Glutamate Transporter Excitatory Amino Acid Experiment

Glutamate Transporter stimulative Amino Acid ExperimentAbstractN-(2-18F-Fluoropropionyl)-L-glutamate(18F-FPGLU) is a potential amino group deadly tracer for tumor imagery with positron emission imagination ( c aress). In this study, therelationship between glutamate conveyor belt excitant amino acid postman 1 (EAAC1) cheek and 18F-FPGLU usance in squealer C6 glioma cubicles tonal pattern and gentleman SPC-A-1 lung adenocarcinoma cells line was investigated. The white plague of 18F-FPGLU in C6 cells increased signifi whoremastertly after induce by ATRA for 24, 48, and 72 h, which was closely related to conceptualisation of EAAC1 in C6 cells (R=0.939). Compared with the SPC-A-1(NT) guard cells, the uptake of 18F-FPGLU on EAAC1 knock-down SPC-A-1(shribonucleic acid) cells significantly change magnitude to 64.0%. In the caress resourcefulness of 18F-FPGLU of SPC-A-1 and EAAC1 knock-down SPC-A-1(shRNA)-bearing mice models, the uptake of 18F-FPGLU in SPC-A-1(shRNA) xenografts was significantly lower than that in SPC-A-1 xenografts, with Tumor/Muscle ratio of 1.67 0.1 vs. 3.01 0.3 at 60 min post-injection. The results suggest that transport mechanism of 18F-FPGLU in glioma C6 and lung adenocarcinoma SPC-A-1 cells lines principally involves in glutamate transporter EAAC1, which is an important transporter of 18F-FPGLU in tumor cells and may be a sweet hallmark of tumor glutamate metabolism caress imaging.Keywords N-(2-18F-fluoropropionyl)-L-glutamate tumor imaging glutamate transporter excitatory amino acid carrier 1IntroductionAs the most commonly determinationd positron emission tomography (PET) tracer for tumor diagnosis, 18F-fluoro-2-deoxy-D-glucose (18F-FDG) also has certain faux negative and false positive results(Shreve et al. 1999 Fletcher et al. 2008). It has been reported that 18F-FDG negative tumors may use a different metabolic pathway cal conduct glutaminolysis(DeBerardinis et al. 2007 Ward et al. 2012). Glutamine and glutam ate play observe roles in the adapted intermediary metabolism of tumors(Gao et al. 2009 Rajagopalan et al. 2011 Shanware et al. 2011). Several 18F-labeled glutamic acid and 18F-labeled glutamine arouse been apply for metabolic PET imaging of tumor in serviceman race (Baek et al. 2013 Venneti et al. 2015). High uptake of these amino acid tracers in tumor cells is apparent related to the increased side of amino acid transporters. For example, the upregulated establishment ASC, oddly ASCT2 might contri howevered to the uptake of 18F-labeled (2S,4R)-4-fluoro-L-glutamine(Ploessl et al. 2012), and 18F-fluoroglutamic acid (BAY 85-8050) transport involved in Na+-dependent XAG- and Na+-independent XC- systems with XC- possibly playing a more(prenominal) dominant role, but both of them showed defluorination in vivo(Krasikova et al. 2011). 18F-labeled (4S)-4-(3-18Ffluoropropyl)-L-glutamate (BAY 94-9392), another derivative of glutamic acid, whose transport was due cistronrally to u pregulation of system XC-, a potential biomarker for tumor oxidative stresscan be useful for detecting system XC- activity in vivo and is considered to be a potential tracer for tumor imaging(Koglin et al. 2011).Our recently developed novel N-18F-labeled glutamic acid, N-(2-18F fluoropropionyl)-L-glutamate (18F-FPGLU), seemed to be a potential amino acid PET tracer for tumor metabolic imaging, with high tumor-to-background contrast in several tumor-bearing mice models. precedent studies showed that 18F-FPGLU wasprimarily transported through Na+-dependent high-affinity glutamate transporter system XAG-(Hu et al. 2014), but the accurate transport mechanism is unknown. Glutamate transport system includes Na+-dependent excitatory glutamate transporter XAG- system and Na+-independent glutamate transporter XC- system(Avila-Chvez et al. 1997). System XC- (xCT) is everywhereexpressed on tumor c ells and is a potential biomarker for tumor oxidative stress. As an important genus Phallus o f XAG- system, excitatory amino acid carrier 1 (EAAC1), also called excitatory amino acid transporter 3 (EAAT3), localizes to the post-synaptic structure of neurons and surrounding glial cells as regulator of excitatory neurotransmission, and also exists in peripheral wanders, possibly as metabolic regulators(Bailey et al. 2011). The mirror image of EAAC1 was known to be regulated by several mechanisms that modify carrier abundance on the plasma membranes and was markedly induced by all tans-retinoic acid (ATRA) in rat C6 glioma cells, which led to strikingly stimulate amino acid influx(Bianchi et al. 2008). However, EAAC1 transporter may be a potential biomarker for tumor molecular imaging. It has not been reported so far. This study investigated the relationship between EAAC1 expression and 18F-FPGLU uptake in C6 rat glioma cells line and SPC-A-1 human lung adenocarcinoma. The uptake of 18F-FPGLU was assessed in ATRA- interact and un inured C6 cells lines, and also in shRNA-med iated EAAC1 knock-down SPC-A-1 cells and the non-targeted (NT) apply cells in vitro. Further prospective researches of PET imaging of tumor-bearing mice models with C6, SPC-A-1 and EAAC1 knock-down SPC-A-1(shRNA) xenografts were performed to reveal the coefficient of correlation between the uptake of 18F-FPGLU and the expression of EAAC1.Materials and methodsMaterials any reagents, unless otherwise specified, were of analytical grade and commercially available. alone chemicals obtained commercially were employ without further purification. Inveon small-animal PET/computed tomography (CT) scanner was purchased from Siemens (Germany).Synthesis of 18F-FPGLUThe synthetic thinking of 18F-FPGLU from 4-nitrophenyl-2-18F-fluoropropionate (18F-NFP) via a twain-step reaction sequence has been set forth in degree by the earlier paper(Hu et al. 2014).Cell civilisation and living creature ModelsThe C6 rat glioma cells, SPC-A-1 human lung adenocarcinoma cells were obtained from Shanghai Institute of Cellular Biology of Chinese honorary society of Sciences (Shanghai, China). The cells were cultured in culture flasks containing DMEM medium(for C6 cells) or RPMI 1640 medium (for SPC-A-1) supplemented with 10%FBS and 1% penicillin/streptomycin at 37oC in a humidified atmosphere of 5% CO2 and 95% air. 24 hours before the experiments in vitro, C6 cells lines or SPC-A-1 cell lines were trypsinized and 2105 cells per rise up were sown into 24-well plates. All animal experimental studies were approved by the Institutional Animal Care and Utilization Committee (IACUU) of the First Affiliated infirmary, Sun Yat-Sen University (approval No.2013A-173). All efforts were made to minimize animal suffering, to reduce the number of animals used, and to use alternatives to in vivo techniques, if available. The nude mice were obtained from Laboratory Animal Center of the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). The C6, SPC-A-1 and EAAC1 knock-down SPC-A -1(shRNA) tumor models were made apply previously described methods(Deng et al. 2011). Tumor cells (2-5-106) were injected subcutaneously and allowed to grow for 1 to 3 weeks. When the tumor reached 6-10 mm (diameter) micro PET/CT scans were done.C6 induced by ATRAThe rat glioma C6 cells were hardened by all trans-retinoic acid (ATRA) 12 h after the passage. Culture medium was substituted with fresh medium (containing DMEM medium supplemented with 10% FBS) in the absence seizure or in the present of ATRA at a concentration of 10 M from a 10 mM stock solution in DMSO according to the literature16. After the treatment of ATRA for 24, 48 and 72 h, decimal real time polymerase image reaction (qRT-PCR) and westward blotting were used to monitored the template RNA and protein expression levels of EAAC1 in ATRA treated C6 and non-treated C6 cells.Generation of shRNA-mediated EAAC1 knock-down cells.The method of generation of shRNA-mediated EAAC1 knock-down cells was uniform to the l iterature(Youland et al. 2013). SPC-A-1 human lung adenocarcinoma cells was used for shRNA-mediated EAAC1 knock-down experiment. SPC-A-1 cells were transduced with lentivirus ecoding EAAC1-targeted short hairpin RNAs (shRNA). shRNA sequences were selected from human EAAC1 messenger RNA NM_004170 and the shRNA fragments were cloned in a lentivirus vector pGLV3 plasmid with the sequence 5-GCATTACCACAGGAGTCTTGG-3. A non-specific targeting (NT) shRNA for control was cloned in the same lenvirus plasmid backbone. Lentiviral packaging was performed with trans-lentiviral packaging mix in 293T cells according to the manufacturers instructions. SPC-A-1 cells were plated on 6-well plates at 2-105 cells per well. After 24 hours, medium was aspirated and replaced with coulomb L of virus-containing solution was added to each well and incubated at 37oC for 24 h. Cells were selected with puromycin and monitored for car park fluorescence protein (GFP) expression. The EAAC1 mRNA expression level wa s monitored by quantitative real-time polymerase chain reaction (qRT-PCR). The EAAC1 protein expression level was quantized by western blotting.qRT-PCR for the expression of EAAC1Relative expression levels of EAAC1 mRNA in C6 and SPC-A-1 cells were metric victimization the fluorescence quantitative real-time polymerase chain reaction (qRT-PCR) (Stratagene Mx3000P Real time PCR, Agilent). Total cellular RNA was isolated with the Rneasy mini Kit (TAKARA). 1 g of RNA was synthesized to cDNA in a 20 L reaction system with reverse transcriptase buffer, RT Enzyme Mix and primer MIX (Bestar qPCR RT kit, DBI). Conditions for reverse agreement were 5 min at 65oC, 5 min on ice, thusly 60 min at 37oC and 10 min at 98oC. Oligodeoxynucleotide primers of EAAC1 gene for PCR amplification was5-AGTTCAGCAACACTGCCTGT-3 (forward) and (5-GTTGCACCAACGGGTA ACAC-3(reverse). PCR was programmed as follows 2 min at 94oC, 20 s at 94oC, 20 s at 58oC becausece 20 s at 72oC for 40 cycles. Glyceraldehyde-3-p hosphate dehydrogenase (GAPDH) was used as a initial control and each sample was amplified in triplicate. The sexual relation expression of EAAC1 mRNA compared with GAPDH was calculated by comparative threshold method (2 -Ct ).Western blotting for EAAC1Cells were lysed in a detergent-containing buffer with protease inhibitors for 20 min at 4oC. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) was used as a reference protein. After solubilization, cell lysates were collected and centrifuged at 14000 rpm for 10 min. The supernatants were transferred into new tubes, quantification of proteins was performed with Pierce BCA Protein Assay Kit (Thermo) and aliquots of 25 g were loaded on an 10% gel for SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoridePVDFmembranes (Millipore) . The membranes with EAAC1 or GAPDH were departed at the middle position, and were stop and incubated with deferent antibody, respectively. Non-specific binding sites were block ed with an incubation in Tris-buffer saline containing 5% of bovine serum albumen (BSA) for 1h at room temperature. Then the blots were exposed to EAAT3 antibody (rabbit monoclonal antiserum, 11000, Abcam) or anti-GAPDH rabbit monoclonal antibody(13000, Abcam) dilute in blocking solution for at 4oC overnight. After washing, the blots were exposed for1h at room temperature to goat anti-rabbit IgG HRP diluted 15000 in blocking solution.Cellular uptake of 18F-FPGLUCells were plated in 24-well plates (2x105cells/well) and uptake studies were performed at 24 h after the passage. The cellular uptake of 18F-FPGLU studies was exchangeable to the methods described previously(Krasikova et al. 2013). The medium was aspirated and the cells were washed 3 times with 1 mL warm PBS. 18F-FPGLU was dissolved in PBS solution and was added to each well (74-111 KBq/0.2 mL/well). After incubated with 18F-FPGLU at 37oC for 30 min, the radioactive medium was outback(a) and the cells were washed 3 times with ice-cold PBS. Then, the cells were dissolved in 0.5 mL of 1 N NaOH and the activity was measured by counter (GC-1200, USTC Chuangxin Co. Ltd. Zonkia Branch, China). The cell lysate (25L) was used for determination of protein concentration by BCA protein assay. The uptake data are based on the amount of activity added to each well and the resume amount of protein in each well. Each experiment was done in triplicate, averaged and was repeated 5 times on different days. The uptake of 18F-FPGLU was assessed on the ATRA-treated or untreated C6 cells, and on EAAC1 knock-down SPC-A-1(shRNA) cells or SPC-A-1(NT) control cells. The intercourse uptake ratios were calculated compared to the control cells.Small-animal PET-CT imagingSmall-animal PET-CT imaging studies with tumor-bearing mice were carried out using the Inveon small-animal PET/CT scanner (Siemens). 3.7-7.4 MBq of 18F-FPGLU were injected intravenously in conscious animals via the stool vein. The mice were anesthetized with 5% chloral hydrate solution (6 mL/kg) and were unbroken warm end-to-end the procedure. Imaging started with a low-battery-acid CT scan, immediately followed by a PET scan. PET images were acquired at 30, 60, 90, 120 min post-injection. For a comparative study, mice were kept fasting for 4 h and were anesthetized with 5% chloral hydrate solution (6 mL/kg) and imaged with 18F-FDG (3.7 MBq) at 60 min after intravenous injection. The images were reconstructed by two-dimensional ordered-subsets expectation maximum (OSEM). For each small-animal PET scan, ROIs were drawn over the tumor and go through of the thigh on decay-corrected whole-body coronal images using Inevon research Workplace 4.1 software. The quantification was performed according the methods described previously(Hu et al. 2014). Radioactivity concentration within a tumor or other tissue was converted to MBq/g and then divided by the administered activity to obtain an imaging ROI-derived percentage of injected dose per g ram of tissue (% ID/g). Then, the ttumor/muscle (T/M) and tumor/brain (T/B) uptake ratios were calculated, respectively.Immunohistochemistry preparation of EAAC1 was assessed by immunohistochemistry on formalin-fixed paraffin embedded rat brain tissues and C6 xenograft samples. Immunohistochemistry experiments were carried out according to the literature(Wang et al. 2013). Normal rat brain tissues and C6 glioma tissues were fixed in 10% neutral buffered formalin overnight at room temperature. Tissues were then dehydrated, embedded in paraffin, and cut into 3-m sections. After antigen retrieval, tissue sections were work to immunohistochemical incubated with antibodies against EAAC1(Abcam), DAB was stained before mounted onto microscope slides. Tissues were analyzed with a Nikon E800M microscope.statistical analysesData were expressed as mean+/-SD. Statistical analysis was performed with SPSS software, variate 16.0 (SPSS Inc.), for Windows (Microsoft). Student t test was used to as sess differences in the magnitudes of samples from two measurements. A P values of less than 0.05 was considered to indicate statistical significant. A scatter plot was drawn with the relative mRNA expression and the relative uptake of 18F-FPGLU in C6 cells treated with ATRA for 24h, 48h, 72h. Spearman correlation analysis and a one-dimensional regression analysis was performed between them.ResultsEAAC1 expression and 18F-FPGLU uptake in C6 cells induced by ATRAThe EAAC1 mRNA relative expression levels in ATRA-treated C6 cells assessed by quantitative real-time polymerase chain reaction (qRT-PCR) are shown by Figure 1A. Compared with the untreated C6 cells, the EAAC1 mRNA relative expression level in ATRA-treated C6 cells treated with ATRA at 10 M for 24, 48 and 72 h was increased to 1.72 0.113.22 0.224.0 0.21 times, respectively( Fig. 1A). Meanwhile, the western blotting results also showed that EAAC1 protein expression in ATRA-treated C6 cells was increased gradually(Fig. 1B). Corresponding with the high EAAC1 expression in ATRA-treated C6 cells, 18F-FPGLU uptake was significantly increased to 1.47 0.112.14 0.292.12 0.16 times in C6 cells treated by ATRA for 24, 48 and 72 h, respectively(Fig. 1C). There was a high correlation between the relative EAAC1 mRNA expresion and the relative 18F-FPGLU uptake in ATRA treated C6 cells (R = 0.939, Fig. 1D). To summarize, EAAC1 expression was markedly induced by ATRA in C6 cell lines. As a result, there was more 18F-FPGLU uptake in ATRA-treated C6 cells line which has more EAAC1 expression at both mRNA and protein levels.Figure 1PET imaging on C6 glioma-bearing miceSmall-animal PET-CT scan was performed on C6 glioma-bearing nude mice models 1h post-injection of 18F-FPGLU. PET-CT fusion imaging of the mice models demonstrated that 18F-FPGLU could intensely accumulate in C6 glioma (Fig. 2A). The tumor/brain uptake ratio of 18F-FPGLU on C6 glioma-bearing mice was higher than that of 18F-FDG at 1h post-injection of r adiotracers(n = 3, P 0.05, Fig. 2B). However, the tumor/muscle uptake ratio of 18F-FPGLU in C6 glioma-bearing mice was lower than that of 18F-FDG (n = 3, P 0.05). Immunohistochemistry showed that widely spread out EAAC1 transporter staining was shown in C6 glioma, however there was minimal EAAC1 staining in normal rat brain write matter tissue (Fig. 2C).Figure 2EAAC1 expression and 18F-FPGLU uptake in EAAC1 knock-down SPC-A-1human lung adenocarcinoma cellsThe influence of EAAC1 expression on 18F-FPGLU uptake was specifically investigated using RNA interference-mediated EAAC1 knock-down SPC-A-1 human lung adenocarcinoma cells. Lentivirally delivered shRNA significantly reduced EAAC1 mRNA expression in SPC-A-1(shRNA) cells, as compared to the non-targeted (NT) shRNA control cells (SPC-A-1(NT) cells), EAAC1 shRNA reduced EAAC1 mRNA expression by 72% in SPC-A-1(shRNA) cells (P 0.01) (Fig. 3A). At the protein expression level, EAAC1 shRNA significantly diminish EAAC1 expression in SPC-A-1(shRNA) cells by 59.6% (P 0.01) (Fig. 3B). Knock-down of EAAC1 expression was associated with a significantly lower 18F-FPGLU uptake by 36% in SPC-A-1(shRNA) cells (P