MTX and MTXPGs block the activity of the key enzyme

MTX and MTXPGs block the activity of the key enzyme DHFR (Fig. 1), which converts folates to their active forms – dihydrofolate (DHF) and tetrahydrofolate (THF). MTXPGs also potently inhibit thymidylate synthase (TS). Furthermore, during dTMP synthesis, TS utilizes the cofactor 5,10-methylene THF, which serves as a donor of the ‐CH2OH group. As a result of this reaction, 5,10-methylene THF is oxidized to DHF, which cannot be reduced back to THF due to the inhibition of DHFR [21]. In addition, MTXPGs and DHF polyglutamates that are accumulated after DHFR inhibition exert an inhibitory effect on GAR transformylase (GART). MTXPGs further inhibit 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase (ATIC). The inhibition of ATIC promotes the accumulation of AICAR, a potent inhibitor of adenosine deaminase (ADA) [7]. Overall, it is well known that MTX interferes with purine and pyrimidine synthesis, which is required for DNA replication and cell proliferation [30]. The inhibition of DHFR and other Rufinamide mg by MTX results in the depletion of reduced forms of DHF and nucleotides, which strongly affects the proliferation of treated cell populations and also induces cell death [33], [35]. However, there are also many non-DHFR-mediated effects of MTX (Fig. 1), which are discussed below.
Oxidative stress Although the cytotoxic effects of MTX are often induced by nucleotide depletion, non-DHFR-mediated effects of MTX are also important, as MTX can interfere with glyoxalase and antioxidant systems. It has been shown that MTX affects α-oxoaldehyde metabolism. The inhibition of glyoxalase I (Glo1) by MTX leads to the accumulation of methylglyoxal, a highly reactive α-oxoaldehyde, which causes glycation of biomolecules. This action contributes to the anticancer activity and toxicity of MTX [4]. Regarding oxidative stress, some reports show that MTX-induced anti-proliferation and pro-apoptotic effects depend on alterations of the intracellular reactive oxygen species (ROS) levels [13], [34]. Indirect evidence for MTX-induced actions through increased ROS production was demonstrated by studying the role of ornithine decarboxylase. The proposed mechanism of action is that MTX indirectly inhibits polyamine-producing enzymes. As a consequence, decreased polyamine production leads to increased intracellular ROS levels [51]. Furthermore, MTX is able to induce both apoptosis, through oxidative stress by reducing NO and increasing caspase-3 levels [8], and oxidative DNA damage, which can be lethal to tumor cells with defects in the MSH2 DNA mismatch repair gene [23].
Cell differentiation It has also been described that MTX acts as a strong differentiation factor for immature and undifferentiated monocytic cells [39] and is able to induce differentiation in human keratinocytes [38]. Moreover, MTX has the ability to trigger cellular differentiation in tumor cells, including human and rat choriocarcinoma cells, HL-60 human promyelocytic cells and other human leukemia cell lines, LA-N-1 human neuroblastoma cells, HT29 colon cancer cells and A549 human lung adenocarcinoma cells [30]. Recently, it has been described that in human melanoma cells, MTX promotes differentiation and prevents invasion [37], whereas it also induces differentiation of human osteosarcoma cells [44].