Screening CD13 expression in vitro using 131I-NGR peptide

Abstract

Objective To synthesize TM I-NGR peptide and use it to screen CD13 expression in dif- ferent tumor cell lines in vitro. Methods NGR peptide was synthesized and labeled with 131I by the Iodo- gen method. The radioehemical purity was evluated with TLC. Stability was tested in double volume of nor- mal saline and fresh human serum at room temperature and 37 ~C at 2, 12 and 24 h after labeling. Immuno- fluorescence and Western blot experiments were used to detect CDI3 expression on HT-1080, human umbili- cal veins endothelial cell （HUVEC） , HepG2, U87Mg, PC-3, HT-29 and MCF-7 tumor cell lines. Binding affinity of CD13 positive cell lines with 1311-NGR was detected by a radiochemica] receptor assay. The lethal effect of various dosages of NaTM I, NGR and 1311-NGR was measured by an 3-（4,5-dimethyhhiazol-2-yl） -2, 5-diphenyhetrazolium bromide （MTr） assay on HT-1080 and HT-29 cells lines after 24, 48 and 72 h incu- bation. The cell inhibitory rate was analyzed by one-way analysis of variance. Results NGR was success- fully synthesized and labeled with 131I. Optimized labeling conditions were 18.5 MBq 131I, 10 μg NGR and 20 Ixg Iodogen. The labeling yield of 131I-NGR was （93.7 ±2.5）% , and specific activity was 1.41 TBq/mmol. 131I-NGR was stable with a radiochemical purity of more than 87% at 24 h. Immunofluorescence experi- ments and Western blot demonstrated that HT-1080, HUVEC, HepG2, U87Mg and PC-3 cell lines were positive for CD13 expression, while MCF-7 and HT-29 ceils lines were negative. The binding affinity of 131I- NGR with CD13 in HT-1080 cells was examined by a receptor binding assay and resulted in Kd = 7.3 nmol/L and Bin= = 0. 302 nmol/L. MTT assay showed that 131I-NGR had a much stronger growth inhibitory effect on HT-1080 ceils than HT-29 cells. The inhibitory rate of ^131I-NGR （3700 MBq/L） on HT-1080 was （67.9 ± 3.4） % at 72 h; while on HT-29 cells, the rate was merely （4.0 ± 0.5 ）% at the same time point. Con- clusions 131I-NGR can be efficiently prepared and very specifically targeted to CD13 positive human tumor cell lines. Tumor-targeted imaging and internal radiotherapy with 131I -NGR needs further research. Key words: NGR; Iodine radioisotopes; Isotope labeling ; Tumor cells, cultured ; Aminopeptidases