?-Glutamyl hydrolase modulation and folate influence chemosensitivity of cancer cells to 5-fluorouracil and methotrexate.
ABSTRACT: ?-Glutamyl hydrolase (GGH) regulates intracellular folate and antifolates for optimal nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. The modulation of GGH may therefore affect chemosensitivity of cancer cells, and exogenous folate levels may further modify this effect.We generated a novel model of GGH modulation in human HCT116 and MDA-MB-435 cancer cells and investigated the effect of GGH modulation on chemosensitivity to 5-fluorouracil (5FU) and methotrexate (MTX) at different folate concentrations in vitro and in vivo.Overexpression of GGH significantly decreased chemosensitivity of MDA-MB-435 cells to 5FU and MTX at all folate concentrations as expected. In contrast, in HCT116 cells this predicted effect was observed only at very high folate concentration, and as the folate concentration decreased this effect became null or paradoxically increased. This in vitro observation was confirmed in vivo. Inhibition of GGH significantly increased chemosensitivity of both cancer cells to 5FU at all folate concentrations. Unexpectedly, GGH inhibition significantly decreased chemosensitivity of both cancer cells to MTX at all folate concentrations. In both GGH modulation systems and cell lines, the magnitude of chemosensitivity effect incrementally increased as folate concentration increased.Modulation of GGH affects chemosensitivity of cancer cells to 5FU and MTX, and exogenous folate levels can further modify the effects.
Project description:?-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.
Project description:We evaluated the pharmacogenetic influence of genetic polymorphisms in folate pathway genes in Indian rheumatoid arthritis patients receiving methotrexate (MTX).Twelve polymorphisms within nine folate pathway genes were analyzed for association with MTX response in 322 Indian rheumatoid arthritis (RA) patients and MTX pharmacokinetics in 94 RA patients.Polymorphisms in GGH, SHMT1 and TS were associated with MTX-related adverse events while SNPs in MTHFR and RFC1/SLC19A1 were associated with MTX efficacy. TS5'UTR and SHMT1 polymorphisms were associated with higher plasma levels of MTX.Polymorphisms in folate-MTX pathway genes contribute to MTX response and affect MTX concentrations in Indian RA patients. A toxicogenetic index could identify patients who develop adverse events to MTX.
Project description:Folylpolyglutamate synthase (FPGS) plays a critical role in intracellular folate homeostasis. FPGS-induced polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence FPGS modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression and aberrant DNA methylation is mechanistically linked cancer development. We investigated whether FPGS modulation would affect global and gene-specific promoter DNA methylation with consequent functional effects on gene expression profiles in HCT116 colon and MDA-MB-435 breast cancer cells. Although FPGS modulation altered global DNA methylation and DNA methyltransferases (DNMT) activity, the effects of FPGS modulation on global DNA methylation and DNMT activity could not be solely explained by intracellular folate concentrations and content of long-chain folylpolyglutamates, and it may be cell-specific. FPGS modulation influenced differential gene expression and promoter cytosine-guanine dinucleotide sequences (CpG) DNA methylation involved in cellular development, cell cycle, cell death and molecular transport. Some of the altered gene expression was associated with promoter CpG DNA methylation changes. In both the FPGS-overexpressed HCT116 and MDA-MB-435 cell lines, we identified several differentially expressed genes involved in folate biosynthesis and one-carbon metabolism, which might in part have contributed to the observed increased efficacy of 5-fluorouracil in response to FPGS overexpression. Our data suggest that FPGS modulation affects global and promoter CpG DNA methylation and expression of several genes involved in important biological pathways. The potential role of FPGS modulation in DNA methylation and its associated downstream functional effects warrants further studies.
Project description:Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to ?-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy.
Project description:We investigated major determinants of the intracellular concentrations of methotrexate polyglutamates (MTXPGs) in patients with rheumatoid arthritis (RA). In 271 RA patients on stable oral low dose weekly pulse MTX therapy, the concentrations of MTXPGs in red blood cells (RBCs) were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to determine the genotypes of solute carrier family 19 member 1 (SLC19A1), folylpolyglutamate synthase (FPGS), and gamma-glutamyl hydrolase (GGH). The mean total MTXPG concentration and the concentrations of individual MTXPGs increased dose-dependently, but reached a plateau at MTX doses >10?mg weekly. The MTXPG3-5/1-2 ratio was lower in patients with adverse events related to MTX than in patients without adverse events. Three polymorphisms of FPGS significantly influenced the MTXPG3-5/1-2 ratio in RBCs, while polymorphisms of SLC19A1 and GGH had no impact. The minor allele frequencies of 2 FPGS genotypes were significantly increased in our patients compared with a Caucasian population. FPGS may have a major role in regulating intracellular polyglutamation of MTX in RA patients receiving low-dose weekly MTX therapy.
Project description:Gamma-glutamyl hydrolase (GGH) catalyzes degradation of the active polyglutamates of natural folates and the antifolate methotrexate (MTX). We found that GGH activity is directly related to GGH messenger RNA expression in acute lymphoblastic leukemia (ALL) cells of patients with a wild-type germline GGH genotype. We identified two CpG islands (CpG1 and CpG2) in the region extending from the GGH promoter through the first exon and into intron 1 and showed that methylation of both CpG islands in the GGH promoter (seen in leukemia cells from approximately 15% of patients with nonhyperdiploid B-lineage ALL) is associated with significantly reduced GGH mRNA expression and catalytic activity and with significantly higher accumulation of MTX polyglutamates (MTXPG(4-7)) in ALL cells. Furthermore, methylation of CpG1 was leukemia-cell specific and had a pronounced effect on GGH expression, whereas methylation of CpG2 was common in leukemia cells and normal leukocytes but did not significantly alter GGH expression. These findings indicate that GGH activity in human leukemia cells is regulated by epigenetic changes, in addition to previously recognized genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.
Project description:Folate is an essential nutrient that supports nucleotide synthesis and biological methylation reactions. Diminished folate status results in chromosome breakage and is associated with several diseases, including colorectal cancer. Folate status is also inversely related to plasma homocysteine concentrations -- a risk factor for cardiovascular disease.We sought to gain further understanding of the genetic determinants of plasma folate and homocysteine concentrations. Because folate is required for the synthesis of thymidine from uracil, the latter accumulating and being misincorporated into DNA during folate depletion, the DNA uracil content was also measured.Thirteen single nucleotide polymorphisms (SNPs) in genes involved in folate uptake and metabolism, including folate hydrolase (FOLH1), folate polyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH), methylene tetrahydrofolate reductase (MTHFR), methionine synthase (MTR), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC1), were studied in a cohort of 991 individuals.The MTHFR 677TT genotype was associated with increased plasma homocysteine and decreased plasma folate. MTHFR 1298A>C and RFC1 intron 5A>G polymorphisms were associated with significantly altered plasma homocysteine concentrations. The FOLH1 1561C>T SNP was associated with altered plasma folate concentrations. The MTHFR 677TT genotype was associated with a approximately 34% lower DNA uracil content (P = 0.045), whereas the G allele of the GGH -124T>G SNP was associated with a stepwise increase in DNA uracil content (P = 0.022).Because the accumulation of uracil in DNA induces chromosome breaks, mutagenic lesions, we suggest that, as for MTHFR C677T, the GGH -124 T>G SNP may modulate the risk of carcinogenesis and therefore warrants further attention.
Project description:AIMS: We investigated whether several polymorphisms within the methotrexate (MTX) pathway genes were related to the toxicity and efficacy of MTX in 92 Japanese patients with articular-type juvenile idiopathic arthritis (JIA). METHODS: Eight gene polymorphisms within the MTX pathway genes, namely, RFC, BCRP, MTHFR (two), FPGS, ?-glutamyl hydrolase (GGH; two) and ATIC, were genotyped using TaqMan assays. Liver dysfunction was defined as an increase in alanine transaminase to five times the normal upper limit. Non-responders to MTX were defined as patients refractory to MTX and were therefore treated with biologics. RESULTS: The non-TT genotype at GGH T16C was associated with a high risk of liver dysfunction (P=0.028, odds ratio=6.90, 95% confidence interval 1.38-34.5), even after adjustment for the duration of MTX treatment. A longer interval from disease onset to treatment (8.5 and 21.3 months, P=0.029) and rheumatoid factor positivity (P=0.026, odds ratio=2.87, 95% confidence interval 1.11-7.39) were associated with lower efficacy of MTX. CONCLUSIONS: The non-TT genotype at GGH T16C was associated with a high risk of liver dysfunction, presumably because the C allele of GGH C16T may reduce the activity of GGH. The time interval before MTX treatment and rheumatoid factor positivity were associated with the efficacy of MTX treatment. The pharmacogenetics of the MTX pathway genes affects the toxicity and efficacy of MTX in Japanese JIA patients.
Project description:Colorectal cancer (CRC) shows highly ineffective therapeutic management. An urgent unmet need is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III CRC patients without any predictive factor of efficacy. In the field of drug discovery, a critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We proposed a patient-derived 3D preclinical model for drug evaluation that could mimic in vitro the patient's disease. Surgically resected CRC tissue and adjacent healthy colon mucosa were decellularized by a detergent-enzymatic treatment. Scaffolds were recellularized with HT29 and HCT116 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences, scanning electron microscopy, and DNA amount quantification. A chemosensitivity test was performed using an increasing concentration of 5-fluorouracil (5FU). In vivo studies were carried out using zebrafish (Danio rerio) animal model. Permeability test and drug absorption were also determined. The decellularization protocol allowed the preservation of the original structure and ultrastructure. Five days after recellularization with HT29 and HCT116 cell lines, the 3D CRC model exhibited reduced sensitivity to 5FU treatments compared with conventional 2D cultures. Calculated the half maximal inhibitory concentration (IC50) for HT29 treated with 5FU resulted in 11.5 µM in 3D and 1.3 µM in 2D, and for HCT116, 9.87 µM in 3D and 1.7 µM in 2D. In xenograft experiments, HT29 extravasation was detected after 4 days post-injection, and we obtained a 5FU IC50 fully comparable to that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that the drug diffused through the repopulated 3D CRC scaffolds and co-localized with the cell nuclei. The bioengineered CRC 3D model could be a reliable preclinical patient-specific platform to bridge the gap between in vitro and in vivo drug testing assays and provide effective cancer treatment.
Project description:Background:Polyglutamylation is a reversible protein modification that commonly occurs in tumor cells. Methotrexate (MTX) in tumor cells is polyglutamylated and strongly binds to dihydrofolate reductase (DHFR) without competitive inhibition by leucovorin. Therefore, tumor cells with high polyglutamylation levels are supposed to be selectively killed, whereas normal cells with lower polyglutamylation are rescued by leucovorin. This study investigated the combined effects of MTX plus histone deacetylase inhibitors (HDACIs), which upregulate MTX polyglutamylation, in primary central nervous system lymphoma (PCNSL). Methods:We evaluated cell viability after MTX treatment and leucovorin rescue and compared the expression of folylpolyglutamate synthetase (FPGS), ?-glutamyl hydrolase (GGH), and DHFR in 2 human PCNSL-derived cell lines (HKBML and TK) and a human Burkitt lymphoma cell line (TL-1). Combination treatments were created using 4 HDACIs: panobinostat, vorinostat, sodium butyrate, and valproic acid. The expression of DHFR was examined as well as ratios of FPGS/GGH expression. The combined effects of MTX plus HDACIs were evaluated using a cell viability assay, mass spectroscopy imaging, and subcutaneous and intracranial xenograft models. Results:HDACIs upregulated the ratio of FPGS/GGH expression resulting in increased polyglutamylation of MTX, but also downregulated expression of the target molecule of MTX: DHFR. The combination of MTX and vorinostat decreased cell viability in vitro (P < .05) and tumor volumes in a subcutaneous model (P < .0001), and prolonged survival in an intracranial model (P < .01), relative to controls. Conclusion:HDACIs enhanced the therapeutic effect of MTX through increased polyglutamylation of MTX and concomitant downregulation of DHFR expression.