Smoking and polymorphisms in folate metabolizing genes and their effects on the histological stage and grade for bladder tumors

Folates are the common sources of DNA synthesis and methylation. Cigarette smoking and genetic susceptibility of folate enzymes are two suspected factors most closely associated with bladder cancer development. This study sought to determine the effect of smoking and genetic polymorphisms in folate metabolizing enzymes on the histological stage and grade of bladder tumors in Tunisian patients. A total of 130 patients with urothelial cell carcinomas were examined with respect to smoking status, MTHFR (5,10-methylenetetrahydrofolate reductase), MTR (methionine synthase), MTRR (methionine synthase reductase) and TYMS (thymidylate synthase) genotypes distribution. Our data have reported that tobacco, MTHFR, MTR and MTRR genotypes were not associated with bladder tumor stage. Only TYMS 3R*G/3R*C genotype was associated with increased risk of developing invasive tumors compared to reference group (RR = 1.74; 95% CI: 0.97-3.12). When we studied the superficial bladder tumor group, we have shown a significant statistical differences for the TYMS 3R*G/2R genotype. This genotype presented a 1.68-fold increased risk of developing high grade tumors compared to reference group (RR = 1.68; 95% CI: 1.12-2.54). Moreover, we have shown that patients having at least one copy of 2R allele were at 4.23-fold increased risk for developing high grade tumors compared to reference group (P = 0.022).     

<Emphasis Type="Italic">PTR1</Emphasis>-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite <Emphasis Type="Italic">Leishmania major</Emphasis>

Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1 ⁻ null mutants were 18-fold more sensitive to H₂O₂ than PTR1-overproducing lines, and significant three- to fivefold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H₂O₂ susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H₄B metabolism specifically. Neither H₂O₂ consumption nor the level of intracellular oxidative stress was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols, these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.     

Molecular basis of antifolate resistance

Folates play a key role in one-carbon metabolism essential for the biosynthesis of purines, thymidylate and hence DNA replication. The antifolate methotrexate has been rationally-designed nearly 60 years ago to potently block the folate-dependent enzyme dihydrofolate reductase (DHFR) thereby achieving temporary remissions in childhood acute leukemia. Recently, the novel antifolates raltitrexed and pemetrexed that target thymidylate synthase (TS) and glycineamide ribonucleotide transformylase (GARTF) were introduced for the treatment of colorectal cancer and malignant pleural mesothelioma. (Anti)folates are divalent anions which predominantly use the reduced folate carrier (RFC) for their cellular uptake. (Anti)folates are retained intracellularly via polyglutamylation catalyzed by folylpoly-γ-glutamate synthetase (FPGS). As the intracellular concentration of antifolates is critical for their pharmacologic activity, polyglutamylation is a key determinant of antifolate cytotoxicity. However, anticancer drug resistance phenomena pose major obstacles towards curative cancer chemotherapy. Pre-clinical and clinical studies have identified a plethora of mechanisms of antifolate-resistance; these are frequently associated with qualitative and/or quantitative alterations in influx and/or efflux transporters of (anti)folates as well as in folate-dependent enzymes. These include inactivating mutations and/or down-regulation of the RFC and various alterations in the target enzymes DHFR, TS and FPGS. Furthermore, it has been recently shown that members of the ATP-binding cassette (ABC) superfamily including multidrug resistance proteins (MRP/ABCC) and breast cancer resistance protein (BCRP/ABCG2) are low affinity, high capacity ATP-driven (anti)folate efflux transporters. This transport activity is in addition to their established facility to extrude multiple cytotoxic agents. Hence, by actively extruding antifolates, overexpressed MRPs and/or BCRP confer antifolate resistance. Moreover, down-regulation of MRPs and/or BCRP results in decreased folate efflux thereby leading to expansion of the intracellular folate pool and antifolate resistance. This chapter reviews and discusses the panoply of molecular modalities of antifolate-resistance in pre-clinical tumor cell systems in vitro and in vivo as well as in cancer patients. Currently emerging novel strategies for the overcoming of antifolate-resistance are presented. Finally, experimental evidence is provided that the identification and characterization of the molecular mechanisms of antifolate-resistance may prove instrumental in the future development of rationally-based novel antifolates and strategies that could conceivably overcome drug-resistance phenomena.     

Genetic diversity of folate profiles in seeds of common bean,lentil, chickpea and pea

Folates are water-soluble B vitamins and act as cofactors in many metabolic functions in the human body. Pulses have traditionally been considered as a good dietary source of folates. The objectives of this study were (1) to determine the concentration of folates in four cultivars each of common bean, lentil, chickpea and pea, and (2) to determine the effect of growing location on folate concentration. Six folate monoglutamates were quantified by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC–MS/MS). Total folate concentration ranged from 351 to 589 μg/100 g in chickpea, 165 to 232 μg/100 g in common bean, 136 to 182 μg/100 g in lentil, and 23 to 30 μg/100 g in pea. The 5-methyltetrahydrofolate (5-MTHF) and 5-formyltetrahydrofolate (5-FTHF) folates were most abundant in common bean, lentil and chickpea, whereas 5-MTHF and tetrahydrofolate (THF) were the predominant forms in pea. Significant differences were detected among cultivars for all folates across the pulses, except for 5,10-methenyltetrahydrofolate (5,10-MTHF) in lentil, 5-MTHF in chickpea, and 5,10-MTHF and folic acid (FA) in pea. Significant effects for location and cultivar by location were also observed for the majority of the folates.     

In vitro and in vivo targeting of different folate receptor-positive cancer cell lines with a novel 99mTc-radiofolate tracer

PURPOSE: For the assessment of folate-based radiopharmaceuticals, human nasopharyngeal KB carcinoma cells are traditionally used although nasopharyngeal cancer is rare. On the other hand, the folate receptor (FR) is frequently overexpressed on diverse cancer types, the highest frequency (>90%) being on ovarian carcinomas. The goal of our study was the in vitro and in vivo assessment of different FR-positive human carcinoma cells. In addition, a murine sarcoma cell line was assessed as a pre-clinical alternative to human xenograft models. METHODS: FR-positive human nasopharyngeal, cervical, ovarian and colorectal cancer cell lines and the transgenic mouse sarcoma (24JK-FBP) cell line were targeted with a novel 99mTc-tricarbonyl folate derivative 2. Comparative in vitro cell binding studies were carried out under standardised folate-deficient conditions. In vivo studies were performed in nude mice and C6 black mice. RESULTS: The in vitro cell experiments revealed only FR-specific binding (unspecific <0.02%), ranging from 3.5% to 52% of complex 2 owing to variable levels of FR expression of the cell lines. In vivo tumour uptake of radiotracer 2 varied less than in vitro. It ranged from 0.66+/-0.17% ID/g (LoVo) through 1.16+/-0.64% ID/g (IGROV-1) and 1.55+/-0.43% ID/g (24JK-FBP) to 2.33+/-0.36% ID/g (KB) 4 h p.i. CONCLUSION: These pre-clinical studies indicate that in vitro data obtained in FR-positive cancer cells do not necessarily correspond with or predict in vivo radiofolate uptake in corresponding (xeno)grafts. In addition, the murine 24JK-FBP cell line proved to be a valuable pre-clinical alternative to human tumour models.     

Cerebral folate deficiency: life-changing supplementation with folinic acid

Cerebral folate deficiency is characterized by low cerebrospinal fluid (CSF) concentrations of 5-methyltetrahydrofolate and a broad spectrum of clinical signs and symptoms. A patient with progressive spasticity, gait disturbance, speech difficulties, initially diagnosed as a recessive spastic paraplegia recovered on folinic acid (15-30 mg/day) and her 5-methyltetrahydrofolate in CSF normalized. This report demonstrates the importance of CSF investigation in the diagnosis of cerebral folate deficiency and efficiency of folinic acid (5-formyltetrahydrofolate) supplementation.     

Presence of methylenetetrahydrofolate reductase of American Leishmania species and its absence from Trypanosoma species

5,10-Methylenetetrahydrofolate reductase was studied in several American trypanosomatids; Leishmania spp., including L. braziliensis, L. mexicana, and L. garnhami. They displayed strong enzymatic activity. In contrast, Trypanosoma cruzi and T. rangeli under identical assay conditions showed no activity. The Leishmania enzyme has an optimum pH of 6.5, is dependent on flavin-adenine dinucleotide, is inhibited by S-adenosylmethionine, and has an apparent K_m value of 25 μM. Due to an excess of flavin-adenine dinucleotide in standard assay conditions, menadione was not an absolute requirement for the Leishmania enzyme assay, however with low concentrations of the coenzyme (5–20 μM), menadione significantly increased enzyme activity, although oxygen contributed about 20% to oxidation of reduced flavin-adenine dinucleotide. There were significant variations in the specific activity of methylenetetrahydrofolate reductase between the 10 different American Leishmania strains studied, but the meaning of these results is not clear because they showed no correlation either with taxonomy or infectivity.     

Disposition of leucovorin and its metabolites in the plasma,intestinal epithelium,and intraperitoneal L1210 cells of methotrexate-pretreated mice

Leucovorin (LV or 5-CHOFH₄) has had longstanding clinical use as a rescue agent from the systemic toxic effects of methotrexate (MTX). Because the mouse has been the animal model most used to investigate MTX therapy, direct tissue assessment of LV and its reduced-folate metabolites was undertaken in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of MTX-pretreated mice using a ternary-complex-based assay method. The results show that total folate accumulation and depletion in tissues is closely related to plasma levels, with somewhat greater persistence occurring in tissues, presumably due to polyglutamylation. Examination of individual folates in plasma showed that the combined 5,10-methylenetetrahydrofolate (CH₂FH₄) plus tetrahydrofolate (FH₄) pool was the most extensively elevated pool other than that of the parent compound [S]-5-formyltetrahydrofolate ([S]-5-CHOFH₄). The dihydrofolate (FH₂) also became elevated, whereas the 5-methyltetrahydrofolate (5-CH₃FH₄) remained unchanged. Individual folates that were elevated in tissues were generally the same as those elevated in plasma, the exception being a significant accumulation of 10-formyltetrahydrofolate (10-CHOFH₄) in both intestinal epithelial and L1210 cells. The elevation of FH₂ in L1210 cells was greater and persisted longer than that in intestinal epithelium, whereas the opposite was true for CH₂FH₄+FH₄. This differential effect in tumor versus epithelial tissue is consistent with the selective rescue of normal tissue by LV.Abbreviations MTX methotrexate - LV or 5-CHOFH₄, leucovorin or 5-formyltetrahydrofolate - FU 5-fluorouracil - FdUMP 5-fluoro-2-deoxyuridine-5-monophosphate - FH₂ dihydrofolate - FH₄ tetrahydrofolate - CH₂FH₄ 5,10-methylenetetrahydrofolate - 5-CH₃FH₄ 5-methyltetrahydrofolate - 10-CHOFH₄ 10-formyltetrahydrofolate This research was supported in part by ACS grant CH461A (to D.G.P.) and USPHS grants CA22754 (to D.G.P.), CA08748 (to F.M.S.), CA22764 (to F.M.S.), CA18856 (to F.M.S.) and CA46153 (to F.M.S.) awarded by the National Cancer Institute     

叶酸干预血管内皮功能障碍的研究进展

血管内皮功能障碍与诸多心血管疾病的发生、发展有密切联系,被认为是心血管疾病危险的终点替代指标。内皮功能障碍的研究和血管内皮的保护成为目前研究的热点。叶酸,一种经济无毒的药物,能明确改善血管内皮功能。其可能机制有降低血浆同型半胱氨酸、抗氧化作用、对内皮型一氧化氮合酶作用等,其对心血管疾病的预防具有重要意义。