Receptor binding characteristics and cytotoxicity of insulin-methotrexate

AIM:To characterize the receptor binding affinity andcytotoxicity of insulin-methotrexate (MTX) for the potentialutilization of insulin as carriers for carcinoma target drugs.METHODS:MTX was covalently linked to insulin.Insulin-MTX conjugate was purified by Sephadex G-25 columnand analyzed by high performance liquid chromatography.Hepatocellular carcinoma cell membrane fractions wereisolated by sucrose density gradient centrifugation.Competitive displacement of ~(125)I-insulin with insulin andinsulin-MTX binding to insulin receptors were carried out.Cytoreductive effect of insulin-MTX on human hepatomaBEL7402 cells and human hepatocyte cell line HL7702 wasevaluated using the MTT assay.RESULTS:Insulin-MTX competed as effectively as insulinwith ~(125)I-insulin for insulin receptors.The values of Kd forinsulin-MTX and insulin were 93.82 19.32 nmol/L and5.01 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 growthof human hepatoma cells (BEL7402) almost as potently asMTX.The inhibitory effect reached a peak on the 5 th daywhen the growth of cells was inhibited by 79% at aconcentration of 5.0μg/mL insulin-MTX.Treatment with5.0μg/mL of MTX and 5.0μg/mL of insulin-MTX merelyresulted in inhibition of HL7702 cells by 31.5% and 7.8%on the 5 th day.CONCLUSION:Insulin-MTX specifically recognizes insulinreceptors and inhibits the growth of BEL7402 cells.Theseresults suggest that insulin can be used as a carrier inreceptor mediated carcinoma-targeting therapy.