Experimental Studies On Killing Effect And Radionuclide Imaging Of Receptor-hsstr2Gene Transfected Tumor Cells

ObjectiveTo introduce human somatostatin receptors subtype-2(hSSTR2) gene into lung carcinoma A549cell which does not express the gene, investigate the binding characters between the radioiodinated somatostatin analog125I-RC-160and hSSTR2expressed on the transfected A549lung carcinoma cells (A549-hSSTR2), and to observe the lethal effect of131I-RC-160on A549-hSSTR2cells in vitro or in vivo and tumor scintigraphy mediated by the receptorMethodsAn enkaryotic expression vector including hSSTR2was constructed. In vitro human lung adenocarcinoma A549cells were transfected with the vector. The expression of SSTR2in the resulting cell clone was investigated by RT-PCR and flow cytometry and the clones overexpressing SSTR2were selected for in vitro radioligand binding assay with125I-RC-160. The lethal effect of various dosage of131I-RC-160、Na131I、RC-160on A549-hSSTR2after different incubation periods was observed by3-(4,5-dimethylthiazol-2. yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Established the xenograft tumor models in nude mice with A549-SSTR2by incubation subcutaneously. SPECT was performed in nude mice bearing A549tumors overexpressing SSTR2. The in vivo lethal effect of131I-RC-160、Na131I、RC-160on the nude mice were also investigated。ResultsA549cell clones stably transfected with hSSTR2was generated, and the expression of SSTR2in those clones was confirmed by RT-PCR and flow cytometry. SSTR2expressed on membrane of the above cells showed high affinity and specificity. Scatchard analysis of125I-RC-160and tumor cell revealed that KD=5.65x10-10mol/L, Bmax=2.01x104/cell. In competitive binding assay, IC5o=1.88x10-9mol/L. The inhibition effect of131I-RC-160on the growth of A549-hSSTR2cell is much stronger than A549-pcDNA3cells, and is concentration-dependent and time-dependent. The in vitro inhibition ratio of A549-hSSTR2cells is78.8±5.9%after incubated96h with7.4MBq/ml of131I-RC-160. Tumor lesions in nude mice was detected with99mTc-RC-160and clear images were achieved at0.5-1h postinjection. At24h postinjection images were showed again. The in vivo inhibition ratio of A549-hSSTR2cells is with131I-RC-160is75.1±4.2%。Conclusion125I-RC-160can bind with hSSTR2expressed on the cells transfected with hSSTR2gene; and the lethal effect of131I-RC-160on A549-hSSTR2 cells is enhanced obviously than that on control cells. This study demonstrates that it is available to transfer hsstr2to tumor cell line which does not express the gene and detect the tumor by the receptor-mediated SPECT. These provide an experimental evidence for the concept of targeted intemalradiation therapy of human cancer mediated by exogenous receptor gene.