Insulin effects on methotrexate polyglutamate synthesis and enzyme binding in cultured human breast cancer cells.

Previous studies have demonstrated that insulin augments methotrexate transport and enhances its cytotoxicity to human breast cancer cells. We therefore investigated the effects of insulin on methotrexate polyglutamate synthesis and binding to dihydrofolate reductase (DHFR) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. Cells were exposed to 2 microM [3H]MTX and varying insulin concentrations for the desired time before determination of the polyglutamate content by high-performance liquid chromatography (HPLC). DHFR-bound drug was separated from free intracellular drug by chromatography on DEAE-Sephacel minicolumns prior to HPLC analysis. Incubation of MCF-7 cells with 2.5 nM insulin for 48 h before exposure to 2 microM [3H]MTX for a further 24 h resulted in a significant increase in both total drug and total polyglutamates compared with control cells. Increasing the insulin concentration in the medium yielded further increases in polyglutamylation so that at 250 nM insulin and above total polyglutamates were increased by 64% compared with control cells. Further evaluation of the effects of physiologic insulin levels on polyglutamate synthesis revealed that 2.5 nM insulin caused an increase in the net glutamylation rate for each polyglutamate derivative during the final 12 h of a 24 h exposure to MTX. Analysis of the effects of insulin on polyglutamate binding to DHFR revealed that exposure to 2.5 nM insulin resulted in the preferential binding of higher polyglutamates to DHFR. In MDA-231 cells, a breast cancer cell line with a poor capacity for polyglutamate synthesis, insulin exposure resulted in an increase in the cellular accumulation of each polyglutamate derivative, with the greatest proportionate increases occurring in the cellular levels of higher polyglutamates. These data suggest that insulin augmentation of MTX polyglutamate synthesis may account for its previously observed ability to enhance MTX cytotoxicity.