Cellular Uptake Of Methotrexate Research Articles

Methylation analysis of theSLC19A1promoter region in Chinese children with acute lymphoblastic leukaemia

Reduced folate carrier 1 (RFC1), which is encoded by the human solute carrier family 19 member 1 (SLC19A1) gene, plays an essential role in the cellular uptake of methotrexate (MTX). RFC1 expression is regulated by genetic variations and epigenetic modifications. The aim of the present study was to investigate the methylation status of the SLC19A1 promoter in peripheral blood and its association with MTX levels and toxicities in children with acute lymphoblastic leukaemia (ALL). Serum MTX concentrations were measured using a fluorescence polarization immunoassay. Methylation quantification for SLC19A1 promoter region #17 was performed by Sequenom MassARRAY in 52 paediatric ALL patients. Overall, the investigated region of the SLC19A1 promoter was in a hypermethylated state. No significant associations were detected between the methylation levels of six CpG units in the SLC19A1 promoter region #17 and clinical parameters of patients with ALL, including sex, age, immunotype and risk stratification. The methylation level of CpG_10 showed a significant positive correlation with MTX 24hours after the initiation of infusion. No significant differences in the methylation levels of six CpG units were observed between patients with and without MTX toxicities. Due to the small sample size of this study, there was a high chance of false-positive results. A large-scale study would be required to confirm these preliminary results. Our preliminary results suggested the hypermethylated status of the SLC19A1 promoter in children with ALL. The methylation levels of the SLC19A1 promoter might affect MTX exposure. These findings have implications for the mechanisms underlying the variability of MTX responses in childhood ALL.

Self-assembled peptide nanoparticles for efficient delivery of methotrexate into cancer cells

The low cellular uptake of Methotrexate (MTX), a commonly used anticancer drug, is a big challenge for efficient cancer therapy. Self-assembled peptide nanoparticles (SAPNs) are one of the major classes of peptide vectors that have gained much attention toward novel drug delivery systems. In the present study, different sequences of cell-penetrating peptides including R2W4R2 and W3R4W3 and their SAPNs (R2W4R2-E12 and W3R4W3-E12) were designed for efficient delivery of MTX into MCF7 breast cancer cells. Based on electron microscopy results, the obtained SAPNs were in nano scale with spherical shape. There was a positive relationship between the free energy of water to octanol transferring and cellular penetration of designed nanostructures. The R2W4R2 possessed proper free energy and ability to form a spherical structure and hydrophobic–hydrophobic interactions, therefore, exhibited more cellular penetration than W3R4W3. The cellular uptake of obtained nanoparticles was examined by flow cytometry and fluorescence microscopy, in which, R2W4R2 and R2W4R2-E12 showed more appropriate penetration into MCF7 cells than W3R4W3 and W3R4W3-E12. The cytotoxicity of MTX-loaded peptides and SAPNs was examined by MTT assay. As a result, at higher concentrations, the R2W4R2 and R2W4R2-E12 showed higher cytotoxic behavior than their counterparts. Despite their enhanced cellular internalization, the cytotoxic behavior of MTX-loaded SAPNs at lower concentrations was relatively less than free MTX, which could be ascribed to the gradual nature of drug detachment from these conjugates. Therefore, R2W4R2 could be considered as an efficient choice to enhance the therapeutic efficiency of MTX in cancer treatments.

Novel biosurfactant and lipid core-shell type nanocapsular sustained release system for intravenous application of methotrexate

In an attempt to prepare novel core shell nanocapsules, lipid and Stearic acid-Valine conjugate (Biosurfactant) based nanosystem was prepared to attain high drug loading of hydrophilic drug methotrexate (MTX), with sustained release. Antisolvent nanoprecipitation technique was employed for the formulation of nanoparticles (NPs). Optimized formulation depicted 209.6 ± 31.3 nm particle size, 0.209 ± 0.072 PDI and 14.98 ± 1.33 %w/w drug loading. In vitro release depicted biphasic release for 12 h with initial burst phase followed by sustained release phase. In vitro Haemolytic study on RBCs revealed haemocompatible nature of MTX-Biosurfactant NPs compared to Biotrexate® (Zydus). In vitro cell culture studies showed 3.33 folds and 3.50 folds increase in cellular uptake of MTX at 10 µg/ml and 15 µg/ml concentration respectively for developed nanoparticles with 4.16 folds decrease in IC50 value. Higher apoptosis and increased lysosomal membrane permeability were obtained in MTX-Biosurfactants NPs. AUC and T1/2 was found to increase by 2.55 and 3.25 folds respectively in pharmacokinetic study. Significant reduction in tumor burden and serum toxicity marker level depicted efficacy and safety respectively of the formulation as compared to Biotrexate®. RBCs morphology was retained after MTX-Biosurfactants NPs exposure proving its haemocompatibility in vivo.

Lipid and TPGS based novel core-shell type nanocapsular sustained release system of methotrexate for intravenous application.

Core shell nanocapsules present an interesting system for attaining high loading of drug. In an attempt, lipid and TPGS based novel core-shell nanocapsule were prepared to achieve high drug loading with sustained release of model hydrophilic drug methotrexate (MTX). Antisolvent nanoprecipitation was utilized for the formulation of nanoparticles. Optimized formulation depicted 223.6 ± 24.1 nm particle size, 0.243 ± 0.034 PDI, zeta potential -2.07 ± 0.51 mV and 15.03 ± 1.92%drug loading. In vitro release showed biphasic release for 12 h with initial burst phase followed by sustained release phase. Haemolytic study on RBCs revealed haemocompatible nature of MTX-TPGS nanoparticles compared to Biotrexate® (Zydus). In vitro cell culture studies depicted 3 folds and 2.66 folds increase in cellular uptake of MTX at 10 μg/ml and 15 μg/ml respectively for developed nanoparticles with 3.81 folds decrease in IC50 value as compared to Biotrexate®. Higher apoptosis and increased lysosomal membrane permeability were also depicted by MTX-TPGS nanoparticles. 2.45 folds increase in AUC and 3.68 folds increase in T1/2 was achieved in pharmacokinetic study. Significant reduction in tumor burden and serum biochemical parameters depicted efficacy and safety respectively of the formulation as compared to Biotrexate®. RBCs morphology was retained after MTX-TPGS exposure proving its haemocompatibility in vivo.

Association between a microRNA binding site polymorphism in SLCO1A2 and the risk of delayed methotrexate elimination in Chinese children with acute lymphoblastic leukemia

Organic anion-transporting polypeptide 1A2 (OATP1A2) is involved in the cellular uptake of methotrexate (MTX). Genetic variation in solute carrier organic anion transporter family member 1A2 (SLCO1A2, the coding gene of OATP1A2) has important implications for the elimination of MTX. We investigated the association between a microRNA (miRNA) binding site polymorphism (rs4149009 G > A) in the 3′-untranslated region (3′-UTR) of SLCO1A2 with the serum MTX concentrations in Chinese children with acute lymphoblastic leukemia (ALL). Genotyping for SLCO1A2 rs4149009 G > A in 141 children with ALL was performed using the Sequenom MassARRAY system. Serum MTX concentrations were determined by fluorescence polarization immunoassay. The percentages of MTX level ≥1 μmol/L at 42 h were compared among the AA, GA, and GG genotypes. The minor allele frequency observed in this study (33.0%) was significantly lower than that in the African samples reported in the 1000 Genomes Project (57.4%, P = 0.00). The incidence rate of delayed MTX elimination was significantly higher in patients with the GG genotype (23.1%) compared with the AA genotype (0.0%, P = 0.03). Bioinformatics tools predicted that the rs4149009 A allele would disrupt the putative binding sites of hsa-miR-324-3p and hsa-miR-1913. These results indicate that the rs4149009 G > A polymorphism might affect MTX pharmacokinetics by interfering with the function of miRNAs.

Enhanced systemic availability of methotrexate in the presence of morin in rats

The present study aimed to investigate the effect of morin on the pharmacokinetics of methotrexate (MTX) in rats. Pharmacokinetic parameters of MTX were determined in rats following an intravenous administration of MTX (2 mg/kg) in the presence and absence of morin (25 mg/kg, p.o.). The cellular accumulation of MTX was also examined by using MDCK cells stably overexpressing hOAT1 or hOAT3. Compared with the control given MTX alone, pretreatment with morin 15 min prior to MTX administration significantly altered the pharmacokinetics of MTX in rats. Renal clearance and total clearance of MTX were reduced by 42% and 58%, respectively, in the presence of morin. Accordingly, the systemic exposure of MTX in the rats pretreated with morin was significantly higher than that from the control group. The mean residence time (MRT) and terminal plasma half-life of MTX were prolonged by 3.3- and 2.4-fold, respectively, by the concurrent use of morin. The cellular uptake of MTX (20 microM) was significantly reduced by the co-incubation with morin (100 microM) in MDCK-hOAT1 cells but not in MDCK-hOAT3 cells. Taken together, morin appeared to be effective in altering the pharmacokinetics of MTX in rats, likely by the inhibition of OAT1-mediated renal excretion.

Anticancer Drug-Inorganic Nanohybrid and Its Cellular Interaction

An anticancer drug, methotrexate (MTX), has been successfully hybridized with layered double hydroxide (LDH) through co-precipitation route to produce MTX-LDH nanohybrids (MTX-LDH). According to the X-ray diffraction and FT-IR spectroscopy, it was confirmed that MTX molecules are stabilized in the interlayer space of LDHs by electrostatic interaction, maintaining their functional groups and structural integrity. According to the drug release study, the total amount of released MTX from the LDH lattice was determined to be larger under a simulated intracellular lysosomal condition (pH = 4.5) than simulated body fluid one (pH = 7.4). It is, therefore, expected that the MTX molecules in MTX-LDH can be effectively released in lysosomes, since the MTX release could be accelerated via ion-exchange reaction and dissolution of LDH in an acidic lysosomal condition. We also examined the anticancer efficacy of MTX-LDH in human breast adenocarcinoma MCF-7 cells. The cellular uptake of MTX was considerably higher in MTX-LDH-treated cells than in free MTX-treated cells, giving a lower IC50 value for the former than the latter. All the results demonstrated that the MTX-LDH nanohybrid allows the efficient drug delivery in cells, and thus enhances drug efficacy.

Transcriptional Regulation of the Human Reduced Folate Carrier in Childhood Acute Lymphoblastic Leukemia Cells

The transcriptional regulation of the human reduced folate carrier (hRFC), involved in cellular uptake of methotrexate and reduced folates, was studied in childhood acute lymphoblastic leukemia (ALL). The hRFC gene is regulated by six noncoding exons (A1/A2 and A to E) and multiple promoters. In ALL, hRFC-A1/A2 and hRFC-B are the major transcript forms. RNAs from 18 ALL lymphoblast specimens and 10 nonobese diabetic/severe combined immunodeficient ALL xenografts were assayed by real-time reverse transcription-PCR for hRFC-A1/A2 and hRFC-B transcripts and for transcripts encoding USF1, GATA1, Sp1, and Ikaros transcription factors. For the xenografts, gel shift and chromatin immunoprecipitation assays assessed transcription factor binding to the hRFC-A1/A2 and hRFC-B promoters. CpG methylation density within a 334-bp region, including the core hRFC-B promoter, was established by bisulfite sequencing. hRFC-A1/A2 and hRFC-B promoter polymorphisms were assayed by DNA sequencing. For the 28 ALLs, hRFC-A1/A2 and hRFC-B transcripts spanned a 546-fold range. By chromatin immunoprecipitation and gel shift assays, binding was confirmed for USF1 and GATA1 for hRFC-A1/A2, and for Sp1, USF1, and Ikaros for hRFC-B. hRFC transcript levels correlated with those for GATA1 and USF1 for hRFC-A1/A2 and with Sp1 and USF1 transcripts for hRFC-B. CpG methylation in ALL did not correlate with hRFC-B transcripts. In 40 ALL and 17 non-ALL specimens, 2 cosegregating high-frequency polymorphisms (T-1309/C-1217 and C-1309/T-1217; allelic frequencies of 36% and 64%, respectively) were detected in the A1/A2 promoter; none were detected in promoter B. The hRFC-A1/A2 polymorphisms only slightly affected promoter activity. Our results show a complex regulation of hRFC in ALL involving the hRFC-A1/A2 and hRFC-B promoters and noncoding exons. Although Sp1, USF1, and GATA1 levels are critical determinants of hRFC transcription in ALL, neither DNA methylation nor promoter polymorphisms contribute to differences in hRFC expression.

Density-dependent inhibition of expression of syncytiotrophoblastic markers by cultured human choriocarcinoma (BeWo) cells.

In the presence of methotrexate, cultured human choriocarcinoma (BeWo) cells undergo a differentiative response that resembles normal trophoblastic development. In the current study, the effects of cell number and population density on drug-induced conversion of BeWo cells from the cytotrophoblastlike to the syncytiotrophoblastlike phenotype were investigated using as markers of differentiation formation of "giant" cells, a process shown to require exogenous purines, and expression of placental (heat-stable) alkaline phosphatase. Giant cell formation, assessed by determination of cell volumes, was reduced in crowded cultures, and addition of hypoxanthine to growth media partially restored methotrexate-induced cell enlargement. Cellular uptake of methotrexate, assessed by following the loss of methotrexate from cell culture fluids during drug exposures, was two-threefold greater in sparsely populated than in densely populated cultures. Although the concentration of methotrexate in culture fluids of crowded cultures declined during exposures of 48 hr, the amount of extracellular drug remaining at 48 hr was well above the threshold for induction of the differentiative response. When culture population was held constant and population density was manipulated by varying the substratum available to cells, methotrexate-induced cell enlargement was inversely related to population density. Expression of placental alkaline phosphatase, salvage of exogenous hypoxanthine, and synthesis of RNA were also reduced at high population densities. These results indicate that expression of markers of methotrexate-induced differentiation of BeWo cells was inhibited in a density-dependent manner that may have been related to reduced cellular uptake of the inducing agent and of exogenous nutrients (purines) from culture fluids.

The effect of trimethoprim on cellular transport of methotrexate and its cytotoxicity to human lymphoblastoid cells in vitro.

A study has been made of the effects of the antifolate drug trimethoprim (2,4-diamino-5-(3'4',5'-trimethoxybenzyl)pyrimidine) on the cellular transport of natural folates and of methotrexate in a human lymphoblastoid cell line grown in culture. The results indicated that cellular uptake of 5-methyltetrahydrofolate, folic acid and methotrexate could be inhibited by trimethoprim; the inhibition, for methotrexate at least, was competitive, but it was in every case rather weak. Calculations showed that under conditions used during therapy with methotrexate, trimethoprim could be employed as an antibacterial agent without the cellular uptake of methotrexate being impaired by more than 1%. The cytotoxic effect of trimethoprim against the cell line used was 2000 times less, on a molar basis, than that of methotrexate. However, the degree of growth inhibition caused by the two agents in combination was greater than the sum of the individual toxicities. Again, this effect was not large at clinically useful concentrations of trimethoprim.