New Mechanisms for an Old Drug; DHFR- and non-DHFR-mediated Effects of Methotrexate in Cancer Cells

Summary

Methotrexate, a structural analogue of folic acid, is one of the most frequently used chemotherapeutics, especially in haematological malignancies, various solid tumours and also inflammatory disorders. Methotrexate interferes with folate metabolism, mainly by inhibition of dihydrofolate reductase, resulting in the suppression of purine and pyrimidine precursor synthesis. The depletion of nucleic acid precursors seems to be responsible for the cytostatic, cytotoxic and differentiation effects of methotrexate. Methylation of biomolecules represents another folate-dependent pathway that is also affected by methotrexate. Furthermore, metho­trexate is able to modify metabolic pathways and cellular processes independently of folate metabolism. Based on the similar structure of methotrexate and of functional groups of certain histone deacetylase inhibitors, the ability of methotrexate to inhibit histone deacetylases was predicted and consequently verified. Recently published findings also suggest that metho­trexate affects glyoxalase and antioxidant systems. Although methotrexate has been used as a folate metabolism antagonist in anticancer therapy for more than 60 years, the identification of its’ other molecular targets in cellular metabolism still continues.

Key words:
methotrexate – folate metabolism – dihydrofolate reductase – methylation – histone deacetylase inhibitors – glyoxalase system – oxidative stress

Introduction

Methotrexate (MTX; amethopterin; 4-amino-10-methylfolic acid), a structural analogue of folic acid, is one of the most frequent chemotherapeutic drugs [1]. MTX is used in the treatment of haematological malignancies, various types of solid tumours and also of inflammatory disorders. This large group of MTX-treated diseases includes leukaemia, breast cancer, colorectal cancer, head and neck cancer, lymphoma, osteo­genic sarcoma, urothelial cancer, choriocarcinoma, psoriasis and rheumatoid arthritis [2]. This review is focused on the various mechanisms of MTX action at the cellular level.

Folate Metabolism

The main biochemical function of folate, especially of its reduced form tetrahydrofolate (THF), is to serve as a co-factor//co-enzyme and to transfer one-carbon groups. THF acts as a donor of these groups in several interconnected metabolic pathways in the cytoplasm (Fig. 1). Three of one-carbon substituted THF derivatives are associated with crucial metabolic pathways: 5-methyl THF, which is required for synthesis of methionine; 5,10-methylene THF, which is essential for the synthesis of deoxythymidylate (dTMP), a pyrimidine component of DNA; and 10-formyl THF, which serves as co-factor for purine synthesis [3,4].

MTX as Inhibitor of Nucleotide Biosynthesis

The enzyme dihydrofolate reductase (DHFR) is the key intracellular target of MTX in folate metabolism. DHFR catalyses the reduction of folate to THF in two steps. The inhibition of DHFR by MTX is competitive with dihydrofolate (DHF) and results in THF depletion, leading to the inhibition of purine and pyrimidine precursor synthesis [5].

The lack of 5,10-methylene THF is a cause of the reduced synthesis of pyrimidine precursors, because thymidylate synthase (TS) is not able to catalyse methylation of dUMP to dTMP without 5,10-methylene THF. Moreover, TS is directly blocked by MTX and by un-metabolised dihydrofolate [6]. A severe lack of dTTP can lead to the phenomenon called ”thymineless stress” followed by “thymineless death” due to the inhibition of DNA synthesis. Preceding thymineless death, a large increase in dUTP concentration and its incorporation into DNA instead of dTTP can be found. Activation of the DNA excision repair ­pathway is the next step; however, this process cannot run correctly and apoptosis is induced by DNA damage [7]. Alternatively, changes in the ratio of intracellular concentrations of the nucleotides (i.e. nucleotide pool imbalance) are also able to trigger the mitochondrial pathway of apoptosis [8]. Nevertheless, other studies show that numerous homologous recombinations resulting from single-strand breaks in DNA are responsible for the cell death [9].

Purine precursor biosynthesis is also partially indirectly inhibited by defi­ciency of another folate co-factor, 10-formyl THF. However, it is primarily inhibited directly by the excessive levels of DHF in a cell [10], because during the inhibition of DHFR, the intracellular concentration of 10-formyl THF is maintained up to 80% [11]. In addition, MTX is also a direct inhibitor of AICAR transformylase (ATIC) [12] and GAR transformylase (GART) [13], two pivotal enzymes responsible for purine precursor synthesis. Unlike DHFR, the inhibition of ATIC and GART is markedly improved by polyglutamylation of MTX as MTX polyglutamates are more potent inhibitors – polyglutamylated MTX provides a stronger bond with enzymes [12,14].

MTX as Inducer of Cell Death

The previous data show that the inhibition of dTMP synthesis and de novo purine synthesis, either directly or as a result of the inhibition of DHFR, is the main reason for MTX-induced cell death. The proportion of the inhibition effect of purine or pyrimidine precursor synthesis on cell death may differ among various cell types, as well as between two main ways of cell death – apoptosis and necrosis [15]. Apoptosis is probably initiated during the S-phase of the cell cycle when DNA is synthesised [6], because a blockade of transition from G1 to S phase prevents MTX-induced apoptosis [16].

Surprisingly, MTX can also induce apoptosis in post-mitotic cells, in which DNA replication does not occur. For example, this phenomenon was described in post-mitotic pulmonary artery endothelial cells [17], or in rodent cortical neurons [18]. Kruman et al [18] found that MTX induces cell cycle re-entry in neurons; it was confirmed by the incorporation of BrdU (5-bromo-2‘-deoxyuridine) into newly synthesised DNA. Subsequently, affected cells can undergo apoptosis. The same effect was shown by homocysteine (Hcy), which additionally increased expression of p53 and cdc25 required for a progression from G1 to the S phase.

MTX as Inducer of Differentiation

Besides the cytostatic and cytotoxic effects of MTX, there was also described a differentiation effect of this compound. MTX was found to be a potent differen­tiation inducer in HL-60 human promyelocytic leukaemia cells [19], LA-N-1 human neuroblastoma cells [20], human neonatal foreskin keratinocytes [21], U937 human monocytic cells [22], human and rat choriocarcinoma cells [23,24], HT29 colon cancer cells [25], A549 adenocarcinoma cells [26], human APL (acute promyelocytic leukaemia) and ALL (acute lymphoblastic leukaemia) cell lines, and patients’ ALL blasts [27].

The cause of the induced differentiation is not still fully understood. In some cases, differentiating effects of MTX result from thymine nucleotide depletion, because the addition of thymidine is able to prevent MTX-induced differentiation [28]. On the contrary, cell differentiation arises apparently due to the deprivation of purines in HT29 human colon cancer cells [25].

In both cases, the differentiating effect of MTX is linked to the nucleotide precursor synthesis arrest. This phenomenon was also observed in mouse [29] and human [30] embryonic stem cells, when they were intravitreally transplanted to induce neuronal differentiation in murine retinas. Furthermore, intravitreally or intraperitoneally administered MTX decreased proliferative activity and tumourigenic potential of transplanted embryonic stem cells and it also induced neuronal differentiation.

MTX as Inhibitor of Methylation of Biomolecules

One of the important folate metabolites is 5-methyl THF, which is – together with homocysteine – necessary for the endogenous synthesis of methionine. Methionine reacts with ATP and S-adenosyl methionine (SAM) is formed. SAM functions as donor of methyl groups for protein methylation (including histones), cytosine bases in DNA (CpG islands), neuro­transmitters, phospholipids and other small molecules [31]. MTX decreases the level of 5-methyl THF in a cell via the functional suppression of DHFR [32,33]. Moreover, MTX directly inhibits the expression and activity of the methionine S-adenosyltransferase (MAT), which is a key enzyme catalysing the synthesis of SAM from methionine [34].

At the molecular level, Ras protein was identified to be a subject of MTX-induced hypomethylation [35]. Ras hypomethylation results in the mis-localisation of this protein from the plasma membrane to the cytoplasm, as well as a decrease of activation of ERK and AKT kinases that play a significant role in cell proliferation and differentiation. How­ever, the inhibition of Ras methylation by MTX is not direct. It is caused by the suppression of isoprenylcysteine carboxyl methyltransferase, which is the enzyme blocked by S-adenosyl homo­cysteine (SAH). SAH arises in a reversible reaction from homocysteine, which cannot be methylated to methionine due to the inhibition of folate metabolism.

MTX also acts as a demethylating agent in highly methylated cutaneous T-cell lymphoma (CTCL) lines and in circulating tumour cells from a patient with leukemic CTCL [36]. In these cells, MTX reduced the methylation of CpG islands in the Fas promoter leading to its higher expression and increased sensitivity to Fas-mediated apoptosis.

Generally, the reduction of DNA methylation after the treatment with MTX usually occurs in intensively rapidly proliferating cells, such as during physiological processes of embryonic development, haematopoiesis and tissue regeneration, but also in transformed cells. In case of an insufficient pool of methyl donors, hemimethylated spots arise in DNA after mitotic division and after the next cycle there are no methyl templates on both strands of DNA of daughter cells. This process can lead to the loss of DNA methylation patterns and consequently to changes in gene expression [37].

Based on the findings mentioned above, MTX is considered to be a methylation inhibitor that could be used in the treatment of cancers with a specific DNA methylation pattern. Hypermethylated CpG sites in genes (and/or their promoters) regulating tissue development, differentiation and tumourigenesis were described in rhabdomyosarcoma [38], medulloblastoma [39], glioma [40,41] and other human cancers [42].

MTX as Inhibitor of Histone Deacetylases (HDAC)

Due to the similar structure of MTX and of functional groups of certain HDAC inhibitors (HDACi), it was predicted that MTX may have the ability to inhibit HDAC [43]. Some known HDACi, such as trichostatin (TSA) and suberoylanilide hydroxamic acid (SAHA) contain a hydrophobic group (benzyl) in their molecule. This group is connected by a short spacer (aliphatic group) with a functional group (hydroxamic acid) that acts as a chelator of Zn ion in the active site of zinc-dependent HDAC [44,45]. In contrast to TSA and SAHA, butyrate, the smallest HDACi, consists of 3-carbon chain linked to a carboxyl group.

MTX contains a pteridine ring, which is the hydrophobic group. Additionally, the residue of p-aminobenzoic acid is structurally similar to the TSA and SAHA. Furthermore, the end of the MTX molecule contains the residue of butyrate. It was demonstrated by computer modelling that MTX is able to bind into the binding site of HDAC homolog (HDAC-like protein) and to interact with the zinc ion and the surrounding structures of this protein. The inhibition of HDAC was also shown under in vitro conditions in cell lines derived from lung cancer, cervical or stomach cancer; an increase in the acetylation status of histone H3 was also described in these cell lines [43].

In addition to the acetylation of histone H3, MTX has the ability to induce the acetylation of p53 protein at residues Lys373/382 [46]. However, this post­translational modification was not observed if other HDACi were applied. Simultaneously with the acetylation, MTX induced the phosphorylation of p53 protein at Ser15 that leads to the accumulation and increasing stability of p53 protein because acetylated sites are used in the process of its ubiquitination. HDAC-inhibiting activity of MTX resulted in down-regulation of the histone-lysine N-methyltransferase (EZH2), which is the catalytic core protein in the Polycomb Repressor Complex 2 (PRC2). PRC2 catalyses the addition of three methyl groups to Lys27 of histone 3 and mediates gene silencing of the tumour suppressor genes [47]. The epigenetic suppression of EZH2 expression by MTX resulted in the increasing expression of E-cadherin, which participates in the reduced cell migration and restricts a neoplastic transformation of epithelial cells [46].

Although the application of HDACi is a promising strategy to counter epigenetic changes associated with tumourigenesis [48,49], combination of these compounds with MTX has a different effect depending on the inhibitor type. For example, SAHA and sodium butyrate (NaBu) seem to be suitable HDACi for combination with MTX in ALL cell lines. These inhibitors increase both the cytotoxicity of MTX and the induction of apoptosis by modulation of the expression of enzymes involved in folate metabolism. After treatment with NaBu or SAHA, DHFR and TS expression decreased and the expression of the folylpoly-γ-glutamate synthetase (FPGS) was enhanced [50]. FPGS is the key enzyme which links glutamate residues to MTX and prevents MTX exclusion from the cell and increases its efficiency [4].

Nevertheless, the main problem of combined treatment with HDACi and MTX seems to be the sequence of their administration because the effects can be opposite [51,52]. Some HDACi (e.g. valproate or MS275) can even enhance the resistance of cells to MTX by up-regulation of thymidylate synthase expression; it was demonstrated in mouse choroid plexus carcinoma cell lines [53].

MTX as Inhibitor of the Glyoxalase System

Recently, it was also found that MTX affects the glyoxalase system. This three-step metabolic pathway is localised in the cytoplasm and it is considered to be the main pathway of methylglyoxal detoxification. Methylglyoxal, a secondary product of glycolysis or lipid peroxidation, is converted to D-lactate via the intermediate S-d-lactoylglutathione. The glyoxalase system consists of two enzymes, glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) and a catalytic amount of reduced glutathione [54].

Enhanced activity or expression of Glo1 was described as a marker of many human neoplasias. This metabolic change is associated with increased invasiveness, metastatic potential and multidrug resistance [54]. Moreover, amplification of the gene encoding Glo1 was identified in some types of primary solid tumours [55].

Bartyik et al [1] showed that MTX inhibits Glo1 in vitro; confirmed indirectly by detection of decrease in plasma D-lactate following MTX treatment in ALL patients. Inhibition of Glo1 elevates the intracellular methylglyoxal level that causes glycation of biomolecules [56,57], production of ROS, or genotoxic damage in tumour cells [58,59]. All these changes can lead to the enhancement of antitumor effects of MTX.

Thus, the glyoxalase system, namely the Glo1 enzyme, represents another target of the anti-neoplastic actions of MTX and expands the range of MTX effects on various metabolic pathways.

MTX as Inductor of Oxidative Stress

Several studies have confirmed the role of oxidative stress in the cytotoxic effect of MTX [60–62]. It was demonstrated that some NAD(P)H-dependent dehydrogenases, namely 2-oxoglutarate, iso-citrate, malate and pyruvate dehydrogenases, are inhibited by MTX [63]. Inhibition of these enzymes can induce a decrease in the NADPH levels; NADPH is required to reduce oxidised glutathione (GSSG) to the reduced form (GSH). GSH acts as cytoplasmic antioxidant and its MTX-induced decrease leads to a reduced effectiveness of the antioxidant defence system [64]. At the tissue level, a decline of GSH, superoxide dismutase and catalase activities were observed after MTX application in rat cerebellum [65].

Association of MTX-induced apoptosis and MTX-induced ROS generation was depicted in HL-60 and Jurkat T human leukaemia cells [2]. Cell death was me­diated by the mitochondrial pathway accompanied with a disruption of the mitochondrial membrane potential and subsequent activation of caspases. Another study showed that MTX activates JNK kinase through production of ROS resulting in induction of pro-apoptotic target genes and increased sensitivity to apoptosis [66].

Conclusion

Recent promising strategies in cancer treatment are based on the administration of drugs in combination and with different modes of action (cytostatics, differentiation inducers and angiogenic growth factors) [67] or on the new compounds affecting multiple, sometimes unrelated, cancer cell targets [68], because drugs designed exclusively against individual molecular targets usually cannot combat complex diseases such as cancer [69].

Although MTX has been used as a folate metabolism antagonist in cancer therapy for more than 60 years, identification of the whole spectrum of its’ molecular targets in cellular metabolism still continues. MTX inhibits not only synthesis of nucleotides and methylation of biomolecules, but also negatively regulates acetylation of histones, glyoxalase metabolism and antioxidant systems. Interventions in all of these metabolic pathways can induce changes in gene expression and consequently can lead to differentiation or cell death of cancer cells.

This study was supported by European Regional Development Fund and the State Budget of the Czech Republic for RECAMO (CZ.1.05./2.1.00/03.0101) and by internal project of Masaryk University No. MUNI/C/0803/2011.

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers.

Jakub Neradil, RNDr., Ph.D.

Laboratory of Tumor Biology

Institute of Experimental Biology School of Science

Masaryk University

Kotlarska 2

611 37 Brno

Czech Republic

e-mail: jneradil@sci.muni.cz

Submitted: 1. 10. 2012

Accepted: 6. 11. 2012