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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.
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 column and analyzed by high performance liquid chromatography. Hepatatocellular carcinoma cell membrane fractions wereisolated 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 BEL 7402 cells and human hepatocyte cell line HL7702 wasevaluated using the MTT assay .RESULTS: Insulin-MTX competed as effectively as insulin with 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 growth of human hepatoma cells (BEL7402) almost as potently asMTX.The inhibitory effect reached a peak on the 5th daywhen the growth of cells was inhibited by 79% at aconcentration of 5.0 μg / mL insulin-MTX. Treatment with 5.0 μg / mL of MTX and 5.0 μg / mL of insulin-MTX was onlyresulted in inhibition of HL7702 cells by 31.5% and 7.8% on the 5th day. CONCLUSION: 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.