Sequential methotrexate and 5-fluorouracil: mechanisms of synergy.
The finding that 5-fluorodeoxyuridylate (FdUMP), the active metabolite of 5-fluorouracil (5-FU) or 5-fluorodeoxyuridine (FUdR), requires the folate cofactor N5,N10-methylene tetrahydrofolate for tight binding to thymidylate synthetase (TS) had important potential consequences for the clinical use of these drugs. The lack of sufficient folates in the tumor cell, especially N5,N10-methylene tetrahydrofolate, would thus result in less than optimal cell kill by FUdR and possibly by 5-FU. Methotrexate (MTX) pretreatment would also decrease the level of this coenzyme by reducing tetrahydrofolate synthesis and thus could antagonize 5-FU and FUdR action. However, MTX and its polyglutamate forms also enhance binding of FdUMP to TS. In addition, we have shown that dihydrofolate polyglutamates also markedly enhance binding of FdUMP to TS. Thus, in mice bearing the sarcoma 180 tumor, pretreatment with MTX results in synergy; the opposite sequence gives less than additive antitumor effects. MTX also enhances 5-FU uptake into cells, as a consequence of increased FUra nucleotide formation that results from increased levels of intracellular phosphoribosylpyrophosphate (PRPP); PRPP is generated due to inhibition of purine synthesis by MTX. The increase in 5-FU nucleotide levels results in elevated levels not only of FdUMP but also of fluorouracil triphosphate (FUTP); this latter compound is incorporated into RNA. In addition, deoxyuridylatetriphosphate (dUTP) is incorporated into DNA when dUMP and dUTP levels increase as a consequence of MTX and/or 5-FU treatment. Biochemical data are thus beginning to accumulate, providing an understanding of the MTX/5-FU synergy that has been well documented in several experimental systems.