The Experimental Study On Radionuclide Imaging And Therapy Of Nasopharyngeal Carcinoma Transferred Human Sodium/Iodide Symporter Gene

ObjectiveIn order to provide a new gene therapy strategy for monitoring and treatment of nasopharyngeal carcinoma (NPC), we construct the technology of radionuclide imaging and therapy NPC transferred human sodium/iodide symporter gene (hNIS). To investigate whether DW-MRI can early monitor the therapy effect of gene therapy and provide a new method for monitoring therapy effect of targeted radionuclide treatment, DW-MRI will be used to assess the therapy effect of NPC xenografts transferred hNIS after 131I treatment. Furthermore, basing on relative apoptosis genes, the mechanisms of 131I treatment NPC mediated transfection of hNIS gene will be investigated.Methods1. In vitro:The recombinant expression plasmid pCMV-Tag2-hNIS was constructed. The plasmid pCMV-Tag2-hNIS was then transferred into NPC cell line CNE-2 by lipofectamine 2000, and stably expressing hNIS cell line CNE-2-hNIS was got by screening with G418. Subsequently the biologic functions including radioiodide uptake assay,125I influx-course, and 125I efflux-course were investigated. And proliferation and apoptosis of NPC cells after treatment with 131I in vitro were detected by CCK-8 cell proliferation, colony formation assay, Hoechst 33342 fluorescence staining and Annexin V-FITC/PI double-labeled flow cytometry.2. In vivo:Xenografted NPC model were established in nude mice. When xenograft tumor had reached 10 mm in diameter, the radioactivity of different organs of nudes was counted in 0.5,1,2,6 and 24 h using gamma counter after i.p. injections 125I. Radioactive isotope 99mTc imaging of nude mice was performed using SPECT after i.p. injections 99mTcO4-.3. Basing on the establishment of NPC xenograft model in nude mice, the changes of xenograft tumor volume were observed in 3,6,12,18,24 days by MRI scan after after i.p. injections 131I in the experimental group and control group, respectively. The changes of ADC value of xenograft tumor were observed by DW-MRI in 3,6,12,18 and 24 days after treatment with 131I. The correlations of changes of ADC with pathological TUNEL, Caspase-3 immunohistochemistry of apoptosis, and Ki-67 proliferation detection were further investigated.4. The protein expression level of p53, Bax, Bcl-2, Caspase-3 and Surviving were detected by Western blot after 131I treatment.Results1. We successfully constructed the recombinant expressive plasmids pCMV-Tag2-hNIS. Stable expressing hNIS NPC cells CNE-2-hNIS were obtained, and hNIS protein was identified by Western blot and immunocytochemistry method. 125I uptake experiments showed that the uptake of 125I was 17.1±2.3 fold higher in CNE-2-hNIS than in those of CNE-2(P<0.05), and the accumulation and efflux of 125I was rapid in CNE-2-hNIS, reaching peak state within 30 minutes, and its efficient half life was about 8.45 minutes. With the increase of the concentration of 131I, the CNE-2-hNIS proliferation rate and cloning efficiency detected by CCK-8 cell proliferation assay and colony formation experiments were gradually decreased, whereas cell apoptosis rate detected by hoechst 33342 staining and Annexin V-FITC/PI flow cytometry was gradually increased.2. NPC xenograft nude mice model was successfully established. Biodistribution of 125I in tumor-bearing nude mice model showed that the uptake of 125I capacity of tumor in experimental group was enhanced, and then arrived peak within 1h, which still maintained a high level in 2h. However, amount of 125I in the experimental group tumors was 10.71±0.35 fold higher than the control group tumor. The 125I biological half-life in the tumor transferred hNIS was about 1.56 h.99mTc imaging study showed that the tumor of experimental group accumulated 99mTc leading to scintigraphic visualization clearly through SPECT in 1～2h after i.p. injections 99mTcO4- in nude mice, whereas the control tumor was not visualized.3. The changes of NPC xenograft tumor volume were further evaluated by MRI scan. Compared with the control group, tumor volume of the experimental group was significantly reduced compared with that of the control group after 131I treatment (P< 0.05). DW-MRI showed that in the 3th day after 131I treatment, ADC values increased in the experimental group, and ADC values reached the peak in the 6th and 12th day after 131I treatment, then ADC values were decreased. ADC values afer treatment was positively correlated with apoptotic index (r=0.72,P<0.05) and caspase-3 positive rate (r=0.65, P<0.05) and there was a negative correlation with Ki-67 proliferation index (r=-0.71, P<0.05).4. After 131I treatment NPC cells transferred hNIS,131I up-regulated of the expression of p53 and Surviving protein, and actived Caspase-3, whereas down-regulated of the expression of Bcl-2 proteins, which has no effect on the expression of Bax.ConclusionWe successfully construct technology of radionuclide imaging and therapy of NPC transferred hNIS, which will provide a new gene therapy strategy for monitoring and treatment of NPC. For the first time, we use DW-MRI technology to early non-invasively monitor effect of targeted radionuclide therapy of tumor in the micro level from the water molecules, showing the application prospect of DW-MRI in monitoring the efficacy of gene therapy.131I can restrain the growth of NPC xenografts transferred hNIS. The mechanisms are involved in up-regulated of expression p53 protein, down-regulated of the expression Bcl-2 proteins and associated with promotion of Caspase-3 proteins activation.