Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells
Fang Tang, Le Zou, Jingyao Chen, Fanqi Meng
Abstract
Background and purpose
Clinical studies showed that prolonged infusion of methotrexate (MTX) leads to more severe adverse reactions than short infusion of MTX at the same dose. We hypothesized that it is the saturation of folate polyglutamate synthetase (FPGS) at high MTX concentration that limits the intracellular synthesis rate of methotrexate polyglutamate (MTX-PG). Due to a similar accumulation rate, a longer infusion duration may increase the concentration of MTX-PG and, result in more serious adverse reactions. In this study, we validated this hypothesis.
Introduction
Methotrexate (MTX) is a folic acid analog with broad-spectrum antineoplastic activity, which is used in the treatment of various solid tumors [1]. High dose methotrexate (HD-MTX) can effectively prevent cell resistance and extramedullary recurrence. It also plays an important role in combined chemotherapy [2]. MTX and its intracellular active metabolite, methotrexate polyglutamate (MTX-PG), can competently inhibit intracellular dihydrofolate reductase, block folate metabolism, inhibit purine synthesis and promote of cancer cell apoptosis [3]. However, after tumor cell death, MTX causes side effects such as mucosal damage, myelosuppression, and liver and kidney damage [4, 5]. In clinical practice, it was observed that the severity of MTX toxicity is correlated with the infusion time. At the same dose, the side effects caused by slower infusion of MTX are more serious, and the regularity is contrary to the general rule of clinical medication [6].The range of HD-MTX infusion time is quite wide (2 to 48 hours). There is a lack of awareness abourt the effects of the infusion rate, and clinicians usually slow down the infusion rate to reduce side effects, which leads to more serious side effects [7]. Related studies [8] have shown that slow administration of MTX increases MTX-PG levels in the kidney and other tissues, enhancing intracellular oxidative stress and leading to tissue damage.
Materials and methods
2.1 Chemicals and reagents
Methotrexate (CAS: 59-05-2) was obtained from the National Institutes for Food and Drug Control (Beijing, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagles medium (DMEM), RPMI-1640, trypsin containing 0.25% EDTA, and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The total protein extraction kit was supplied by Bestbio Co., LTD (Shanghai, China). Bradford protein quantitation assay kit was obtained from Beyotime Biotech Co., LTD (Nanjing, China). Trypsin Gold, Mass Spectrometry Grade was purchased from Promega Corporation (Madison, WI, USA). The synthetic peptide SGLQVEDLDR and YLESAGAR (≥99.5% purity) and the stable isotope-labeled internal standard (SIL-IS) SGL(13C,15N)QVEDLDR and YLESAGAR(13C,15N) (≥99.5% purity) were synthesized by Shanghai Science Peptide Biological Technology Co.,LTD (Shanghai, China). HPLC-grade acetonitrile, formic acid and methanol were obtained from Merck KGaA (Darmstadt, HE, DE). Human serum albumin (HSA), ammonium bicarbonate (NH4HCO3), dithiothreitol (DTT) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water was purified and deionized with a Milli-Q Direct 8 system manufactured by Merck Millipore (Billerica, MA, USA).
Results
3.1 Validation of quantitative methods for FPGS and GGH
The established method quantified the abundance of FPGS (Y = 0.00194X-0.000237, R2 = 0.9993, weight: 1/X2) and GGH (Y = 0.000465X+0.000316, R2 = 0.9992, weight: 1/X2). The detection signals of both peptides (SGLQVEDLDR and YLESAGAR) were linear at the range of 0.1–100 ng/mL and the lowest limit of quantification (LLOQ) was 0.1 ng/mL. Since the FPGS and GGH depleted cell matrix was not available, 5% human serum albumin (HSA) in PBS was applied as a surrogate matrix in which all peptides showed good specificity. For both two peptides, the intra-day and inter-day precisions (%RSD) were within ±10% and accuracy (% RE) was within ± 15%. All samples were stable under different conditions (24h at room temperature, 20d at -20°C, and 3 freeze-thaw cycles at -20°C).
UPLC-MS/MS is high-throughput, sensitive, accurate and precise, and has been successfully applied in detecting trace proteins in complex matrices. The diversity of intracellular proteins can affect the precision of results. The separation and purification of total cellular proteins by electrophoresis can improve the accuracy of quantitative results.
Discussion
The model parameter vmax-FPGS represents the maximum rate of intracellular MTX-PG generation when FPGS is saturated. The abundance of FPGS was strongly correlated with vmax-FPGS, indicating that the abundance of FPGS was the key factor determining the maximum rate of intracellular MTX. The parameter kGGH represents the first-order kinetic constant of GGH catalyzing the hydrolysis of MTX-PG2-7. The correlation between the abundance of GGH and kGGH was relatively weak, indicating that the abundance of GGH dose not determine kGGH. It was reported [22] that the activity of the same enzyme was not exactly the same in different cell lines. Therefore, kGGH may be affected by both enzyme abundance and unit enzyme activity. However, when FPGS is saturated, the differences in enzyme activity can be neglected, and the abundance of the enzyme limits the rate of MTX-PG production. In summary, the abundance of FPGS determines the synthesis rate of MTX-PG in different cell lines. FPGS can be saturated with high dose of substrates, which limits the rate of intracellular MTX-PG synthesis. The abundance of GGH affects the hydrolysis rate of MTX-PG. The hydrolysis process can be also affected by the substrate concentration. Therefore, compared with FPGS, the effect of GGH on the intracellular concentration of MTX-PG is relatively small. In high-dose MTX treatment, FPGS is usually saturated and the intracellular MTX-PG synthesis rate is limited. The difference in plasma concentration between fast/slow infusion at the same dose has little effect on the synthesis rate of MTX-PG. During slow infusions, longer duration of exposure to MTX results in higher concentrations of MTX-PG and more serious side effects. The results suggest that the abundance of the intracellular metabolic enzyme FPGS can be used as an important indicator for ndividualized treatment with MTX. It can be used for predicting the accumulation of intracellular MTX-PG and drug efficacy.
Conclusion
In this study the cytopharmacokinetic mechanism leading to more severe adverse reactions after slower MTX infusion at the same dose was determined in A549, BEL-7402 and MHCC97H cell lines. FPGS was saturated and the accumulation rate of MTX-PG was limited when excessive MTX was administered. The exposure time of MTX was prolonged under slow infusion, increasing the accumulation of MTXPG, which aggravated side effects. This study provides strategies for adjusting the MTX regimen and predicting efficacy/side effects. This study provides a new method for studying the intracellular metabolism of drugs.
Citation: Tang F, Zou L, Chen J, Meng F (2024) Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells. PLoS ONE 19(6): e0302663. https://doi.org/10.1371/journal.pone.0302663
Editor: Yawei Du, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, CHINA
Received: February 4, 2024; Accepted: April 9, 2024; Published: June 4, 2024
Copyright: © 2024 Tang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0302663#abstract0
