Low-Dose Methotrexate Inhibits Methionine S-Adenosyltransferase In Vitro and In Vivo
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Methionine S-adenosyltransferase (MAT) catalyzes the only reaction that produces the major methyl donor in mammals. Low-dose methotrexate is the most commonly used disease-modifying antirheumatic drug in human rheumatic conditions. The present study was conducted to test the hypothesis that methotrexate inhibits MAT expression and activity in vitro and in vivo. HepG2 cells were cultured under folate restriction or in low-dose methotrexate with and without folate or methionine supplementation. Male C57BL/6J mice received methotrexate regimens that reflected low-dose clinical use in humans. S-adenosylmethionine and MAT genes, proteins and enzyme activity levels were determined. We found that methionine or folate supplementation greatly improved S-adenosylmethionine in folate-depleted cells but not in cells preexposed to methotrexate. Methotrexate but not folate depletion suppressed MAT genes, proteins and activity in vitro. Low-dose methotrexate inhibited MAT1A and MAT2A genes, MATI/II/III proteins and MAT enzyme activities in mouse tissues. Concurrent folinate supplementation with methotrexate ameliorated MAT2A reduction and restored S-adenosylmethionine in HepG2 cells. However, posttreatment folinate rescue failed to restore MAT2A reduction or S-adenosylmethionine level in cells preexposed to methotrexate. Our results provide both in vitro and in vivo evidence that low-dose methotrexate inhibits MAT genes, proteins, and enzyme activity independent of folate depletion. Because polyglutamated methotrexate stays in the hepatocytes, if methotrexate inhibits MAT in the liver, then the efficacy of clinical folinate rescue with respect to maintaining hepatic S-adenosylmethionine synthesis and normalizing the methylation reactions would be limited. These findings raise concerns on perturbed methylation reactions in humans on low-dose methotrexate. Future studies on the clinical physiological consequences of MAT inhibition by methotrexate and the potential benefits of S-adenosylmethionine supplementation on methyl group homeostasis in clinical methotrexate therapies are warranted.
Folate serves as a key metabolic carrier in one-carbon metabolism. 10-Formyl tetrahydrofolate is essential for de novo purine synthesis, and 5,10-methylenete-trahydrofolate assists in thymidine synthesis. In the form of 5-methyltetrahy-drofolate (5-methylTHF), folate transfers the methyl groups to methionine synthase, generating methionine from homocysteine remethylation. Methionine can be further utilized for synthesis of the universal methyl donor S-adenosyl-methionine.
Low-dose methotrexate (MTX) is one of the most common immunosuppressants used in rheumatic and other inflammatory conditions. MTX is a potent inhibitor of dihydrofolate reductase and thymidylate synthase, which can deplete folate and inhibit DNA synthesis. MTX also interferes with the enzymes methyl-enetetrahydrofolate reductase and methionine synthase, resulting in reduced cellular 5-methylTHF levels and decreased homocysteine remethylation (1,2). By decreasing 5-methylTHF, both folate depletion and antifolate treatments can impair 5-methylTHF-dependent methionine synthesis (3), reduce S-adenosylmethionine and affect methylation reactions (4). In HT29 colon cancer cells, high-dose MTX (in µmol/L) markedly decreases intracellular adenosine, S-adenosylmethionine and polyamine content (5). A previous study found that prolonged incubation of low-concentration MTX (1–10 nmol/L) inhibited in vitro monocyte chemotaxis and superoxide production; such inhibition was augmented by a low methionine condition, but was abolished by folinate and S-adenosylmethionine treatments (6). These findings suggested that, in addition to the inhibition of nucleotide synthesis, the efficacy of low-dose MTX may be linked to its inhibition of methionine synthesis and/or the depletion of S-adenosylmethionine.
MAT [ATP: L-methionine S-adenosyltransferase (EC 188.8.131.52)] catalyzes the only reaction that produces the major methyl donor and precursor of polyamine synthesis—S-adenosylmethionine (7). MAT proteins are encoded by two genes (MAT1A and MAT2A), and a third gene (MAT2β) regulates the MAT2A-encoded isoenzyme (7, 8, 9). The activity of MAT is regulated by the methionine concentration, but the Michaelis constants of this enzyme to methionine differ among these MAT proteins. The Km is lowest for MAT II (expressed ubiquitously in all tissues), intermediate for MAT I and highest for MAT III (both expressed exclusively in the liver) (10, 11, 12, 13, 14). It is plausible that during antifolate treatments such as MTX, liver and nonhepatic tissues have distinctive responses to methionine depletion/supplementation with respect to S-adenosylmethionine synthesis and availability.
The liver plays an essential role in methyl group homeostasis. By decreasing 5-methylTHF, MTX could impair 5-methylTHF-dependent methionine synthesis and reduce S-adenosylmethionine. Although the efficacy of low-dose MTX in monocytes can be altered by folate and S-adenosylmethionine, the impacts of these metabolites on liver transmethylation in humans on low-dose MTX are less clear because the liver is not the main target for immunosuppressants. If S-adenosylmethionine depletion resulting from MTX treatments is mainly due to reduced folate-dependent methionine synthesis brought on by the inhibition of methylenetetrahydrofolate reductase and methionine synthase, then methionine supplementation would be equally, if not more, effective than folate supplementation to restore S-adenosylmethionine synthesis. However, our preliminary experiments suggested otherwise (15). Neither folinate nor methionine supplementation normalized S-adenosylmethionine levels in cells treated with low-dose MTX. These observations led to our postulation that MTX might suppress S-adenosylmethionine production by inhibition of MAT independent of folate depletion. The present study was conducted to test the hypothesis that MTX inhibits MAT expression and activity in vitro and in vivo.
Chemicals and Cell Culture
All chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise specified. HepG2 human hepatocellular carcinoma cell line (FIRDI, Hsinchu, Taiwan) was grown in minimum essential α medium (α-MEM) with 10% (v/v) bovine calf serum. The medium was replaced every 72 h.
Effects of Folate Restriction and MTX Treatment on Folate Status and S-Adenosylmethionine Production
The experimental conditions were based on previous experiments from our group and others. To study the impacts of low-dose MTX and compare them to the impact of folate-depletion conditions on folate and S-adenosylmethionine status, HepG2 cells were either cultured in folate-restricted medium (10 nmol/L folinate for 144 h) (16) or treated with 50 nmol/L MTX for 72 h (17). Control cells were cultured in regular α-MEM medium that contained 2.2 µmol/L folic acid in all experiments (Gibco Invitrogen, Carlsbad, CA, USA). In arthritis patients taking low-dose MTX for immunosuppression, intracellular MTX concentration was approximately 34 nmol/L (18–51 nmol/L) in erythrocytes (16,18). In the folate restriction experiments, cells were cultured in αMEM with 10 nmol/L folinate with 10% folate-depleted fetal calf serum that was prepared by dialyzing serum against 10 volumes of phosphate-buffered saline (PBS) at 4°C for 24 h with buffer changes every 4 h (16,19). These cells were harvested for further analysis of folate, S-adenosylmethionine and MAT activity, as previously described (16,20,21).
Effects of Folate and Methionine Supplementations on Intracellular S-Adenosylmethionine Production in MTX Pretreated Cells
The experimental conditions were based on our previous experiments. To investigate whether folate or methionine supplementation could ameliorate the S-adenosylmethionine depletion caused by MTX, cells were pretreated with 50 nmol/L MTX for 72 h and then supplemented with either folinate (100 nmol/L) or methionine (500 µmol/L) for 48 h. In a separate experiment, cells were concomitantly cotreated with MTX and folinate (100 nmol/L) or methionine (500 µmol/L) for 72 h. The control cells were cultured in the regular α-MEM media containing 100 µmol/L methionine. After these treatments, cells were collected for intracellular S-adenosylmethionine determinations as previously described (4,16,22).
Effects of Methotrexate on MAT Expression and Activities In Vivo
The animal experiment was approved by the Institutional Animal Care and Use Committee of National Chung Hsing University. To study the impacts of MTX on MAT expression and activity, 12 male C57BL/6J mice (3 wks of age) were obtained from the National Lab of Animal Care (NLAC, Taipei, Taiwan). The mice were fed an amino acid-defined diet containing 2 mg folic acid/kg diet (23) with 1% succinyl sulfathiazole (Dyets Inc., Bethlehem, PA, USA). All mice were housed in specific pathogen-free filtered cages (2 mice per cage) at 22°C and experienced a 12-h light/darkness cycle (0700 to 1900 h). After adaption at the facility for 1 wk, the mice were equally divided into 2 groups by body weight. MTX treatment (0.3 mg MTX/kg/2 d, dissolved in PBS with 0.01% dimethyl sulfoxide [DMSO]) was freshly prepared prior to injection and began when mice were 4 wks old. The mice were injected intraperitoneally once on alternate days with either PBS (0.01% DMSO) (control group, n = 6) or low-dose MTX (MTX group, n = 6) for 4 wks. This dose was within the range (0.1–0.5 mg/kg/d) of those used in previous studies in rodent arthritis models. MTX treatment at the dose of 1 mg MTX/kg/2 d (0.5 mg/kg/d) significantly decreased the arthritis score and increased locomotion in the mouse collagen arthritis model (24). At the dose of 0.75 mg/kg body weight per wk (0.11 mg/kg/d), MTX intraperitoneal injections significantly reduced paw volume in the adjuvant-induced arthritis (25). At a dose of 0.3 mg/kg/2 d (0.15 mg/kg/d), MTX significantly decreased the arthritic score and improved histological and radiological scores in rats with collagen-induced arthritis (26). Furthermore, the dose used in the present study (0.15 mg/kg/d in mice) was comparable to the clinical use of low-dose MTX in treating human rheumatoid arthritis. MTX was generally administered to rheumatoid arthritis patients at a dosage of 7.5–15 mg/wk (18,27,28). Considering the normal life expectancy of mice, the 4-wk duration reflected approximately 3 years of MTX treatment in humans. The median duration of MTX treatment in our human subjects from previous studies was approximately 36 months (95% CI 45.7–77.8) (27, 28, 29, 30), and the mean weekly dose was 12.5 mg (95% CI 10.5–12.4 mg) in arthritis patients (31). The mice were sacrificed at the age of 8 wks and tissue samples were stored at − 80°C for analyses of levels of MAT gene and protein expression and MAT activity.
Effects of MTX on Glucocorticoid-Induced MAT2A Expression
To investigate whether MTX suppresses glucocorticoid-induced MAT2A expression, the HepG2 cells were synchronized in serum-free media for 16 h, then treated with either MTX (50 nmol/L for 72 h), dexamethasone (100 nmol/L for 72 h), actinomycin D (5 µg/mL for 12 h just before cells were harvested), dexamethasone combined with MTX (cotreated for 72 h) or dexamethasone combined with actinomycin D (dexam-ethasone treatment for 72 h; at 60 h, actinomycin D was added for an additional 12 h just before cell harvest). The performance of these experiments was based on previous experimental conditions (17,32).
Effects of Concomitant and Post-MTX Folinate Supplementation on MAT2A Gene Expression
Because concomitant folinate supplementation with MTX effectively restored S-adenosylmethionine levels, we next investigated whether concurrent folinate supplementation could rescue MTX-induced MAT2A gene inhibition in HepG2 cells. MAT2A gene expression levels were compared between cells that received concurrent MTX (50 nm for 72 h) and folinate treatments (100 nmol/L for 72 h) and cells pretreated with the same dose of MTX followed by folinate supplementation for various time periods.
Determination of MAT Gene Expression
Total RNA was isolated from the livers (expressing MAT1A) and kidneys (expressing MAT2A) of the mice or MTX-treated cells with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA). The integrity of RNA was checked by electrophoresis. A total amount of 2 µg RNA was treated with DNase I for eliminating DNA, and then RNA was converted to cDNA by reverse transcription-polymerase chain reaction (PCR) by use of Moloney murine leukemia virus RNA reverse transcriptase (Promega, Madison, Wisconsin, USA) and oligo-dT as primers. Human MAT 2A and 18S gene expression levels were determined by quantitative real-time PCR ABI7000 using commercial TaqMan probes (Applied Biosystems Inc., Foster City, CA, USA). Mouse gene expression levels (Mus MAT 1A, Mus MAT 2A, Mus 18S) were determined by quantitative realtime PCR by using SYBR Green reagents (Applied Biosystems). The expression of MAT genes was calculated by normalizing the threshold cycle value of the target gene to that of the control housekeeping gene (18 sRNA) (20).
Determination of MAT Protein Expression
After the incubation period, cells were harvested, washed and pelleted. At termination, mouse tissues were immediately frozen at -70°C until analyses. Cell pellets or mouse tissues were homogenized in ice-cold radioimmunoprecipitation assay buffer (25 mmol/L Tris-HCl pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) with protease inhibitor (CalBiochem, Darmstadt, Germany). The lysates were centrifuged and the supernatants were used for Western blot assay. A total amount of 60 µg protein (measured by the bicinchoninic acid method) was used for SDS polyacrylamide gel electrophoresis (SDS-PAGE) and further immunoblotting. Protein lysates from tissue or cells were denatured and separated on a 12% SDS-PAGE gel by using a Minigel apparatus, and then transferred onto a polyvinylidene difluoride membrane by using a transfer cell (Bio-Rad, Hercules, CA, USA). After blocking with Tris-buffered saline (TBS) containing 10% skim milk, the lysates were incubated with rabbit polyclonal antibody (1:1000) against MAT II (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in HepG2 cells or mouse kidney samples. A mouse monoclonal antibody was used for the determination of MAT I/III (Abnova Inc., Taipei, Taiwan) in mouse liver samples. Membranes were washed three times with TBS containing 0.1% Tween 20 and then covered with horseradish peroxidase-linked anti-rabbit IgG for MAT II or anti-mouse IgG for MAT I/III (1:5000) at room temperature. The immunoblots were visualized by use of an enhanced chemiluminescence kit (New England Biolabs, Beverly, MA, USA). The expression level of MAT protein was calculated by normalizing the quantity of target protein to that of the internal control β-actin.
Determination of MAT Activity
Harvested samples from cells or animal tissues were homogenized in an ice-cold buffer consisting of 0.154 mol/L KCl/50 mmol/L Tris-HCl and 1 mmol/L EDTA (pH 7.4). MAT activity was determined by quantifying S-adenosylmethionine production by 300 µg of cellular protein. The reaction mixture consisted of 80 mmol/L Tris-HCl/50 mmol/L KCl (pH 7.4), 5 mmol/L ATP, 40 mmol/L MgCl2 and cellular protein of enzyme solution (20). Methionine (5 mmol/L) was added to initiate the enzyme reaction after preincubation of the cellular protein and the reaction mixture for 3 min. The reaction was carried out at 37°C for 1 h and then terminated by 10% ice-cold perchloric acid. MAT activity was calculated as the S-adenosylmethionine production (the concentrations of these reactions were 0.4–1.0 nmol/L in the cell experiments and 18–30 nmol/L in the tissues) after subtracting the baseline S-adenosylmethionine level (<0.01 nmol/L) from the supernatant fraction without adding methionine, and expressed as nanomoles S-adenosylmethionine formed per milligram protein per hour (20).
In separate experiments, we investigated whether MTX treatments inhibit MAT activity in cell-free protein lysates prepared from untreated cells and healthy animal tissues. Protein lysates extracted from normal mouse liver or HepG2 cells were preincubated with various concentrations of MTX (500–2500 µmol/L) at room temperature for 20 min, then the assay reagents were added for determination of MAT activity as previously described (33).
Comparisons of means between the control and the MTX treatment groups or the control and the folate-deficient groups were determined by using the Student t test. Results are expressed as mean ± standard deviation. The expression level of MAT2A and concentrations of S-adenosylmethionine among cells treated with MTX for different periods with and without folinate supplementation were examined by analysis of variance. All statistical analyses were performed with SYSTAT 11.0 for Windows™ (Systat Software Inc., Richmond, CA, USA). For all analyses, the results were considered statistically significant if P values were <0.05.
Folate Restriction and MTX Treatment Both Reduced Intracellular Folate and S-Adenosylmethionine in HepG2 Cells
Folinate or Methionine Supplementation Restored S-Adenosylmethionine Levels in Folate-Depleted Cells but Not in MTX-Pretreated Cells
To establish reliable experimental conditions and determine the optimal duration of methionine supplementation for S-adenosylmethionine production, we incubated these cells with excessive methionine for various time periods in a pilot experiment. S-adenosylmethionine levels reached a plateau within 90 min and remained at that level for at least 48 h when the incubation time was prolonged. In the experiments in which cells were pretreated with the same dose of MTX, the S-adenosylmethionine level remained suppressed even 48 h after loading with excessive methionine.
These results suggested that the mechanism by which MTX caused S-adenosylmethionine depletion was not simply due to folate deficiency or methionine depletion, and that an additional mechanism was likely involved. These observations provided initial evidence that MTX may inhibit MAT; and MTX-induced S-adenosylmethionine depletion was investigated further.
Effects of Concomitant Treatments with Folinate or Methionine on S-adenosylmethionine Status
When methionine was supplemented concomitantly (co-Met sup) with MTX treatment, intracellular S-adenosylmethionine remained reduced (Figure 2B), supporting our postulation that impaired methionine synthesis is not the main cause for MTX-induced S-adenosylmethionine depletion. However, when folinate was supplemented concomitantly (co-FA sup) with MTX, intracellular S-adenosylmethionine was restored to a normal level similar to that of the untreated control cells (Figure 2B). The impacts of concomitant and post-MTX folinate supplementations were investigated further.
MTX Inhibited MAT Activity and Protein and Gene Expressions In Vitro Independent of Folate Depletion
MTX Significantly Reduced MAT Activity and MAT Protein and Gene Expression In Vivo
MTX Does Not Affect MAT Activity in Cell-Free Protein Lysates
MTX Inhibited Glucocorticoid-Induced MAT Gene Expression
Only Concomitant Folinate Supplementation Can Alleviate MAT2A Inhibition by MTX Treatments
In summary, folinate rescue could not restore S-adenosylmethionine levels or MAT2A expression in cells preexposed to MTX. However, at the same treatment dose, concomitant folinate supplementation with MTX treatment could effectively normalize MAT2A mRNA expression and maintain optimal S-adenosylmethionine status.
MTX is a potent inhibitor of numerous enzymes in folate and methionine metabolism, including dihydrofolate reductase, thymidylate synthase and methylenete-trahydrofolate reductase. MTX also reduces methionine synthase activity due to folate depletion (1,2). Here, we have demonstrated novel findings that an additional key enzyme in the transmethylation cycle—MAT—is inhibited by MTX. At a dose comparable to clinical use in treating human inflammation, MTX inhibits MAT gene and MAT I/III and MAT II protein expression, as well as MAT enzyme activity both in vitro and in vivo independent of folate depletion. The evidence for these findings was as follows. First, MTX treatment in vitro inhibited MAT gene expression, which was the opposite of folate depletion alone, which induced MAT gene expression. Second, MTX treatment decreased MAT protein expression and reduced MAT activity, which also was different from folate depletion. Third, although MTX treatments caused folate depletion and S-adenosylmethionine reduction, folate and methionine supplementation could not restore the S-adenosylmethionine level in MTX-pretreated cells. Fourth, low-dose MTX inhibited the expressions of MAT1A and MAT2A genes and MATI/II/III protein, as well as MAT enzyme activities in mouse liver and kidney. Fifth, MTX inhibited glucocorticoid dexamethasone-induced MAT2A expression. The inhibition of MAT by low-dose MTX raised concerns as to the adverse effects of S-adenosylmethionine depletion and perturbed methylation reactions in the clinical use of MTX.
An interesting observation from this study was that the timing of folinate rescue determined its efficacy on S-adenosylmethionine production and MAT2A gene expression. Concomitant administration of folinate with the MTX treatment maintained the optimal S-adenosylmethionine status, but at the same dose folinate supplementation was unable to ameliorate the S-adenosylmethionine status in MTX pretreated cells. If MTX directly inhibited MAT, how did folinate rescue S-adenosylmethionine synthesis when it was coadministered with MTX? The observation that concomitant methionine supplementation was completely ineffective in restoring S-adenosylmethionine indicated that concomitant folinate rescue on S-adenosylmethionine production was independent of folate-dependent methionine synthesis. We speculated that this was due to the competition between folinate and MTX in their uptake and polyglutamation. Folate receptors have a lower affinity for MTX (0.15–1.7 µmol/L) (34, 35, 36) but a high affinity for folic acid (~1 nmol/L) and reduced folates, including folinate (5-formyltetrahydrofolate) and 5-methylTHF (10–40 nmol/L). In clinical conditions, folates have been coadministered with some antifolate drugs to reduce toxicity, and natural folate has been shown to diminish the activity of agents that undergo polyglutamation by suppression of the formation of these agents at the level of folylpoly-glutamate synthetase (FPGS) (37). Folinate is a better substrate of FPGS than MTX. As a potent competitor of glutamylation (38), folinate at low concentrations could prevent formation of cytotoxic polyglutamates of MTX. We speculate that when high levels of folinate are coadministered with MTX, they may compete with each other for folate receptors and FPGS; as a result, the inhibition of MAT is alleviated by blocking MTX uptake/retention. This assumption is partly supported by our observation that folinate supplementation rescued S-adenosylmethionine production and MAT mRNA expression only when it was concurrently administered with MTX; the same high dose folinate failed to restore S-adenosylmethionine once those cells had already been exposed to MTX due to MAT inhibition.
Although a single dose of concomitant folinate supplement coadministered with MTX treatment could successfully maintain optimal S-adenosylmethionine levels in vitro, it was clear that folinate could not rescue S-adenosylmethionine production if those cells had already been exposed to MTX. In other words, once MTX had entered and become polyglutamated in the cell, a high dose of folinate was ineffective in alleviating the inhibition of MAT by MTX treatment. It should be noted that clinical low-dose MTX used in immunosuppression is a long-term regimen in that MTX is polyglutamated and gradually accumulates in the cells over a period of time (39). Low-dose MTX is usually given once weekly as an oral dose started at around 7.5 mg weekly and is increased by 2.5 mg over few a months as needed. Folate rescue is usually taken at 5–10 mg weekly or as two tablets of Pregaday daily. Many rheumatologists avoid applying folate rescue on the same day as the MTX, because higher doses of folate may reduce the efficacy of MTX. Folate rescue is commonly recommended to be taken 1–2 d after the MTX (40, 41, 42, 43).
Our findings in a cell line and in animal models may or may not reflect what happens in humans. If clinical use of MTX indeed inhibits MAT action in humans, as we have demonstrated in the animal experiments, then S-adenosylmethionine-dependent methylation reactions could be impaired in patients continuously taking long-term low-dose MTX. Such perturbations may not be fully rescued by folate supplements. The effects of the clinical use of MTX on S-adenosylmethionine and transmethylation reactions during chronic inflammation must be considered more carefully, and studies should be conducted to investigate this in humans.
Studies are also required to evaluate the efficacy of the coadministration of folinate with MTX in each regimen, specifically for S-adenosylmethionine synthesis and methylation reactions under clinical conditions. Low-dose MTX, a traditional folate antagonist, and DMARD administered weekly either alone or as combination therapy, are widely used in treating human rheumatoid arthritis (44). In the blood and liver of humans receiving low-dose MTX treatments, MTX is predominantly in the polyglutamated form with hepatic folate stores depleted compared with baseline specimens (22). Our finding that MTX inhibited MAT activity implied that patients on long-term low-dose MTX are susceptible to hepatic S-adenosylmethionine deficiency that may not be rescued by folinate supplementation. Direct inhibition of MAT by MTX could be more harmful than we previously realized because MAT is the required enzyme that catalyzes the only reaction that produces the S-adenosylmethionine (7) needed for global methylation reactions and polyamine synthesis. Humans with mutations in the MAT1A gene that result in a MAT I/III deficiency have persistent hypermethioninemia without elevated homocysteine (45). Although MTX-treated humans may not have elevated methionine due to the inhibition of methionine synthase, chronic depletion of S-adenosylmethionine and perturbed methylation status in the liver by MAT inhibition are of concern. Increased risk of liver pathogeneses has been reported in the case of extreme MAT deficiency in humans and mouse models. In MAT I/II-deficient patients, the irregular formation of S-adenosylmethionine in the liver leads to low delivery of S-adenosylmethionine from the liver to plasma and tissues (46). In animal models of alcoholic liver injury, depletion of the hepatic S-adenosylmethionine level could induce global DNA hypomethylation and increase c-myc expression and genome-wide DNA strand breaks (47). These changes are considered to lead to predisposition to pathological conditions of the liver and malignant degeneration (47). Chronic hepatic S-adenosylmethionine deficiency in the MAT1A knockout (−/−) mouse model was shown to predispose the organ to further injury, spontaneous development of steatohepatitis and hepatocellular carcinoma (48, 49, 50). By inhibiting MAT, long-term exposure to MTX might increase the susceptibility to liver injury and/or promote the progression of existing liver diseases. More importantly, because polyglutamated MTXs tend to stay in the hepatocytes and directly inhibit MAT in the liver, the efficacy of folinate supplements with respect to rescuing S-adenosylmethionine synthesis and normalizing the methylation reactions would be limited. Direct S-adenosylmethionine supplementation might be an option for optimizing S-adenosylmethionine status as well as methylation homeostasis in patients taking MTX. However, well-designed clinical trials would be required.
In conclusion, we have provided novel in vitro and in vivo evidence that MTX directly inhibited MAT mRNA expression and reduced MAT I/II/III protein levels, which significantly decreased MAT activity in different tissues. Future studies on the clinical physiological consequences of MAT inhibition by MTX and the potential benefits of S-adenosylmethionine supplementation on methyl group homeostasis in clinical MTX therapies are warranted.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This project was supported in part by the National Science Council (NSC98-2320-B005-004MY3 and NSC100-2628-B-005-002-MY4 to E-PI Chiang and NSC101-2911-I-005-301). This work was also supported in part by the Ministry of Education, Taiwan, R.O.C. under the Aim for Top University (ATU) plan. Both authors read and approved the final manuscript. These funding organizations were not involved in study design, data collection and analysis, decision to publish or preparation of the manuscript.
This work was presented in part at the 2008 American Association of Cancer Research Annual meeting (#3342), San Diego, CA, USA.
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