Transfection Of The Sodium Iodide Symporter Gene Into Human Large Cell Lung Cancer For ¹³¹I Therapy

Objective:Lung cancer is a kind of common diseases which harms people's health or even lives severely.Currently,the morbidity and mortality of lung cancer are ascending all over the world,accounting for 38.08%of malignant tumor caused death in male and 16%in female in cities,ranking top in both sex.Nowadays depending on the biological behaviors,researchers trend to categorize lung cancers to small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).The therapy of NSCLC in particular has not been obviously improved for many years.On the one hand, biological characters of NSCLC are complicated,its malignancy is high and multi-drug resistant occurrence is common.On the other hand,when were diagnosed 80%of NSCLC patients have been in advanced stage and lost chances of operation. So chemotherapy can not be replaced for the patients.However,multi-drug resistance seriously influences the effect of chemotherapy.Recently,the effectiveness of combined chemotherapy is only 20%-40%.Therefore,we conceived to combine transgenic technology and radionuclide therapy together,in this experiment,human sodium/iodide(hNIS)gene was transfected into one of human NSCLC—large cell lung cancer cell line H460.This method enabled H460 cells to uptake radioactive iodide(¹³¹I).Therefore,¹³¹I could inhibit cells proliferation and destroy tumor cells finally.Methods:1.We created the recombinant expression plasmids(pcDNA3.1-hNIS). Transfected hNIS gene into human large cell lung cancer cell line H460 by recombinant expression plasmids with lipofectamine 2000-plasmid complexes. Selected the stably expressing hNIS gene cell lines(hNIS-H460)with G418,and selected the control cell line(H460).Subsequently we investigated the biologic functions of stably expressing hNIS gene cell line(hNIS-460),including ¹²⁵I uptake assay of transiently and stably hNIS-gene-expressing cell lines,¹²⁵I influx-course and ¹²⁵I-efflux-course,perchlorate(NaClO₄)inhibition.And drew the curves of time-cpm.2.Xenografted large cell lung cancer were established in nude mice by subcutaneous injection of 10⁷/0.1mL hNIS-H460 cells on the shoulder flank,and controls were injected 10⁷/0.1mL wild type H460 on the same position.At the same time of the establishment of large cell lung cancer nude mice model,the thyroid gland blocking was performed by L-T₄(5mg/L)supplementation.When the xenografts reached 15mm in diameter,radioactive isotope ^99mTcO₄^-imaging in transfection group was successfully performed using ECT imaging system.For quantitative analysis of the amount of ¹²⁵I in the tumors or other organs,their radioactivities were quantified by a well-gamma counter.For ¹³¹I therapy studies,mice were divided into transfected group and control group,both of which were injected with ¹³¹I and imaged. Then Xenografted tumors' volumes and pathologic morphological changes were observed in order to investigate ¹³¹I treatment effect on xenografted large cell lung cancer in vivo.Results:1.The recombinant expression plasmids(pcDNA3.1-hNIS)were cut at Bglâ…¡sites and electrophoreses were performed by 1%Agrase.The result was that the recombinant expression plasmids was cut into gene fragments of 5502bp and 1922bp.The sequences were as the same as the prediction results.2.hNIS gene was successfully transfected into human large cell lung cancer cell line H460 by recombinant expression plasmids(pcDNA3.1-hNIS)with lipofectamine 2000 plasmid complexes.Selecting the stably expressing hNIS gene cell lines(hNIS-H460)with G418,the level of G418 was 700μg/ml.And the control cell line(H460)haven't the resistance of G418.3.Investigated radioiodide uptake assay of transient expressing hNIS gene cell lines:Transfected hNIS gene into human large cell lung cancer cell line H460 with lipofectamine 2000 plasmid complexes for 24h.The uptake abilities of ¹²⁵I was 3.04 times higher in hNIS-H460 cells than control group H460 cells(P＜0.05).4.¹²⁵I influx experiments:¹²⁵I accumulated quickly in stably-hNIS-gene-expressing cells(hNIS-H460),and reached the stable state 60-90min after ^I25I was added.Control group cells(H460)couldn't accumulate ¹²⁵I.5.Investigated ¹²⁵I uptake assay of stably-hNIS-gene-expressing cells:The uptake of ¹²⁵I was 50.97 times higher in hNIS-H460 cells than control group H460 cells(P＜0.05).6.¹²⁵I efflux experiment:The efflux of ¹²⁵I was rapid in hNIS-H460 whose effective half life was about 6 min.7.Perchlorate inhibition experiment:Uptaking was measured by incubating stably-hNIS-gene-expressing cells(hNIS-H460)with 0.5μCi ¹²⁵I and NaClO₄.The uptake abilities of hNIS-H460 cells was inhibited by NaClO₄ 50.31 times than control (P＜0.05).The result clearly demonstrated that the uptake abilities of hNIS-H460 cells depend on the hNIS gene.8.Established the large cell lung cancer nude mice model,the thyroid gland block was performed by L-T₄(5mg/L)supplementation.When the xenografts reached 15mm in diameter,radioactive isotope ^99mTcO₄^-imaging in transfected group was successfully performed using ECT imaging system.In contrast,no ^99mTcO₄^-uptake was found in control group.9.The quantitative analysis results showed the amount of ¹²⁵I in the transfected group tumors was 7.91 times higher than the control group(P＜0.05).The blocked thyroid gland's was 4.37 times higher than the control group.And for other organs, their ¹²⁵I uptake activities were similar to the control group and slightly higher than background. 10.¹³¹I therapy studies:Radionuclide ¹³¹I imaging showed that the xenografts of transfected group could uptake ¹³¹I.In contrast,the control group could not.And the effect of ¹³¹I treatment showed that,the tumors of control group grew quickly continuously.On the other hand,in the transfected group,tumor's growth is slower. And from the biginning of second week on,tumor sizes were beginning to shrink.Conclusion:We transfected hNIS gene into human large cell lung cancer cell line H460 by recombinant expression plasmids.Stably-hNIS-gene-expressing cell line(hNIS-H460) could uptake radioactive iodide on account of hNIS function and then we used radioactive iodide to inhibit tumor cells proliferation and killed tumor cells finally. The objective of this study was to provide ^99mTcO₄^-imaging and ¹³¹I therapy after hNIS gene transfection,the successful results of the present study appears to open a novel potential diagnostic and therapeutic approach in non-thyroid tumor,and the perspective is promising.