Transfer Of The Sodium Lodide Symporter Gene Into Gliomas For Radioiodine Therapy In Vitro

Objective:The most frequent brain tumors are the gliomas,Glioblastomas, due totheir rapid growth, diffusely infiltrate the brain, aggressive infiltration and highvascularity is up to date still incurable. Reasons why current therapies hadlimited impact on the mortality of patients with malignant gliomas are included:1)their property to rendering complete resection impossible, 2) the exstenceof the blood-brain barrier that limits the penetration of chemotherapeuticagents into the tumor and 3) the limited dose of irradiation which can bedelivered to the brain. Therefore, the development of new therapeuticstrategies for these incurable tumors is essential.Radioactive iodide treatment is an effective cancer therapy ofhyperthyroidism and I—transporting thyroid carcinoma. Radioactive iodidecan get into the body by mouth, by metastases as the same as the normaliodide, and concentrate into I—transporting organisms. The radioactiveiodide can releaseβenergy.βenergy can interfere metastases of tumor cellsby the direct and indirect effect of radiation and lead tumor cells to die.Radioactive iodide treatment have another advantage, which the effect ofradiation has a certain circumscription, such asβenergy from ¹³¹I can travel1.2mm from the point of radionuclide decay in tissues. So radioactive iodidecan not only destroy a cell, but also cell group.Therefore, we take Gene Therapy and Radioactive Iodide Treatment tocombine together, transected Human Sodium/Iodide (hNIS) gene into humangtioma celt lines U251 by recombinant expression ptasmids. Stablyexpressing hNIS gene cell lines can intake radioactive iodide by hNIS gene, and then we used radioactive iodide to inhibit cell proliferation and destroytumor cells finally. The objective of this study was to find the possibility oftransecting hNIS gene into human glioma cell lines by recombinantexpression plasmids, and tried to provide an objective evident for radioactiveiodide treatment in nonthyroid tumor in vitro.Methods:We will accredit the recombinant expression plasmids (pcDNA3.1-hNIS).Transecting hNIS gene into human glioma cell lines U251 by recombinantexpression plasmids with lipofectamine 2000-plasmid complexes. Selectionthe stably expressing hNIS gene cell lines (hNIS-U251) with G418, andselection the stably negative control cell lines (pcDNA3.1⁺ - U 251).Subsequently we investigated stably expressing hNIS gene cell lines(hNIS-U251)'s biologic function, including ¹²⁵I uptake assay of transientlyand stably expressing hNIS gene cell lines, ¹²⁵I influx-course and¹²⁵I-efflux-course. Furthermore we investigated ¹³¹I inhibitory effect on kindsof cell proliferation by MTT assay, cell clonogenic assays and FSM.Results:1. The recombinant expression plasmids (pcDNA3.1-hNIS) was cut outat Bqlâ…¡sites, and electrophoresis by 1% Agrase. The result was that therecombinant expression plasmids was cut into gene fragments of 5502bp and1922bp.The sequences were as the same as the prediction results.2. Transecting hNIS gene into human glioma cell lines U251 byrecombinant expression plasmids (pcDNA3.1-hNIS) with lipofectamine2000-plasmid complexes. Selection the stably expressing hNIS gene cell lines(hNIS-U251) with G418, the level of G418 was 900μg/ml; Selection thestably negative control cell lines(pcDNA3.1⁺ - U 251), the level of G418 was700μg/ml; The empty control cell lines (U251) haven't the resistance of G418.3. Investigated radioiodide uptake assay of transiently expressinghNIS gene cell lines: Transected hNIS gene into human glioma cell linesU251 with lipofectamine 2000-plasmid complexes by 24h. The uptakes of ¹²⁵Iwas 4 fold higher in hNIS-U251 cell lines than control groups U251 cell linesand pcDNA 3.1⁺ -U251 cell lines(P＜0.01).4. Investigated radioiodine uptake assay of stably expressing hNISgene cell lines: The uptakes of ¹²⁵I was 117 fold higher in hNIS-U251 celllines than control groups U251 cell lines (P＜0.01), and 106 fold higher thanpcDNA 3.1⁺ -U251 cell lines (P＜0.01).5. ¹²⁵I influx experiments showed: That ¹²⁵I was accumulated quickly instably expressing hNIS gene cell lines (hNIS-U251), and reached the steadystate with 90-120min after iodide is added. Control groups cell lines (U251)couldn't accumulate ¹²⁵I.6. ¹²⁵I efflux experiments showed: The efflux of ¹²⁵I was rapid inhNIS-U251; its effective half life was about 7 min.7. NaClO₄ inhibition experiments showed: Uptake was measured byincubating stably expressing hNIS gene cell lines (hNIS-U251) with 0.5μCi¹²⁵I and NaClO₄. And uptake was inhibited by NaClO₄, 30 fold lower thanbefore (P＜0.001). These results clearly demonstrated hNIS dependent iodideuptake by the tumor cells.8. We demonstrated the clonal forming efficiecy of stably expressinghNIS gene cell lines (hNIS-U251) after incubating with 0.5μCi ¹²⁵I for 12h was 9 fold lower (P＜0.01) than groups which was not incubated with ¹²⁵I in Cellclonogenic assays. In stably expressing hNIS gene cell lines (hNIS-U251)without incubated with ¹²⁵I and control groups cell lines (U251)withoutincubated with ¹²⁵I, the clonal forming efficiecy showed no significantdifference (P=0.82).9. In MTT assay which investigated ¹³¹I inhibitory effect on kinds of cellproliferation, the results showed the proliferous efficiecy of stably expressinghNIS gene cell lines (hNIS-U251) was lower than control groups cell lines(U251) after incubating with ¹²⁵I(P＜0.01), but still high.10. We measured S-phase fraction (SPF) and proliferous index (PI) ofthe cell lines in FSM. The results showed the longer the cells was incubatedby ¹²⁵I, the lower SPF and PI (P＜0.01). And after incubating with ¹²⁵I for thesame 8h, the SPF and Pt of hNIS-U251 was lower than U251 (P＜0.01).Conclusion:We transected hNIS gene into human glioma cell lines by recombinantexpression plasmids. Stably expressing hNIS gene cell lines (hNIS-U251)could intake radioactive iodide by the function of hNIS gene and then wewere used Radioactive Iodide to inhibit tumor cell proliferation and destroytumor cells finally. The objective of this study was to provide an objectiveevident for radioactive iodide treatment in nonthyroid tumor in vitro.1. We transected hNIS gene into human glioma cell lines by recombinantexpression plasmids successfully, and we were obtained the stablyexpressing hNIS gene cell lines: hNIS-U251.2. The stably expressing hNIS gene cell lines (hNIS-U251) could intakeradioactive iodide by hNIS gene. The uptakes of ¹²⁵I were 110 fold higher in hNIS-U251 cell lines than control groups cell lines U251.3. ¹²⁵I was accumulated quickly in stably expressing hNIS gene cell lines(hNIS-U251), and reached the steady state with 90-120min after iodide isadded. The efflux of ¹²⁵I was rapid in hNIS-U251; its efficient half life wasabout 7 min.4. The clonal forming efficiecy of stably expressing hNIS gene cell lines(hNIS-U251) after incubating with ¹³¹I for 12h was obviously lower thangroups which were not incubated with ¹³¹I. And the proliferous efficiecy ofstably expressing hNIS gene cell lines (hNIS-U251) was lower than controlgroups cell lines (U251) after incubating with ¹³¹I for the same times. ¹³¹Ican kill tumor cells surely.