Synthesis, Retention, and Biological Activity of Methotrexate Polyglutamates in Cultured Human Breast Cancer Cells

Abstract

A B S T R A C T To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines converted the drug to y-polyglutamate derivatives in a dose-and timedependent reaction. After 24-h incubations with 2 zM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/ g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH2-10-CH3-PteGlu5 and 47 and 38% of the 4-NH2-10-CH3-PteGlu4 remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. MTX and an additional 24 h in drug-free medium,[3H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [3H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 ,uM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 jM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates.These studies demonstrate that polyglutamate formation allows a prolonged retention of drug in a noneffluxable form and prolonged inhibition of both thymidylate synthesis and cell growth following removal of extracellular drug.

Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.

“…However, after the 24-h incubation in drug-free media, MTX and MTX-Glu2 effluxed readily, and although the small quantities of longer polyglutamates were retained during efflux, their initial synthesis was insufficient to maintain drug in excess of the binding capacity. Closer study of individual polyglutamate metabolites confirmed that, as determined in prior experiments with breast cancer cell lines (13) and small-cell cell lines (17), drug retention correlates with glutamyl chain length. Thus, for all cell lines after a 24-h exposure to 1.0 1sM MTX, only 4-24% of parent drug present at the end of drug incubation remained after 24-h efflux, while 25% of MTX-Glu2, 70% of MTX-Glu3, 75% of MTX-Glu4, and 83% of MTX-Glu5 were retained.…”

“…In the absence of free drug, intracellular physiologic folates can compete for binding sites on DHFR, reversing enzyme inhibition (7)(8)(9). The transformation of MTX to polyglutamyl derivatives results in the formation of active metabolites of the drug that, when present in excess of the intracellular binding capacity, results in prolonged inhibition of DHFR (10)(11)(12)(13)(14)(15)(16)(17). In addition, MTX polyglutamates (MTX-PGs) may have additional sites of action as inhibitors of aminoimidazole carboxamide ribonucleotide transformylase (de novo purine synthesis) (18) and thymidylate synthase (TS) (19).…”

Cell-Cycle-Dependent Pharmacology of Methotrexate in HL-60

“…4. MTX, and folates in general, are metabolized intracellular to polyglutamate by folyl polyglutamate synthetase (34). MTX-polyglutamate is also preferentially retained in cells and has equal affinity with MTX for DHFR but dissociates at a slower rate and crosses cellular membranes less easily.…”

Comparison of plasma and tissue levels of ZD1694 (Tomudex), a highly polyglutamatable quinazoline thymidylate synthase inhibitor, in preclinical models

“…Their measurement in tissue culture cells has been achieved previously after incubation with radiolabelled compounds followed by extraction and HPLC separation (Jolivet et al, 1982;Sikora et al, 1988;Pizzomo et al, 1989;Jackman et al, 1991c;Gibson et al, 1993). Similar methodology has been used to measure the polyglutamated forms of methotrexate in isolated human leukaemic blast cells (Goker et al, 1993).…”