotracer from these cells. In HepG2+ NET cells, 26.0% and 38.6% of radioactivity was released after 1 and 2 h, respectively. The amount of ¹³¹I-MIBG in wild control cells did not significantly change over time. A significant difference in the efflux of ¹³¹I-MIBG was observed between HepG2+ NET + VMAT2 and HepG2+ NET cells (P<0.001 for both cell types).

In vitro competition experiment

In the presence of 30 mmol/L maprotiline, ¹³¹I-MIBG uptake was inhibited by 26.81%±0.66% and 21.03%±0.69% in HepG2+ NET and HepG2+ NET+ VMAT2 cells, respectively (Fig. 3).

In experimental groups treated with tetrabenazine, uptake of ¹³¹I-MIBG in HepG2+ NET+ VMAT2 cells was constantly below the levels observed in untreated cells. After treatment with tetrabenazine, the effluence rate of intracellular ¹³¹I-MIBG was higher than the control group. Therefore, tetrabenazine could inhibit the efflux in cells containing the VMAT2 gene (Fig. 4).

¹³¹I-MIBG uptake and efflux in tumor-bearing mice

As shown in the scintigraphic images of mice (Fig. 5), ¹³¹I-MIBG uptake was visible in both HepG2+ NET+ VMAT2 or HepG2+ NET cell transplanted tumors 30 min after injection, and it was accompanied by highly-colored background of the abdomen. At 2 h, uptake of HepG2+ NET+ VMAT2 tumor on the left flank was higher than in HepG2+ NET tumor on the right. High tracer uptake was also observed in the heart, liver, kidney, bladder, and gastrointestinal tract. Scintigraphic visualization of HepG2 + NET+ VMAT2 tumor tissue was sustained for up to 24 h after tracer administration, whereas ¹³¹I-MIBG uptake in HepG2+ NET tumor declined gradually and was almost scintigraphically invisible at 24 h.

In vitro quantitation of ¹³¹I-MIBG uptake per gram of tumor tissue revealed that the HepG2+ NET+ VMAT2 tumor respectively showed a 1.68- and 1.78-fold higher accumulation of ¹³¹I-MIBG after 2 and 4 h compared with HepG2+ NET tumors. However, at 24 h after ¹³¹I-MIBG administration, only 0.54% and 0.19% of the injected dose per gram of tissue were found in the HepG2+ NET+ VMAT2 and HepG2+ NET tumors (Table 1), respectively.

Discussion

NET gene cloning may open new avenues for development of strategies in cancer gene therapy. In vitro and in vivo¹³¹I-MIBG uptake in treated cells was very efficient when administration of the compound is coupled with virus- or plasmid-mediated delivery of NET gene into non-neuroendocrine tumor cells [ 9, 10, 25]. However, in treated tissue, ¹³¹I-MIBG had a short half-life, which is insufficient for therapy; the radiation doses delivered were within range of those used for treatment of neural crest-derived tumor patients [ 12]. Our preliminary results using gene transfection suggested that this strategy is limited by the lack of intracellular retention of internalized radiolabeled MIBG. Thus, we concluded that cytotoxic efficacy of NET gene transfer may be limited by radiolabeled MIBG efflux, and enhancement of intracellular retention of radiolabeled MIBG may confer a therapeutic advantage [ 11].

Literature provides limited information regarding effective means of extending the retention of MIBG in transformed cells. We hypothesized that we could enhance intracellular retention of radiolabeled MIBG by promoting its transportation into endocytosis vesicles after uptake of radiolabeled MIBG through the NET of plasma membranes and that increased retention would yield more cytotoxicity. To test this hypothesis, we constructed an expression vector encoding for VMAT2 and co-transfected this vector with the NET gene. In the present study, VMAT2 expression was proven by Western blot analysis, and in vitro experiments showed slower effusion of the radiotracer from VMAT2-expressing cells than negative control cells. However, a 25.4% efflux was still observed 2 h after withdrawal of the radiolabeled MIBG. Furthermore, in vivo study demonstrated significant efflux in VMAT2-expressing tumor, with only 0.54% of the injected dose per gram of tissue observed 24 h after tracer administration. Short in vivo biological half-lives of 7.9 and 6.1 h were calculated for HepG2+ NET+ VMAT2 and HepG2+ NET tumors, respectively. Effectiveness of radioiodine therapy depends on the biological half-life of the isotope in tumors. Previous studies showed that when 14.8 MBq of ¹³¹I-MIBG was administrated to NET expressing non-neuroectodermal tumor-bearing mice (corresponding to 2200 MBq/m² in humans), the absorbed dose of radiation in tumor was not therapeutically effective [ 12]. Therefore, co-transfection of NET and VMAT2 gene will not increase the possibility of successful radioiodine treatment through mild expansion of biological half-life of the compound.

To efficiently improve MIBG retention, better solutions should be developed to further understand the mechanisms governing MIBG transport in cells. A positive correlation was observed between the content of chromaffin neurosecretory granules and uptake of radiolabeled MIBG [ 26]. Transmission electron microscopy revealed that after treatment with retinoic acid (RA), neuroblastoma (NB) cell line SH-SY5Y yielded varicosities containing clusters of large dense-core vesicles and smaller clear vesicles [ 27]. Moreover, Iavarone showed stimulation of MIBG uptake and storage in RA-treated NB cells [ 28]. Accordingly, increase in the number of vesicle-containing profiles possibly plays a role in increased efficiency of MIBG storage, as observed in NB cells. In our study, although transfection of VMAT gene led to expression of functional proteins on intracellular compartments (that is, recycling endocytotic vesicles in non-neuroendocrine cell), the number of these vesicles was far less than that of secretory granules in neuroendocrine tumor cells. This process possibly resulted in insufficient activity of intracellular transportation and retention and non-significant extension of MIBG trapping. The mechanism of secretory granule formation is only partially understood. Granins are a family of granule cargo proteins found throughout the endocrine and neuroendocrine systems. These proteins are believed to play a role in granule biogenesis [ 29]. Interestingly, expression of granins is sufficient to generate structures that resemble secretory granule-like structures in various non-neuroendocrine cell lines, such as COS, CV1, or human embryonic kidney cells [ 30– 32]. The accumulations of granins, which morphologically resemble secretory granules on electron microscopy, are membrane-bound. Therefore, co-transfection of granin gene may lead to increased VMAT2 expression and enhancement of MIBG intracellular retention.

In line with previous reports [ 11– 14], the present study demonstrated that transduction of human NET coding sequence into HepG2 cells induced the expression of functional NET, causing active uptake of ¹³¹I-MIBG. In our previous study, we used recombinant adenovirus as vector, which also showed high transfection efficiency [ 11]. In this study, we chose recombinant lentivirus because this vector can stably integrate the target gene into the genome of host cells; the target gene was then expressed permanently with prolonged expression. However, HepG2 cells transfected with NET using recombinant adenovirus vector showed 4.87 times higher ¹³¹I-MIBG uptake ability than wild-type cells in our previous study [ 11], and was superior to LV-NET-infected cells in this study. The discrepancy observed between our results and those of previous reports possibly originated from different transfection efficiency of different vectors. Further work is necessary to address this issue.

In conclusion, this study demonstrated that an increased retention of MIBG level was obtained both in vitro and in vivo in non-neuroectodermal tumor cells co-infected with LV-NET and LV-VMAT2. However, a considerable efflux of the radiotracer was also observed in the cells. Therefore, co-transfer of NET and VMAT2 gene to non-neuroectodermal tumor cells, together with the application of ¹³¹I-MIBG in clinically relevant amounts, is not sufficient in achieving therapeutically useful absorbed doses for recombinant tumors in vivo. Future experiments should focus on developing means to achieve prolonged retention of MIBG in recombinant tumors and to subsequently determine a therapeutically sufficient radiation dose for the possible transfer of multiple genes involved in MIBG trapping.

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