Ability of luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 binding to LHRH receptor on human liver cancer cells

摘要

AIM: To explore the ability of recombinant toxin luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) and LHRH binding to LHRH receptor (LHRHR) on the membrane surface of human liver cancer HEPG cells.METHODS: LHRH was labeled by using ¹²⁵I with enzymatic reaction. The affinity and receptor volume of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of human liver cancer cells were measured with radioligand receptor assay.RESULTS: The specific activity of LHRH labeled with ¹²⁵I was 2.7 × 10⁴ kBq/μL, and its radiochemical purity reached to 99.2%-99.7%. The binding of ¹²⁵I to LHRH was maximal for 240 min in the warm cultivation, and this binding was stabilized. The inhibiting rates of ¹²⁵I-LHRH and LHRH on the proliferation of human liver cancer HEPG cells were not significantly different. On the basis of the saturation curve of ¹²⁵I-LHRH binding to the membrane LHRHR of HEPG cells, ¹²⁵I-LHRH of 1 × 10⁵ cpm was selected for radioligand receptor assay. The affinity constants (Kd) of LHRH-PE40 and LHRH binding to the membrane LHRHR of HEPG cells were 0.43 ± 0.12 nmol/L and 4.86 ± 1.47 nmol/L, respectively, and their receptor volumes were 0.37 ± 0.15 μmol/g and 0.42 ± 0.13 μmol/g, respectively. The binding of LHRH-PE40 to the membrane protein of normal liver cells was not observed.CONCLUSION: The recombinant toxin LHRH-PE40 binding to the membrane surface of LHRHR of human liver cancer HEPG cells was very strong, while the specific binding of it to normal liver cells was not observed. The results provide an important experimental basis for the clinical application of LHRH-PE.