Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells.

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. EXPERIMENTAL APPROACH: A549, BEL-7402 and MHCC97H cell lines were treated with MTX at gradient concentrations. Liquid chromatograph-mass spectrometer (UPLC-MS/MS) was used to quantify the intracellular concentration of MTX-PG and the abundance of FPGS and γ-glutamyl hydrolase (GGH). High quality data were used to fit the cell pharmacokinetic model. KEY RESULTS: Both cell growth inhibition rate and intracellular MTX-PG concentration showed a nonlinear relationship with MTX concentration. The parameter Vmax in the model, which represents the synthesis rate of MTX-PG, showed a strong correlation with the abundance of intracellular FPGS. CONCLUSION AND IMPLICATIONS: According to the model fitting results, it was confirmed that the abundance of FPGS is a decisive factor limiting the synthesis rate of MTX-PG. The proposed hypothesis was verified in this study. In addition, based on the intracellular metabolism, a reasonable explanation was provided for the correlation between the severity of adverse reactions of MTX and infusion time. This study provides a new strategy for the individualized treatment and prediction of efficacy/side effects of MTX.

Interaction between glycolysis‒cholesterol synthesis axis and tumor microenvironment reveal that gamma-glutamyl hydrolase suppresses glycolysis in colon cancer.

Background: Metabolic reprogramming is a feature of cancer. However, colon cancer subtypes based on the glycolysis‒cholesterol synthesis axis have not been identified, and little is known about connections between metabolic features and the tumor microenvironment. Methods: Data for 430 colon cancer cases were extracted from The Cancer Genome Atlas, including transcriptome data, clinical information, and survival outcomes. Glycolysis and cholesterol synthesis-related gene sets were obtained from the Molecular Signatures Database for a gene set variation analysis. The relationship between the genomic landscape and immune landscape were investigated among four metabolic subtypes. Hub genes were determined. The clinical significance of candidate hub gene was evaluated in 264 clinical samples and potential functions were validated in vitro and in vivo. Results: Colon cancer cases were clustered into four metabolic subtypes: quiescent, glycolytic, cholesterogenic, and mixed. The metabolic subtypes differed with respect to the immune score, stromal score, and estimate score using the ESTIMATE algorithm, cancer-immunity cycle, immunomodulator signatures, and signatures of immunotherapy responses. Patients in the cholesterogenic group had better survival outcomes than those for other subtypes, especially glycolytic. The glycolytic subtype was related to unfavorable clinical characteristics, including high mutation rates in TTN, APC, and TP53, high mutation burden, vascular invasion, right colon cancer, and low-frequency microsatellite instability. GGH, CACNG4, MME, SLC30A2, CKMT2, SYN3, and SLC22A31 were identified as differentially expressed both in glycolytic-cholesterogenic subgroups as well as between colon cancers and healthy samples, and were involved in glycolysis‒cholesterol synthesis. GGH was upregulated in colon cancer; its high expression was correlated with CD4+ T cell infiltration and longer overall survival and it was identified as a favorable independent prognostic factor. The overexpression of GGH in colon cancer-derived cell lines (SW48 and SW480) inhibited PKM, GLUT1, and LDHA expression and decreased the extracellular lactate content and intracellular ATP level. The opposite effects were obtained by GGH silencing. The phenotype associated with GGH was also validated in a xenograft nude mouse model. Conclusions: Our results provide insight into the connection between metabolism and the tumor microenvironment in colon cancer and provides preliminary evidence for the role of GGH, providing a basis for subsequent studies.

Ultrasonically Enhanced ZD2767P-Carboxypeptidase G2 Deactivates Cisplatin-Resistant Human Lung Cancer Cells.

The prodrug-enzyme regimen ZD2767P+CPG2 is limited by low efficacy. Here, ultrasound was used to modulate ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against cisplatin-resistant human lung cancer cells. A549 and A549/DDP (resistant subline) cells received ZD2767P+CPG2 or ZD2767P+CPG2+US. Either ZD2767P+CPG2 or ZD2767P+CPG2+US led to cell death and apoptosis, and ZD2767P+CPG2+US produced stronger effects; comet assays revealed that these two means directly caused DNA double-strand break. Z-VAD-fmk and/or ferrostatin-1 increased the cell survival percentage, and Z-VAD-fmk decreased the apoptosis percentage. The level of transferrin was increased in treated cells, but those of ferroportin and glutathione peroxidase 4 (GPX4) were reduced, with higher intracellular levels of reactive oxygen species and of iron. Intracellular pharmacokinetics of ZD2767D (activated drug) indicated that the peak level, area under the drug level vs. time curve, and mean residence time in ZD2767P+CPG2+US were higher than those in ZD2767P+CPG2. Both ZD2767P+CPG2 and ZD2767P+CPG2+US were effective on xenograft tumors in nude mice; inhibitory rates were 39.7% and 63.5% in A549 tumors and 50.0% and 70.1% in A549/DDP tumors, respectively. A higher apoptosis level and a lower GPX4 level were noted in tumors receiving treatments. No severe adverse events were observed. These data demonstrated that ZD2767P+CPG2+US deactivated cancer cells via apoptosis and ferroptosis pathways, being a candidate therapy for cisplatin-resistant lung cancer.

Characterization of a Stable Form of Carboxypeptidase G2 (Glucarpidase), a Potential Biobetter Variant, From Acinetobacter sp. 263903-1.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5-10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20-70 °C compared to PsCPG2. The Km value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90  µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site.

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

2,4-Diamino-N10-methylpteroic acid (DAMPA) crystalluria in a patient with osteosarcoma treated with carboxypeptidase-G2 rescue after high-dose methotrexate-induced nephrotoxicity.

BACKGROUND: High-dose methotrexate (HDMTX) therapy is a key component of many chemotherapy protocols. However, some patients develop HDMTX-induced nephrotoxicity. Carboxypeptidase-G2 (CPDG2) hydrolyses MTX into 2,4-diamino-N10-methylpteroic acid (DAMPA) and glutamic acid, and is used as a rescue agent in patients with nephrotoxicity and delayed elimination. Despite the frequency of HDMTX-induced renal injury, crystalluria is uncommon. Furthermore, crystals are rarely identified by conventional chemical methods. OBJECTIVE: To determine the composition of crystalluria in a patient with osteosarcoma who was treated with CPDG2. METHODS: Crystalluria was evaluated by optical microscopy, and chemical identification was performed by Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and Orbitrap™ high-resolution mass spectrometry (HRMS). RESULTS: The HRMS spectra of the patient's urine sediment showed a main peak at m/z 326.13, corresponding to the molecular mass of DAMPA [(C15H15O2N7) + H+]. The FT-IR spectral patterns of the sediment and DAMPA were not identical. SEM was unable to identify the crystal. CONCLUSION: DAMPA crystalluria was identified by Orbitrap™ HRMS in a patient treated with CPDG2 after HDMTX nephrotoxicity. This case reinforces the need to implement adequate measures to prevent nephrotoxicity. In cases of HDMTX-induced nephrotoxicity, urine sediment analysis should be requested.

Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy.

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.

High pemetrexed sensitivity of docetaxel-resistant A549 cells is mediated by TP53 status and downregulated thymidylate synthase.

The chemoresistance of non-small cell lung cancer (NSCLC) that occurs in docetaxel (DOC) chemotherapy substantially decreases the survival of patients. To overcome DOC-induced chemoresistance, we established DOC-selected A549 lung cancer sublines (A549/D16 and A549/D32) and revealed that both sublines were cross-resistant to vincristine (VCR) and doxorubicin (DXR). Notably, both sublines were more sensitive to pemetrexed (PEM) than parental cells according to MTT and clonogenic assays. The expression levels of thymidylate synthase (TS) and γ-glutamyl hydrolase (GGH) were downregulated in DOC-resistant sublines. When exogenous TS was overexpressed in A549/D16 cells, PEM sensitivity was significantly decreased, however it was not decreased by overexpression of exogenous GGH. PEM treatment induced more apoptotic sub-G1 cells in both DOC-resistant sublines and in the in vivo PEM sensitivities of A549/D16 cells. These findings were further confirmed by a xenografted tumor model. To unmask the mediator of TS downregulation, we investigated human lung cancer cell lines that have various TP53 statuses using DOC treatment. The level of TS protein was significantly decreased in wild-type TP53-containing cells with DOC treatment; TS expression levels were not affected in mutant-TP53 and TP53­null cells under the same conditions. Furthermore, when the expression of TP53 was inhibited in A549 cells, the expression level of TS was increased. Our data indicated that DOC activated wild-type TP53 and suppressed TS expression under continuous DOC exposure. Therefore, the expression of TS remained at low levels in DOC-resistant A549 cancer cells. Our data revealed that for lung cancer with DOC resistance and wild­type TP53 status, the administration of PEM as a second-line agent to overcome DOC-resistance may benefit patients.

High-Level γ-Glutamyl-Hydrolase (GGH) Expression is Linked to Poor Prognosis in ERG Negative Prostate Cancer.

γ-glutamyl-hydrolase (GGH) is a ubiquitously-expressed enzyme that regulates intracellular folate metabolism for cell proliferation, DNA synthesis, and repair. Employing GGH immunohistochemistry on a tissue microarray with 12,427 prostate cancers, we found that GGH expression was negative to low in normal prostate epithelium, whereas 88.3% of our 10,562 interpretable cancers showed GGH expression. GGH staining was considered as low intensity in 49.6% and as high intensity in 38.6% of cancers. High GGH expression was linked to the TMPRSS2:ERG-fusion positive subset of cancers (p < 0.0001), advanced pathological tumor stage, and high Gleason grade (p < 0.0001 each). Further analysis revealed that these associations were merely driven by the subset of ERG-negative cancers, High GGH expression was weakly linked to early biochemical recurrence in ERG negative cancers (p < 0.0001) and independent from established histo-pathological parameters. Moreover, GGH expression was linked to features of genetic instability, including presence of recurrent deletions at 3p, 5q, 6q, and 10q (PTEN, p ≤ 0.01 each), as well as to accelerated cell proliferation as measured by Ki67 immunohistochemistry (p < 0.0001). In conclusion, the results of our study identify GGH as an ERG subtype specific molecular marker with modest prognostic relevance, which may have clinical relevance if analyzed in combination with other molecular markers.

Pharmacogenetic analysis of high-dose methotrexate treatment in children with osteosarcoma.

Inter-individual differences in toxic symptoms and pharmacokinetics of high-dose methotrexate (MTX) treatment may be caused by genetic variants in the MTX pathway. Correlations between polymorphisms and pharmacokinetic parameters and the occurrence of hepato- and myelotoxicity were studied. Single nucleotide polymorphisms (SNPs) of the ABCB1, ABCC1, ABCC2, ABCC3, ABCC10, ABCG2, GGH, SLC19A1 and NR1I2 genes were analyzed in 59 patients with osteosarcoma. Univariate association analysis and Bayesian network-based Bayesian univariate and multilevel analysis of relevance (BN-BMLA) were applied. Rare alleles of 10 SNPs of ABCB1, ABCC2, ABCC3, ABCG2 and NR1I2 genes showed a correlation with the pharmacokinetic values and univariate association analysis. The risk of toxicity was associated with five SNPs in the ABCC2 and NR1I2 genes. Pharmacokinetic parameters were associated with four SNPs of the ABCB1, ABCC3, NR1I2, and GGH genes, and toxicity was shown to be associated with ABCC1 rs246219 and ABCC2 rs717620 using the univariate and BN-BMLA method. BN-BMLA analysis detected relevant effects on the AUC0-48 in the following SNPs: ABCB1 rs928256, ABCC3 rs4793665, and GGH rs3758149. In both univariate and multivariate analyses the SNPs ABCB1 rs928256, ABCC3 rs4793665, GGH rs3758149, and NR1I2 rs3814058 SNPs were relevant. These SNPs should be considered in future dose individualization during treatment.

The 46 kDa dimeric protein from Variovorax paradoxus shows faster methotrexate degrading activity in its nanoform compare to the native enzyme.

Methotrexate degrading enzymes are required to overcome the toxicity of the methotrexate while treating the cancer. The enzyme from Variovorax paradoxus converts the methotrexate in to non toxic products. Methotrexate degrading enzyme from V. paradoxus is a dimeric protein with a molecular mass of 46 kDa and it acts on casein and gelatin. This enzyme is optimally active at pH 7.5 and 40°C and nanoparticles of this enzyme were prepared by desolvation-crosslinking method. Enzyme nanoparticles could degrade methotrexate faster than the native enzyme and they show lower Km compare to the native enzyme. Enzyme nanoparticles show better thermostability and they were stable for much longer time in the serum compare to the native enzyme. Enzyme nanoparticles show better functionality than the native enzyme while clearing the methotrexate added to the serum suggesting their advantage over the native enzyme for the therapeutic and biotechnological applications.

Folate deficiency-induced oxidative stress contributes to neuropathy in young and aged zebrafish--implication in neural tube defects and Alzheimer's diseases.

Folate is a nutrient essential for the development, function and regeneration of nervous systems. Folate deficiency has been linked to many neurological disorders including neural tube defects in fetus and Alzheimer's diseases in the elderly. However, the etiology underlying these folate deficiency-associated diseases is not completely understood. In this study, zebrafish transgenic lines with timing and duration-controllable folate deficiency were developed by ectopically overexpressing a recombinant EGFP-γ-glutamyl hydrolase (γGH). Impeded neural crest cell migration was observed in the transgenic embryos when folate deficiency was induced in early stages, leading to defective neural tube closure and hematopoiesis. Adding reduced folate or N-acetylcysteine reversed the phenotypic anomalies, supporting the causal link between the increased oxidative stress and the folate deficiency-induced abnormalities. When folate deficiency was induced in aged fish accumulation of beta-amyloid and phosphorylated Tau protein were found in the fish brain cryo-sections. Increased autophagy and accumulation of acidic autolysosome were apparent in folate deficient neuroblastoma cells, which were reversed by reduced folate or N-acetylcysteine supplementation. Decreased expression of cathepsin B, a lysosomal protease, was also observed in cells and tissue with folate deficiency. We concluded that folate deficiency-induced oxidative stress contributed to the folate deficiency-associated neuropathogenesis in both early and late stages of life.

Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid.

MAIN CONCLUSION: Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ(9cis,11trans,13cis) ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ(12)-double bond of linoleic acid (18:2Δ(9cis,12cis) ) into conjugated Δ(11trans) and Δ(13cis) -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1∆(9cis) ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.

Folic acid pathway single nucleotide polymorphisms associated with methotrexate significant adverse events in United States veterans with rheumatoid arthritis.

OBJECTIVES: Methotrexate (MTX) is the cornerstone medication in the treatment of rheumatoid arthritis (RA). We examined whether single nucleotide polymorphisms (SNPs) in enzymes of the folic acid pathway (folylpoly-gamma-glutamate synthetase [FPGS], gamma-glutamyl hydrolase [GGH], and methylenetetrahydrofolate reductase [MTHFR]) associate with significant adverse events (SigAE). METHODS: Patients (n=319) enrolled in the Veterans Affairs RA (VARA) registry taking MTX were genotyped for HLA-DRB1-SE and the following SNPs: FPGS (rs7033913, rs10760503, rs10106), GGH (12548933, rs7010484, rs4617146, rs719235, rs11988534), MTHFR (rs1801131, rs1801133). AE were abstracted from the medical record using a structured instrument. SigAE were defined as an AE leading to MTX discontinuation. Covariates included: age, gender, race, RA antibody status, tobacco, RA disease duration between diagnosis and MTX course, Charlson-Deyo comorbidity index, glucocorticoids, use of prior RA medications, and mean 4-variable disease activity score. Cox regression was performed to determine factors associated with time-to-SigAE. A p-value ≤ 0.005 established significance in the final model. RESULTS: The presence of ≥ 1 copy of the minor allele in MTHFR rs1801131 was associated with an increased hazard ratio (HR) of SigAE (HR 3.05, 95% CI 1.48-6.29, p-value 0.003 and HR 3.88, 95% CI 1.62-9.28, p-value 0.002 for heterozygotes and homozygotes for the minor allele, respectively). An interaction term, between FPGS rs7033913 heterozygotes and GGH rs11988534 homozygotes for the minor allele, had a p-value <0.0001. CONCLUSIONS: RA subjects taking MTX may have decreased time-to-SigAE with ≥ 1 copy of the minor allele in MTHFR rs1801131. Further investigation is warranted, as these SNPs may indicate susceptibility to MTX toxicity.

Detection of the prodrug-activating enzyme carboxypeptidase G2 activity with chemical exchange saturation transfer magnetic resonance.

PURPOSE: The purpose of this study is to evaluate if the differential exchange rates with bulk water between amine and amide protons can be exploited using chemical exchange saturation transfer magnetic resonance (CEST-MR) to monitor the release of glutamate induced by carboxypeptidase G2 (CPG2), an enzyme utilized in cancer gene therapy. PROCEDURES: Z spectra of solutions of the CPG2 substrate, 3,5-difluorobenzoyl-L-glutamate (amide), and glutamate (amine) were acquired at 11.7 T, 37 °C, across different pH (5-8). The ability of CEST-MR to monitor CPG2-mediated release of glutamate was assessed in extracts of CPG2-expressing cancer cells and purified solution of CPG2. RESULTS: The addition of CPG2 to a solution containing 3,5-difluorobenzoyl-L-glutamate led to a marked and progressively increasing CEST effect (+3 ppm), concomitant with the time-dependent release of glutamate induced by CPG2. CONCLUSION: CEST-MR allows the detection of CPG2 activity in vitro and supports the translation of CEST-MRI to assess CPG2-based gene therapy in vivo.

γ-Glutamyl hydrolase modulation and folate influence chemosensitivity of cancer cells to 5-fluorouracil and methotrexate.

BACKGROUND: γ-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. METHODS: 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. RESULTS: 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. CONCLUSION: Modulation of GGH affects chemosensitivity of cancer cells to 5FU and MTX, and exogenous folate levels can further modify the effects.

Reduction in γ-glutamyl hydrolase expression is associated with response to uracil and tegafur/leucovorin chemotherapy in patients with colorectal cancer.

To identify the genes and clinical parameters associated with efficacy of uracil and tegafur/leucovorin (UFT/LV) chemotherapy in colorectal cancer (CRC), we compared the levels of reduced folate in tumors between patients receiving LV and those not receiving LV (study I), and explored the changes in the expression levels of 14 genes after two weeks of UFT/LV chemotherapy in 73 patients with CRC (study II). In study I, the reduced folate levels after LV administration were approximately 3-fold higher than those without LV administration in patients with right-sided tumors or aged >75 years old (p=0.019). In study II, the reduction in γ-glutamyl hydrolase (GGH) expression in responders and in patients with right-sided tumors or aged >75 years old was significantly greater than that in non-responders (p<0.001) and in other patients, respectively (p=0.003). Increase of reduced folate and reduction in GGH expression were associated with response to UFT/LV chemotherapy in patients with right-sided tumors or aged >75 years old.

Association of the TYMS 3G/3G genotype with poor response and GGH 354GG genotype with the bone marrow toxicity of the methotrexate in RA patients.

PURPOSE: Gamma-glutamyl hydrolase (GGH), cyclin D1 (CCND1) and thymidylate synthase (TS) genes encode enzymes that are involved in methotrexate (MTX) action. In a group of 184 RA patients treated with MTX, we have investigated whether selected polymorphisms in these genes modulate MTX efficacy and/or have impact on adverse drug effects (ADEs). METHODS: The efficacy of the MTX therapy has been estimated using the disease activity score in 28 joints (DAS28-ESR) based on EULAR criteria and relative DAS28 values (rDAS28). All adverse drug events were recorded. Patients were genotyped for selected polymorphisms of the GGH (-354 G > T and 452 C > T), CCND1 (870 A > G) and TYMS (variable number of tandem repeats, VNTR, and G to C substitution of triple repeat, 3R allele) gene. Association studies have been performed between obtained genotypes and the efficacy and toxicity of MTX. RESULTS: According to the EULAR response criteria, 146 RA patients (79.3 %) were classified as responders (good/moderate response) and 38 (20.7 %) as non-responders (poor response). Higher frequency of the TYMS 3 G/3 G genotype has been found among non-responders as compared to individuals with remaining genotypes (p = 0.02). ADEs were recorded in 53 patients. Among those patients eight experienced bone marrow toxicity, all of them carried GGH -354GG genotype (p = 0.003). No other significant association were observed. CONCLUSION: The 3 G/3 G genotype of the TYMS gene may indicate predisposition of poor response to MTX and GG genotype of GGH -354 T > G polymorphism may have high predictive value for myelosuppression in RA patients.

The impact of GGH -401C>T polymorphism on cisplatin-based chemoradiotherapy response and survival in cervical cancer.

AIMS: Cervical cancer is the third most frequent cancer in women worldwide, mostly treated with cisplatin-based chemoradiotherapy. Since it is known that folate metabolism might interfere with cisplatin effectiveness, we intended to study the influence of the Gamma Glutamyl Hydrolase -401C>T polymorphism in treatment response in cervical cancer. METHODS: We retrospectively reviewed the clinical data of 167 patients with bulky cervical cancer submitted to cisplatin-based chemoradiotherapy. The genotypes of GGH -401C>T SNP were determined by real-time PCR and statistical analysis was performed by χ(2) test and survival analysis. RESULTS: The genotypes of GGH-401C>T were significantly associated with the response to platinum-based chemoradiotherapy. Treatment response was higher in patients carrying the CC genotype, who presented a significant increased chance of treatment response (survival time in months/genotype: 91 for CC Vs 72 for CT/TT; p=0.035, log rank test). A Cox regression analysis accordingly showed that the presence of the T allele was significantly linked to a worse treatment response (HR=3.036; CI 95% 1.032-8.934, p=0.044). CONCLUSIONS: The results of our study suggested the potential interest of GGH -401C>T as a predictive factor of the outcome of cervical carcinoma treated with cisplatin-based chemoradiotherapy.