Receptor binding characteristics and cytotoxicity of insulin-methotrexate.
OBJECTIVE
To characterize the receptor binding affinity and cytotoxicity of insulin-methotrexate (MTX) for the potential utilization of insulin as carriers for carcinoma target drugs.


METHODS
MTX was covalently linked to insulin. Insulin-MTX conjugate was purified by Sephadex G-25 column and analyzed by high performance liquid chromatography. Hepatocellular carcinoma cell membrane fractions were isolated by sucrose density gradient centrifugation. Competitive displacement of (125)I-insulin with insulin and insulin-MTX binding to insulin receptors were carried out. Cytoreductive effect of insulin-MTX on human hepatoma BEL7402 cells and human hepatocyte cell line HL7702 was evaluated using the MTT assay.


RESULTS
Insulin-MTX competed as effectively as insulin with (125)I-insulin for insulin receptors. The values of Kd for insulin-MTX and insulin were 93.82+/-19.32 nmol/L and 5.01+/-1.24 nmol/L, respectively. The value of Kd for insulin-MTX was significantly increased in comparison with insulin (t=7.2532, n=4, P<0.005). Insulin-MTX inhibited the growth of human hepatoma cells (BEL7402) almost as potently as MTX. The inhibitory effect reached a peak on the 5 th day when the growth of cells was inhibited by 79% at a concentration of 5.0 microg/mL insulin-MTX. Treatment with 5.0 microg/mL of MTX and 5.0 microg/mL of insulin-MTX merely resulted in inhibition of HL7702 cells by 31.5% and 7.8% on the 5 th day.


CONCLUSIONS
Insulin-MTX specifically recognizes insulin receptors and inhibits the growth of BEL7402 cells. These results suggest that insulin can be used as a carrier in receptor mediated carcinoma-targeting therapy.
Ou
Xiao-Hong
XH
Department of Nuclear Medicine, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China. ouxiaohong2002@xinhuanet.com
Kuang
An-Ren
AR
Liang
Zheng-Lu
ZL
Peng
Xian
X
Zhong
Yu-Guo
YG
D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology / Q000601:surgery; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004337:Drug Carriers; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000633:toxicity*; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000601:surgery; D008727:Methotrexate / Q000633:toxicity*; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism
D004337:Drug Carriers; D007328:Insulin; D011972:Receptor, Insulin; D008727:Methotrexate