Figure 1.

Methotrexate mode of action. Methotrexate (MTX) is actively transported into the cell by the reduced folate carrier 1 (RFC1; also known as SLC19A1) and is then polyglutamated by folylpolyglutamate synthetase (FPGS) to form MTX polyglutamates (MTX PG), which are kept inside the cell [221] and are responsible for MTX anti-inflammatory intracellular actions [17,174]. Glutamates can be removed by γ-glutamyl hydrolase (GGH) and MTX monoglutamate is rapidly effluxed from the cell via membrane transporters of the ATP-binding cassette (ABC) family [222], especially ABCC1-4 and ABCG2 [223,224]. Inside the cell, MTX PG exert their anti-inflammatory actions through inhibition of essential enzymes of the folate pathway: dihydrofolate reductase (DHFR) [225], blocking the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF) and ultimately leading to depletion of methionine and decreased DNA methylation; thymidylate synthase (TYMS) [226,227], interfering with de novo pyrimidine synthesis; and 5-aminoimidazole-4-carbox-amide ribonucleotide (AICAR) transformylase (ATIC) [148,228], an enzyme of the de novo purine synthesis, causing accumulation of AICAR, which will finally result in increased secretion of adenosine, a strong anti-inflammatory mediator [229,230]. The enzyme 5,10-methylene-tetrahydrofolate reductase (MTHFR) is not directly inhibited by MTX, but is affected by it because of its action in the folate pathway [176]. ADA, adenosine deaminase; AMPd, adenosine monophosphate deaminase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; FAICAR, 10-formyl 5-aminoimidazole-4-carboxamide ribonucleotide; IMP, inosine monophosphate; Methyl-THF, 5-methyl-tetrahydrofolate; Methylene-THF, 5,10-methylene-tetrahydrofolate; MS, methionine synthase; SHMT, serine hydroxymethil transferase.