Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells.
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 (MCF-7 and ZR-75-B) converted the drug to gamma-polyglutamate derivatives in a dose- and time-dependent reaction. After 24-h incubations with 2 muM 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-NH(2)-10-CH(3)-PteGlu(5) and 47 and 38% of the 4-NH(2)-10-CH(3)-PteGlu(4) 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. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX. After 24-h incubation with 2 muM MTX and an additional 24 h in drug-free medium, [(3)H]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, [(3)H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 muM 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 muM 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.
D001943:Breast Neoplasms / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D010452:Peptide Biosynthesis; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000096:biosynthesis*; D013997:Time Factors; D014018:Tissue Distribution
D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008727:Methotrexate