| BackgroundHepatocellular carcinoma is the sixth most prevalent cancer and the third most frequent cause of cancer-related death. Although biopsy is considered to be the gold standard for diagnosis of hepatocellular carcinoma, it is more invasive than serum biomarkers or common imaging techniques. The detection of hepatocellular carcinoma mainly relies on serum alpha-fetoprotein and liver imaging techniques such as B-mode ultrasound, X-ray computed tomography, magnetic resonance imaging and positron emission computed tomography. But when these abnormalities could be detected, the staging of hepatocelluar carcinoma usually goes into an advanced symptomatic stage. Therefore, exploring a new non-invasive technology to detect hepatocellular carcinoma in a very early stage is urgently needed. Molecular imaging is such a technology, which integrates the principles of cell and molecular biology, immunology, nuclear medicine and diagnostic imaging. Then it becomes an issue to find a target molecule which could specifically detect tumor in an early stage.Overexpression of angiotensin.Ⅱ type1receptor (AT1R) in a variety of tumors has been reported recently. AT1R promotes tumor growth and angiogenesis partially through upregulation of vascular endothelial growth factor. We hypothesized that AT1R expression might be upregulated in hepatocellular carcinoma tissue and131I-anti-AT1R mAb might be a new potential molecular imaging agent in tumor. The aim of this study was to validate this hypothesis.MethodsEthics statementThe animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee at School of Medicine, Shandong University.Cell culture and reagentsMurine hepatocellular carcinoma cell line H22, murine liver cell line NCTC clone1469, human cervical cancer cell line Hela and rat adrenal pheochromocytoma cell line PC12were cultured in RPMI1640medium, all supplemented with100unitsmL"1penicillin,100μg·mL-1streptomycin and10%fetal bovine serum at37℃in a95%air/5%CO2humidified atmosphere.Animal modelMale BALB/c mice (6-8weeks old) were purchased from Shandong University Animal Center and were maintained under pathogen-free conditions. The BALB/c mice were injected subcutaneously with1x107H22cells in0.1ml phosphate buffered saline into the right upper back to establish a hepatoma model.Radioiodination of anti-AT1R mAb and isotype IgG50μg anti-AT1R mAb or isotype IgG was iodinated with15μL Na131I(185MBq) using the lodogen method as described previously. Radioiodinated anti-AT1R mAb and isotype IgG were separated from free iodine using size exclusion columns Sephadex G-25.Radiochemical purity and stabilityRadiochemical purity was determined by paper chromatographic method using strips on two-paper sheet. Radiochemical purities were measured at1,6,24,48,72and96hours, respectively, to assess the stability.Radioligand-based binding assayFor saturation studies, a reaction mixture contained200μL H22cells (5×106/mL) and100μL131I-anti-AT1R mAb (0.1-32nM, diluted in1x PBS) in a final volume of500μL.10-1-105nM unlabeled anti-AT1R mAb and12nM131I-anti-AT1R mAb were used for competition binding assay. The mixture was incubated at37℃for2h. The bound radioligand was separated by rapid vacuum filtration through Whatman GF/B filters using a cell harvester followed by3x2mL washes of PBS at room temperature. The radioactivity of filters containing the bound radioligand was assayed in test tube by Wipe Test/Well Counter. The results of saturation and inhibition experiments were subjected to nonlinear regression analysis and the equilibrium dissociation constant (KD), the maximum number of binding sites (Bmax), the inhibitor constant (Ki) and the half maximal inhibitory concentration (IC50) were calculated.Whole-body autoradiography10%potassium iodide was added to drinking water3days before injection of131I-labeled antibody to block the uptake of thyroid gland.12days after injection of H22cells,3.7MBq131I-anti-AT1R mAb or131I-IgG was respectively injected into the mice through tail vein. Whole-body autoradiography was performed at1,6,24,48and72hours after injection, respectively. The anesthetized mice were fixed on the storage phosphor screen plate in supine position with four limbs stretched in order to make the tumor tightly close to the plate. The plate was exposed to a mouse for15minutes. After exposure, the plate was scanned by Cyclone Plus Storage Phosphor System and analyzed using the OptiQuant Acquisition software.Biodistribution of131I-anti-AT1R mAb and131I-IgGSix mice of each group were sacrificed at1,6,24,48and72hours after injection, respectively, and blood, tumor, muscular tissue on the opposite side of tumor and main organs were removed, weighed, and counted radioactivity in the gamma counter. The percent injected dose per gram (%ID/g) and target to non-target ratio (T/NT) were calculated.Pharmacokinetic analysis10μL blood samples were taken from periorbital vein of six mice at0,1,3,6,12,24,48,72,96and120hours after injection of131I-anti-AT1R mAb and then the radioactivity was measured by Gamma Counter. The distribution half-life (T1/2a), the elimination half-life (Ti/2p) and the mean residence time (MRT) were calculated.Real-time PCRThe primer for AT1R was chosen in cDNA portions by accessing mouse sequences in GenBank. The sequences were as follows:sense primer,5’-GAAGAACAAGCCAAGAAATGATG-3’; antisense primer,5’-TTGATGACTCCAGGTTAGCAGAT-3’(887bp). Cycling conditions for amplification were as follows:4min, denaturation step at94℃; followed by35cycles of30s, at94℃,1min, at55℃, and1s, at72℃. Quantitative assessment of relative gene expression levels involved the2-ΔΔCT method.HistologyTissues were fixed in phosphate-buffered4%paraformaldehyde, embedded in paraffin, and cut into4-um thick sections. Sections were deparaffinized and stained with hematoxylin and eosin using a standard protocol to determine morphology. AT1R protein expression was determined by immunostaining with anti-AT1R mAb (1:50) using the streptavidin-biotin method. Image-Pro Plus v5.0.2was used for quantitative assessment of relative AT1R protein expression levels.Western blotProtein extracted from tumor was incubated with anti-AT1R mAb (1:400) or a antibody against (3-actin (1:1000), followed by appropriate horseradish peroxidase-labeled secondary antibodies. Protein levels were normalized to that of p-actin as an internal control. HeLa cells and PC12cells were used as positive controls.Statistical analysisSPSS v11.5was used for statistical analysis. Continuous data were expressed as mean±SEM and compared by one-way ANOVA, followed by unpaired t-test or paired t-test as appropriate. A P value<0.05was considered statistically significant. ResultsRadioiodination of anti-AT1R mAb and isotype IgGThe radiochemical purity of131I-anti-AT1R mAb was92.8%and that of131I-IgG was93.2%. The specific activity of131I-anti-AT1R mAb was35.24±5.76MBq/μmol and that of131I-IgG was38.61±7.18MBq/μmol.The affinity of131I-AT1R mAb against AT1RSaturation assay showed KD and Bmax were1.83±0.48nM and5361±345.3cpm, respectively. Competitive binding assay showed the unlabeled anti-AT1R mAb competed effectively with131I-anti-AT1R mAb binding sites on H22cells at low micromole concentrations and Ki and IC50were9.68±1.33nM and73.13±1.33nM, respectively. These results revealed high affinity of131I-anti-AT1R mAb against AT1R. The stability of131I-AT1R mAb and131I-IgGThe radiochemical purities of131I-anti-AT1R mAb and131I-IgG were still over90%in serum, and declined under80%in saline at72hours, indicating that they maintained more stable in serum than in saline. There was no significant difference between the two imaging agents.The whole-body autoradiography in the hepatoma miceWhole-body autoradiography images showed much clearer for observing hepatocellular carcinoma in the131I-anti-AT1R mAb group than the131I-IgG group24hours after tracers injection and the difference reached a peak at48hours. These data demonstrate that the131I-anti-AT1R mAb appears to be more specific than131I-IgG for targeting hepatocellular carcinoma.Biodistribution of131I-anti-AT1R mAb and131I-IgGIn the131I-anti-AT1R mAb group,%ID/g of the tumor was higher than that of other tissues, and T/NT reached a peak at48hours after injection. In the131I-IgG group, there was no significant increase of%ID/g in the tumor and T/NT remained stable throughout the experiment. The results indicated that hepatocellular carcinoma tissue uptakes more131I-anti-AT1R mAb than other tissues, whereas hepatocellular carcinoma tissue does not selectively uptake 131I-IgG. Thus,131I-anti-AT1R mAb may be a potential imaging agent for targeting hepatocellular carcinoma.Pharmacokinetic analysisPharmacokinetic analysis showed that the pharmacokinetics of131I-anti-AT1R mAb was in accordance with the two-compartment model, with a rapid distribution phase and a slow decline phase. T1/2a and T1/2p were5.7h and156.7h, respectively, and MRT was8.8h.AT1R mRNA and protein expressionThere was a markedly higher AT1R mRNA level in H22cells than that in NCTC clone1469cells. Similarly, there was a significantly higher AT1R mRNA level in hepatocellular carcinoma tissue than that in contralateral muscle (control1) or normal liver tissue (control2). AT1R protein was mainly localized to cell membranes. AT1R protein level was significantly higher in H22cells than that in NCTC clone1469cells. Similarly, AT1R protein level was significantly higher in hepatocellular carcinoma tissue than that in control1or control2. In addition, AT1R protein level was higher in PC12cells than in H22or HeLa cells.ConclusionAT1R expression was up-regulated in hepatocellular carcinoma tissue.131I-anti-AT1R mAb enables non-invasive evaluation of hepatocellular carcinoma specifically and may be a new potential molecular imaging agent for targeting tumor. |
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