| Objective To investigate the internalization of the radioiodinated somatostatin analog 125I-RC-160 by human somatostatin receptors subtype-2 (hSSTR2) transfected A549 lung carcinoma cells (A549-hSSTR2), and the lethal effect of 131I-RC-160 on A549-hSSTR2 cells in vitro,and the inhibition of 131I-RC-160 on A549-hSSTR2 transplantation tumors in nude mice.Methods:①Used radioligand binding assay, A549-hSSTR2 cells or A549-pc3 cells were incubated at 37℃for different times(0.25,0.5,1,4,8,20,24h) together with 125I-RC-160,regarded as radioligand,then determined the internalization radioligand at the end of incubation.②Used 3-(4 ,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT) assay.To observe the lethal effect of various dosage of 131I-RC-160,Na131I or RC-160 on A549-hSSTR2 or A549-pc3 cells after 24h,48h,72h,96h incubation periods.③Established the transplanted tumor models with A549-SSTR2 cells in the right haunch subcutis of nude mice,and with A549-pc3 cells in the left haunch subcutis as control.The inhibition effects of 131I-RC-160,RC-160,Na131I and normal sodium(NS)on the tumors were observed respectively。When the therapy ended up, determined tumor volumes, then the nude mices were sacrificed for obtaining the transplanted tumors to do hematoxylin and eosin stain(HE stain)and fluorescence immunocytochemistry.Results:①125I-RC-160 rapidly combined with the membrane receptor of A549-hSSTR2 and was internalized. The cell-bound radioactivity counting (internalization and membrane-bound),receptor internalization and membrane-bound radioactivity counting are all time-dependent increasing before 1h。And the cell-bound,receptor internalization and membrane-bound radioactivity counting of A549-hSSTR2 cells is (18.2±1.9)%,(8.4±1.3)% and (9.8±1.2)% of total radioactivity counting added while 1hour incubation respectively。It showed time-dependent increasing internalization and time- dependent decreasing membrane binding of 125I-RC-160 by A549-hSSTR2 cells after incubating 1h,and the cell-bound is time-dependent decreasing slightly。The cell-bound,receptor internalization and membrane-bound radioactivity counting of A549-hSSTR2 cells is (16.9±2.2)%,(13.0±1.1)% and (3.9±2.2)% of total radioactivity counting added while incubating 24h respectively。The cell-bound radioactivity counting of A549-pc3 [(5.7±1.4)% while 1h and (5.2±1.3)% while 24h] is lower than that of A549-hSSTR2 cells。②The effect of 131I-RC-160 on the growth of A549-hSSTR2 cells is stronger than that on A549-pc3 cells, and is time-,and concentration-dependent。The inhibition ratio of 3700KBq/ml of 131I-RC-160 is (78.8±5.9)% on A549-hSSTR2 cells and (7.8±4.7)% on A549-pc3 cells after incubating 96h;The effect of 131I-RC-160 is about 3.5 times than that of RC-160 on the growth of A549-hSSTR2 cells;Na131I has no obviously effect on the growth of A549-hSSTR2 or A549-pc3 cells。③In the A549-SSTR2 transplanted tumors,contrast to normal sodium groups,the growth of the transplanted tumors therapied by 131I-RC-160 and RC-160 was inhibited significantly,and the inhibition rate is (75.1±4.2)% and (45.2±3.7)% respectively,HE stain showed that bulk lamellar cellular necrosis occurred in the transplanted tumors therapied by 131I-RC-160, was up to about 80 percent of tumor tissues。The tumors therapied by RC-160 also have lamellar cellular necrosis,but was inferior to the 131I-RC-160 group。Fluorescence immunocytochemistry showed the green fluorescence intensity of the necrosis zones of 131I-RC-160 and RC-160 groups is weaker than the other zones,NS and Na131I groups which showed relucent green fluorescence;In the A549-pc3 transplanted tumors,contrast to normal sodium contral group,slight inhibition effect of 131I-RC-160 and RC-160 on the tumors has no statistical significance(P>0.1), we didn't observe conspicuous lamellar but punctiform cellular necrosis in HE stain and not observe conclusive green light in fluorescence immunocytochemistry.Na131I has no inhibition effent on transfected or non-transfected transplantation tumors.Conclusion:①125I-RC-160 can be internalized by the hSSTR2 transfected A549 cells。②the lethal effect of RC-160 and 131I-RC-160 on A549-hSSTR2 cells or transplantation tumor are enhanced obviously than that on A549-pc3 cells or transplantation tumor;And the lethal effect of 131I-RC-160 is stronger than that of RC-160 on A549-hSSTR2 cells or transplantation tumor。③transfection SSTR2 gene-mediated targeted radiatherapy can provide experimental evidence for the concept of targeted exogenous receptor gene-mediated internal radiatherapy of human cancer,and develop a new therapy method for those SSTR-negative tumors。... |
