Methotrexate Polyglutamate Synthesis Research Articles

Insulin-dependent suppression in glutamyl hydrolase activity and elevated cellular methotrexate polyglutamates

Folates and antifolates are converted to polyglutamates, which are better retained in cells and may also bind more tightly to cellular proteins than the parent compounds. The regulation of the process of polyglutamate formation and breakdown is not fully clarified yet and is being studied by a number of approaches. An early observation concerning the potential regulation of polyglutamate formation was that insulin caused a marked increase in the rate and accumulation of polyglutamates of methotrexate (MTX) in rat hepatoma cells. The present study demonstrated that insulin caused a decrease in the activity of γ-glutamyl hydrolase (GH), the enzyme that degrades polyglutamates, that was inversely commensurate with the increase in the synthesis of MTX polyglutamates. The effects of insulin on GH activity with regard to concentration, time of onset, and the effect of N 6, O 2′ dibutyryl cAMP and theophylline were consistent with the reduction in GH being responsible for the increase in cellular MTX polyglutamate accumulation. Insulin addition also led to an increase in folate polyglutamates. The insulin effects were also seen with H35D cells, a subline with enhanced glutamyl hydrolase activity as a result of having been made resistant to 5, 10-dideazatetrahydrofolic acid. When H35 cells with insulin were compared with H35D cells lacking insulin, there was an 8-fold increase in GH and a 44-fold decrease in the number of γ-glutamate residues added to MTX.

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.

The effect of leucovorin on the synthesis of methotrexate poly-γ-glutamates in the MCF-7 human breast cancer cell line

The modulating effects of leucovorin on the synthesis of methotrexate (MTX) polyglutamates in the MCF-7 human breast cancer cell line have been investigated using a paired-ion high performance liquid chromatography (HPLC) system. Leucovorin decreased the intracellular level of MTX and profoundly affected polyglutamate synthesis irrespective of whether it was administered with or after MTX. Inhibition of MTX polyglutamate synthesis was also observed when concentrations of leucovorin too low to affect intracellular levels of MTX were employed. Leucovorin did not promote efflux of MTX from the MCF-7 cells and did not affect the distribution of the retained drug amongst the various polyglutamate forms.

Pharmacokinetics of methotrexate in erythrocytes in psoriasis.

The intraerythrocytic levels of folate and methotrexate were measured in 25 patients on long-term methotrexate therapy for recalcitrant psoriasis. The mean steady state concentration of methotrexate in erythrocytes was 85 nmol/l and the mean erythrocyte folate concentration was 729 nmol/l. A linear correlation was not observed between these parameters, but the greatest methotrexate accumulation was found in cells at the lower end of the erythrocyte folate concentration range. In 5 patients started on methotrexate therapy the erythrocyte concentrations of methotrexate and folate were followed over 6 months. 3-4 days after the first dose, methotrexate had been accumulated against a concentration gradient in the erythrocytes. The methotrexate concentration increased steadily until the steady state level was reached after 4-6 weeks. The steady state level was maintained during the 6 month observation period. The erythrocyte folate concentration did not change during this period. The data suggest that methotrexate polyglutamate synthesis within the circulating erythrocyte is a major cause of methotrexate accumulation in these cells.

Methotrexate polyglutamate synthesis by cultured human breast cancer cells.

We studied the conversion of methotrexate to poly-gamma-glutamyl derivatives by cultured human breast cancer cells. After incubation with 2 micro M [3',5',9-3H]methotrexate, MCF-7 cells were washed free of extracellular drug and were boiled to lyse cells and to release drug bound to dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The supernatant fraction was chromatographed on Sephadex G-15 to separate parent drug from polyglutamate forms. These cells rapidly and quantitatively converted methotrexate to polyglutamates, such that after 24 hr of incubation, 70 +/- (SEM) 3% of intracellular methotrexate existed as polyglutamates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase indicated that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. These studies demonstrate that methotrexate polyglutamates are readily formed in human tumor cells and bind to dihydrofolate reductase. Because these forms of the drug may be selectively retained within the cell, they may be important determinants of the duration of action and, ultimately, the cytotoxicity of methotrexate in human solid tumors.

Synthesis of Methotrexate Polyglutamates by Bone Marrow Cells from Patients with Leukemia and Lymphoma

Bone marrow cells from children with lymphoblastic leukemia, myeloblastic leukemia and non-Hodgkin's lymphoma were incubated with tritium-labelled methotrexate in short-term cultures. Cells obtained both during disease activity as well as during remission synthesized large quantities of methotrexate polyglutamates. Synthesis was time- and dose-dependent and occurred with concentrations of methotrexate regularly achieved during conventional treatment with this drug.