Experimental Studies On Killing Effect And Radionuclide Imaging Of Receptor-hSSTR2 Gene Transfected Tumor Cells

Objective To introduce human somatostatin receptors subtype-2(hSSTR2) gene to A549 lung carcinoma cell which does not express the gene, investigate the binding characters between the radioiodinated somatostatin analog 125I-RC-160 and hSSTR2 expressed on the transfected A549 lung carcinoma cells (A549-hSSTR2) ,and to observe the lethal effect of 131I-RC-160 on A549-hSSTR2 cells and tumor scintigraphy mediated by the receptor .Methods An enkaryotic expression vector including hSSTR2 was constructed. In vitro human lung adenocarcinoma A549 cells were transfected with the vector. The expression of SSTR2 in the resulting cell clone was investigated by RT-PCR and flow cytometry and the clones overexpressing SSTR2 were selected for in vitro radioligand binding assay with 125I-RC-160. Used 3-(4,5-dimethylthiazol-2.y1)-2,5-diphenyltetrazolium bromide(MTT) assay to observe the lethal effect of various dosage of 131I-RC-160,Na131I,RC-160 on A549-hSSTR2 after different incubation periods. Established the xenograft tumor models in nude mice with A549- SSTR2 by incubation subcutaneously. SPECT was performed in nude mice bearing A549 tumors overexpressing SSTR2.Results A549 cell clones stably transfected with hSSTR2 was generated, and the expression of SSTR2 in those clones was confirmed by RT-PCR and flow cytometry. SSTR2 expressed on membrane of the above cells showed high affinity and specificity. 125I-RC-160 rapidly combined with the membrane receptor of A549-hSSTR2. Scatchard analysis of 125I-RC-160 and tumor cell revealed that KD=5.65×10-10mol/L,Bmax =2.01×104/cell. In competitive binding assay, IC50=1.88×10-9mol/L. The inhibition effect of 131I-RC-160 on the growth of A549-hSSTR2 is stronger than that on A549- pcDNA3 cells, and is time-,and concentration-dependent. The inhibition ratio of A549-hSSTR2 cells is 78.8±5.9% after incubated 96h with 7.4MBq/ml of 131I-RC-160. Tumor lesions in nude mice was detected with 99mTc-RC-160 and clear images were achieved at 0.5-1h postinjection. At 24h postinjection images were showed again.Conclusion 125I-RC-160 can bind with hSSTR2 expressed on the cells transfected with hSSTR2 gene; and the lethal effect of 131I-RC-160 on A549-hSSTR2 cells is enhanced obviously than that on control cells. This study demonstrates that it is available to transfer hsstr2 to tumor cell line which does not express the gene and detect the tumor by the receptor-mediated SPECT.These can provide experimental evidence for the concept of targeted exogenous receptor gene-mediated internalradiation therapy of human cancer.