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The binding mode of antineoplastic antimetabolite, floxuridine (FUDR), with human serum albumin (HSA), the leading carrier in blood circulation, was ascertained using multi-spectroscopic, microscopic, and computational techniques. A static fluorescence quenching was established due to decreased K sv values with rising temperatures, suggesting FUDR-HSA complexation. UV-vis absorption spectral results also supported this conclusion. The binding constant, K a values, were found within 9.7-7.9 × 10 3 M -1 at 290, 300, and 310 K, demonstrating a moderate binding affinity for the FUDR-HSA system. Thermodynamic data (ΔS = +46.35 J.mol -1 .K -1 and ΔH = -8.77 kJ.mol -1 ) predicted the nature of stabilizing forces (hydrogen-bonds, hydrophobic, and van der Waals interactions) for the FUDR-HSA complex. Circular dichroism spectra displayed a minor disruption in the protein's 2° and 3° structures. At the same time, atomic force microscopy images proved variations in the FUDR-HSA surface morphology, confirming its complex formation. The protein's microenvironment around Trp/Tyr residues was also modified, as judged by 3-D fluorescence spectra. FUDR-bound HSA showed better resistance against thermal stress. As disclosed from ligand displacement studies, the FUDR binding site was placed in subdomain IIA (Site I). Further, the molecular docking analysis corroborated the competing displacement studies. Molecular dynamics evaluations revealed that the complex achieved equilibrium during simulations, confirming the FUDR-HSA complex's stability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In cancer cells that need a lot of iron for growth and metastasis, halo-transferrin (TF-containing iron) enters the cell with the help of the transferrin receptor 1 (TFR1) protein. If the anticancer drug can bind to the iron site by interacting with apo-transferrin (iron-free FT), it can enter the cancer cell by the same mechanism. Two iron-related proteins, Bovine liver catalase (BLC) and apo-Transferrin (TF), that are important in cancer patients were selected and their interaction with the anti-cancer drug Floxuridine (FUDR) was investigated. Here, the protective role of FUDR was evaluated by several variables such as drug concentration, interaction time, and temperature-induced degradation of enzyme function. The results showed that the protective effect of the FUDR is greater in high concentrations (in 5 × 10 -5 M:1.78 % and 2.59 % after 24 and 48 h). The interaction of the FUDR with both proteins can reduce the intensity of the fluorescence emission by a static mechanism. The binding strength of the FUDR with both proteins was almost similar and with the order of 10 4 M -1 (K b  = 3.90 ± 0.41 × 10 4 M -1 for BLC-FUDR and 5.01 ± 0.36 × 10 4 M -1 for TF-FUDR at 310 K). The thermodynamic calculations (in agreement with the docking results) indicated that FUDR-protein complex formation was exothermic and the main binding forces in the binding process were van der Waals interactions and hydrogen bonds. Both fluorophores tryptophan (Trp) and tyrosine (Tyr) of both proteins had significant roles in fluorescence quenching and the interaction process, the polarity of their microenvironment changed. CD results showed that the secondary structure changes of TF are slightly more than BLC. Molecular docking showed that the binding of the FUDR to TF is very close to the Fe-specific site and is placed in the cavity among the wrapping domain, N-Terminal arm, and β-barrel in BLC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Reduced glutathione (GSH) plays an essential role in relieving oxidative insult from the generation of free radicals via normal physiological processes. However, GSH can be exploited by bacteria as a signalling molecule for the regulation of virulence. We describe findings arising from a serendipitous observation that when GSH and Escherichia coli were incubated with 5'fluorodeoxyuridine (FUdR)-synchronised populations of Caenorhabditis elegans, the nematodes underwent rapid death. Death was mediated by the production of hydrogen sulphide mainly through the action of tnaA, a tryptophanase-encoding gene in E. coli. Other Enterobacteriaceae species possess similar cysteine desulfhydrases that can catabolise l-cysteine-containing compounds to hydrogen sulphide and mediate nematode killing when worms had been pre-treated with FUdR. When colonic epithelial cell lines were infected, hydrogen sulphide produced by these bacteria in the presence of GSH was also able to inhibit ATP synthesis in these cells particularly when cells had been treated with FUdR. Therefore, bacterial production of hydrogen sulphide could act in concert with a commonly used genotoxic cancer drug to exert host cell impairment. Hydrogen sulphide also increases bacterial adhesion to the intestinal cells. These findings could have implications for patients undergoing chemotherapy using FUdR analogues that could result in intestinal damage., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

A novel theranostic probe called CX-B-DF is constructed for precise chemotherapy guided by near-infrared (NIR) fluorescence imaging. Moreover, the theranostic probe shows high cytotoxicity to cancer cells under dual activation (H 2 O 2 and TP), which causes the accuracy of drug release to be improved and the toxic side effects to be reduced.

The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α-L-rhamnosidase from Alternaria sp. L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2'-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α-L-rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells., (Copyright © 2019. Published by Elsevier Ltd.)

Automated attachment of chemotherapeutic drugs to oligonucleotides through phosphoramidite chemistry and DNA synthesis has emerged as a powerful technology in constructing structure-defined and payload-tunable oligonucleotide-drug conjugates. In practice, however, in vivo delivery of these oligonucleotides remains a challenge. Inspired by the systemic transport of hydrophobic payloads by serum albumin in nature, we report the development of a lipid-conjugated floxuridine homomeric oligonucleotide (LFU20) that "hitchhikes" with endogenous serum albumin for cancer chemotherapy. Upon intravenous injection, LFU20 immediately inserts into the hydrophobic cave of albumin to form an LFU20/albumin complex, which accumulates in the tumor by the enhanced permeability and retention (EPR) effect and internalizes into the lysosomes of cancer cells. After degradation, cytotoxic floxuridine monophosphate is released to inhibit cell proliferation., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ɣ H of curcumin heptadiene chain are closely positioned to the A 16 -H8 and A 17 -H8, while G 12 -H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery., Design: Gemcitabine metabolites were analysed in LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation., Results: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%., Conclusions: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Objective: To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining., Results: BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2'-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61-84%., Conclusion: An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.

Development of gemcitabine (GEM) nanocarriers as theranostic agents for pancreatic cancer chemotherapy has received extensive attention in recent years. A novel enzyme-sensitive albumin-based GEM delivery nanoplatform was developed in this research by simple conjugation of GEM to human serum albumin (HSA) via cathepsin B cleavable peptide GFLG and then complexing with near-infrared (NIR) dye IR780, forming a HSA-GEM/IR780 complex. The successful preparation of HSA-GEM/IR780 complex was confirmed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), UV-vis-NIR absorption spectra and fluorescent emission spectra. The in vivo performance of HSA-GEM/IR780 complex was carried out on BxPC-3 pancreatic tumor xenografted mice. As revealed by in vivo NIR imaging, HSA-GEM/IR780 exhibited enhanced accumulation and long-term retention in tumor tissues compared to free IR780. Meanwhile, compared to free GEM, the deamination of GEM nanovectors into inactive 2',2'-difluorodeoxyuridine (dFdU) can be greatly suppressed, while the concentration of the activated form of GEM (gemcitabine triphosphate, dFdCTP) was significantly increased in tumor tissue, thus exhibiting superior tumor inhibition activity with minimal side effects., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Antimetabolites are incorporated into DNA and RNA, affecting their function. Liquid-chromatography-mass-spectrometry (LC-MS-MS) permits the sensitive, selective analysis of normal nucleosides. The method was adapted to measure the incorporation of deoxyuridine, gemcitabine (difluorodeoxycytidine), its metabolite difluorodeoxyuridine (dFdU), and the novel compound fluorocyclopentenylcytosine (RX3117). DNA was degraded to its deoxynucleotides for quantification by LC-MS-MS, gradient chromatography on a Phenomenex prodigy-3-ODS with positive ionization. The range of deoxyuridine DNA-mis-incorporation varied nine-fold in 27 cell lines (leukemia, colon, ovarian, lung cancer). At low-folate conditions a 2.1-fold increase in deoxyuridine was observed. Global methylation (given as % 5-methyl-deoxycytidine) was comparable between the cell lines (4.6-6.5%). Exposure of A2780 cells to 1 μM gemcitabine (4 hours) resulted in 3.6 pmol gemcitabine/μg DNA, but in AG6000 cells (deoxycytidine-kinase-deficient) no incorporation was found. However, when A2780, AG6000, or CCRF-CEM cells were exposed to 100 μM dFdU we found it as gemcitabine, 20.5, 19.6, and 0.51 pmol gemcitabine/μg DNA, respectively. Preincubation of CCRF-CEM cells with cyclopentenyl-cytosine (a CTP-synthetase inhibitor) increased dFdU incorporation four-fold. Apparently dFdU is activated independently of deoxycytidine-kinase and possibly converted in-situ to dFdCMP. RX3117 was incorporated into both DNA and RNA (0.0037 and 0.00515 pmol/μg, respectively). In summary, a sensitive method to quantify the incorporation of gemcitabine, deoxyuridine, and RX-3117 was developed, which revealed that dFdU was incorporated into DNA as the parent compound gemcitabine.

Purpose: To describe concentration versus time profiles of capecitabine and its metabolites 5'-DFUR, 5'-DFCR and 5-FU, depending on tablet formulation and on frequent and/or relevant genetic polymorphisms of cytidine deaminase, dihydropyrimidine dehydrogenase, thymidylate synthase and methylenetetrahydrofolate reductase (MTHFR)., Methods: In 46 cancer patients on chronic capecitabine treatment, who voluntarily participated in the study, individual therapeutic doses were replaced on four consecutive mornings by the study medication. The appropriate number of 500 mg test (T) or reference (R) capecitabine tablets was given in randomly allocated sequences TRTR or RTRT (replicate design). Average bioavailability was assessed by ANOVA., Results: Thirty female and 16 male patients suffering from gastrointestinal or breast cancer (mean age 53.4 years; mean dose 1739 mg) were included. The T/R ratios for AUC0-t(last) and C max were 96.7 % (98 % CI 90.7-103.2 %) and 87.2 % (98 % CI 74.9-101.5 %), respectively. Within-subject variability for AUC0-t(last) and C max (coefficient of variation for R) was 16.5 and 30.2 %, respectively. Similar results were seen for all metabolites. No serious adverse events occurred. For the MTHFR C677T (rs1801133) genotype, an increasing number of 677C alleles showed borderline correlation with an increasing elimination half-life of capecitabine (p = 0.043)., Conclusions: The extent of absorption was similar for T and R, but the rate of absorption was slightly lower for T. While such differences are not considered as clinically relevant, formal bioequivalence criteria were missed. A possible, probably indirect role of the MTHFR genotype in pharmacokinetics of capecitabine and/or 5-FU should be investigated in further studies.

In this work, an in vitro liver model in a microfluidic device to imitate and detect prodrug metabolism was developed. A widely used prodrug capecitabine (CAP), which needs to be metabolized into active intermediate in the liver and then transformed into final effective drug in tumor cells, was selected as a model compound. The microfluidic device we exploited consists of a cell co-culture section, in which HepG2 cells were cultured to represent liver while MCF-7 cells were used to represent the tumor tissue, and an on-line solid phase extraction (SPE) section connecting to the ionization source of the ESI-Q-TOF mass spectrometer. The prodrug metabolism was realized and confirmed within this in vitro liver model as the intermediate product of the prodrug 5'-deoxy-5-fluorouridine (DFUR) was successfully detected with MS after the conditioning of HepG2 cells, and the anti-tumor effect of the active metabolite was observed through cell vitality assays of MCF-7 cells. The limit of detection (LOD) using on-chip SPE was at 10nM and semi-quantitative analysis could be realized. This system has been proved useful and practical, showing a potential to replace conventional drug screening methods., (Copyright © 2015 Elsevier B.V. All rights reserved.)

5-Fluorouracil (5-FU) and its oral prodrug capecitabine are among the most widely used chemotherapeutics. For cytotoxic activity, 5-FU requires cellular uptake and intracellular metabolic activation. Three intracellular formed metabolites are responsible for the antineoplastic effect of 5-FU: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). In this paper, we describe the development of an LC-MS/MS assay for quantification of these active 5-FU nucleotides in peripheral blood mononuclear cells (PBMCs). Because the intracellular 5-FU nucleotide concentrations were very low, maximization of the release from the cell matrix and minimization of interference were critical factors. Therefore, a series of experiments was performed to select the best method for cell lysis and nucleotide extraction. Chromatography was optimized to obtain separation from endogenous nucleotides, and the effect of different cell numbers was examined. The assay was validated for the following concentration ranges in PBMC lysate: 0.488-19.9 nM for FUTP, 1.66-67.7 nM for FdUTP and 0.748-30.7 nM for FdUMP. Accuracies were between -2.2 and 7.0% deviation for all analytes, and the coefficient of variation values were ≤ 4.9%. The assay was successfully applied to quantify 5-FU nucleotides in PBMC samples from patients treated with capecitabine and patients receiving 5-FU intravenously. FUTP amounts up to 3054 fmol/10(6) PBMCs and FdUMP levels up to 169 fmol/10(6) PBMCs were measured. The FdUTP concentrations were below the lower limit of quantification. To our knowledge, this is the first time that 5-FU nucleotides were quantified in cells from patients treated with 5-FU or capecitabine without using a radiolabel., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The goal of this study was to understand roles of nucleoside and nucleobase transport processes in capecitabine pharmacology in cells derived from human renal proximal tubule cells (hRPTCs) and three human renal cell carcinoma (RCC) cell lines, A498, A704, and Caki-1. Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) mediated activities and a sodium-independent nucleobase activity were present in hRPTCs. In hRPTCs, uptake of 5'-deoxy-5-fluorouridine (DFUR), a nucleoside metabolite of capecitabine, was pH dependent with highest uptake seen at pH 6.0. In RCC cell lines, hENT1 was the major nucleoside transporter. Nucleobase transport activity was variable among the three RCC cell lines, with Caki-1 showing the highest and A498 showing the lowest activities. Treatment of RCC cell lines with interferon alpha (IFN-α) increased thymidine phosphorylase levels and prior treatment of RCC cell lines with IFN-α followed by 5-FU or DFUR resulted in enhanced sensitivity of all cell lines to 5-FU and two of three cell lines to DFUR. We report for the first time a nucleobase transport activity in hRPTCs and RCC cell lines. In addition, our in vitro cytotoxicity results showed that RCC cell lines differed in their response to 5-FU and DFUR and prior treatment with IFN-α potentiated cytotoxic response to metabolites of capecitabine.

In this study we report a high sensitive method for the simultaneous analysis of LY2334737 (2'-deoxy-2',2'-difluoro-N-(1-oxo-2-propylpentyl)-cytidine), an amide prodrug of gemcitabine (2', 2'-difluoro-deoxycytidine), along with its active drug gemcitabine and its major metabolite dFdU (2',2'-difluoro-deoxyuridine) by LC-MS/MS. Quantification of all three analytes within a single analysis was challenging because the physio-chemical properties of LY2334737 were significantly different from gemcitabine and dFdU and was accomplished by incorporating column-switching. The assay was fully validated to quantify LY2334737 from 0.1 to 100ng/mL, gemcitabine from 0.25 to 100ng/mL and dFdU from 1 to 1000ng/mL in order to cover the diverse concentration ranges expected in clinical samples. A 25-fold dilution was also validated to accommodate any samples outside this range. Overall, the assay had good accuracy (ranging from -7.0 to 1.2% relative error) and precision (ranging from 2.1 to 8.4% relative standard deviation). Extraction efficiency was greater than 80% for all three analytes and there were no matrix effects. Plasma samples were stable for 24h at room temperature, 660 days in frozen storage, and at least 4 freeze-thaw cycles, at both -20 and -70°C. Data from clinical trials showed that plasma concentrations for LY2334737, gemcitabine, and dFdU were successfully quantified from a single LC-MS/MS analysis and that the assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis., (Copyright © 2013. Published by Elsevier B.V.)

Human DNA mismatch repair (MMR) recognizes and binds 5-fluorouracil (5FU) incorporated into DNA and triggers a MMR-dependent cell death. Absence of MMR in a patient's colorectal tumor abrogates 5FU's beneficial effects on survival. Changes in the tumor microenvironment like low extracellular pH (pHe) may diminish DNA repair, increasing genomic instability. Here, we explored if low pHe modifies MMR recognition of 5FU, as 5FU can exist in ionized and non-ionized forms depending on pH. We demonstrate that MMR-proficient cells at low pHe show downregulation of hMLH1, whereas expression of TDG and MBD4, known DNA glycosylases for base excision repair (BER) that can remove 5FU from DNA, were unchanged. We show in vitro that 5FU within DNA pairs with adenine (A) at high and low pH (in absence of MMR and BER). Surprisingly, 5FdU:G was repaired to C:G in hMLH1-deficient cells cultured at both low and normal pHe, similar to MMR-proficient cells. Moreover, both hMSH6 and hMSH3, components of hMutSα and hMutSβ, respectively, bound 5FU within DNA (hMSH6>hMSH3) but is influenced by hMLH1. We conclude that an acidic tumor microenvironment triggers downregulation of hMLH1, potentially removing the execution component of MMR for 5FU cytotoxicity, whereas other mechanisms remain stable to implement overall 5FU sensitivity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose: Multiple proteins are involved in activation and inactivation of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC). We aimed to clarify the mechanism of dFdC resistance in a pancreatic cancer cell line by applying a combination of targeted proteomic and metabolomic analyses., Methods: Twenty-five enzyme and transporter proteins and 6 metabolites were quantified in sensitive and resistant pancreatic cancer cell lines, PK9 and RPK9, respectively., Results: The protein concentration of deoxycytidine kinase (dCK) in RPK9 cells was less than 0.02-fold (2 %) compared with that in PK9 cells, whereas the differences (fold) were within a factor of 3 for other proteins. Targeted metabolomic analysis revealed that phosphorylated forms of dFdC were reduced to less than 0.2 % in RPK9 cells. The extracellular concentration of 2',2'-difluorodeoxyuridine (dFdU), an inactive metabolite of dFdC, reached the same level as the initial dFdC concentration in RPK9 cells. However, tetrahydrouridine treatment did not increase phosphorylated forms of dFdC and did not reverse dFdC resistance in RPK9 cells, though this treatment inhibits production of dFdU., Conclusions: Combining targeted proteomics and metabolomics suggests that acquisition of resistance in RPK9 cells is due to attenuation of dFdC phosphorylation via suppression of dCK.

Aim: The aim of this study was to evaluate the association of gemcitabine pathway SNPs with detailed pharmacokinetic measures obtained from solid tumor patients receiving gemcitabine-based therapy., Materials & Methods: SNPs within nine gemcitabine pathway genes, namely CDA, CMPK, DCK, DCTD, NT5C2, NT5C3, SLC28A1, SLC28A3 and SLC29A1 were analyzed for association with gemcitabine pharmacokinetics., Results: Significant association of gemcitabine clearance with SNPs in NT5C2 was identified. Clearance of 2´,2´-difluorodeoxyuridine, a gemcitabine metabolite was significantly predicted by CDA, SLC29A1 and NT5C2 SNPs. This study reports an association of formation clearance of 2´,2´-difluoro-2´-deoxycytidine triphosphate, an active form of gemcitabine with SNPs within uptake transporters SLC28A1, SLC28A3 and SLC29A1., Conclusion: Genetic variation in gemcitabine pathway genes is associated with its pharmacokinetics and hence could influence gemcitabine response. Our study identified pharmacogenetic markers that could be further tested in larger patient cohorts and could open up opportunities to individualize therapy in solid tumor patients.

This work describes an efficient, simple, and green bioprocess for obtaining 5-halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5-fluoro-2'-deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L(-1) min(-1) (60%) of 5-chloro-2'-deoxyuridine in 3 h. These halogenated pyrimidine 2'-deoxynucleosides are used as antitumoral agents., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

Purpose: To determine the correlations/discrepancies of drug stabilities between in the homogenates of human culture cells and of human tissues., Methods: Amino acid/dipeptide monoester prodrugs of floxuridine were chosen as the model drugs. The stabilities (half-lives) of floxuridine prodrugs in human tissues (pancreas, liver, and small intestine) homogenates were obtained and compared with ones in cell culture homogenates (AcPC-1, Capan-2, and Caco-2 cells) as well as human liver microsomes. The correlations of prodrug stability in human small bowel tissue homogenate vs. Caco-2 cell homogenate, human liver tissue homogenate vs. human liver microsomes, and human pancreatic tissue homogenate vs. pancreatic cell, AsPC-1 and Capan-2, homogenates were examined., Results: The stabilities of floxuridine prodrugs in human small bowel homogenate exhibited the great correlation to ones in Caco-2 cell homogenate (slope = 1.0-1.3, r2 = 0.79-0.98). The stability of those prodrugs in human pancreas tissue homogenate also exhibited the good correlations to ones in AsPC-1 and Capan-2 cells homogenates (slope = 0.5-0.8, r2 = 0.58-0.79). However, the correlations of prodrug stabilities between in human liver tissue homogenates and in human liver microsomes were weaker than others (slope = 1.3-1.9, r2 = 0.07-0.24)., Conclusions: The correlations of drug stabilities in cultured cell homogenates and in human tissue homogenates were compared. Those results exhibited wide range of correlations between in cell homogenate and in human tissue homogenate (r2 = 0.07 - 0.98). Those in vitro studies in cell homogenates would be good tools to predict drug stabilities in vivo and to select drug candidates for further developments. In the series of experiments, 5'-O-D-valyl-floxuridine and 5'-O-L-phenylalanyl-L-tyrosyl-floxuridine would be selected as candidates of oral drug targeting delivery for cancer chemotherapy due to their relatively good stabilities compared to other tested prodrugs.

Gemcitabine is an established chemotherapy agent in several solid tumors. Its mechanism of action has been theoretically established and this is supported with strong experimental evidence. However, certain aspects of the resistance mechanism for this agent remain elusive. We present a method of analysis using tandem liquid chromatography and mass spectrometry that provides a broader, yet more focused view of the action of gemcitabine and its primary metabolite, difluorodeoxyuridine in relation to the (deoxy) nucleoside and (deoxy) nucleotide pools in tumor cell lines. Alcoholic cytosole extracts were incubated with alkaline phosphatase reducing the nucleotide pools to their respective nucleosides. Determination of the nucleoside content by a sensitive LCMSMS method before and after incubation enables the calculation of the total amount of phosphorylation of each (deoxy) nucleoside in the cell. Incubation with clinically relevant levels of gemcitabine (dFdC) or difluorodeoxyuridine (dFdU) for 24 hours enabled the determination of the changes in the (deoxy) nucleotide pools in relation to chemotherapeutic and toxicological effects. Confirmation of the presence of dFdC phosphorylation is presented as well as direct evidence of dFdU phosphorylation after both dFdC and dFdU treatment. Differences in the nucleotide pools are presented after dFdC and dFdU incubation, indicating that dFdU might have more chemotherapeutic properties than previously believed.

Purpose: To develop a sensitive analytical method to quantify gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolites 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) simultaneously from tumour tissue., Methods: Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer (KP ( FL/FL ) C and KP ( R172H/+) C) was collected after dosing the mice with gemcitabine. (19)F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue., Results: A (19)F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC-MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour., Conclusion: We have developed a sensitive LC-MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays.

Gemcitabine (dFdC) is a chemotherapeutic nucleoside analog that undergoes uptake via equilibrative nucleoside transporters (hENT) followed by sequential phosphorylation to the active triphosphate moiety (dFdCTP). Its deaminated metabolite, 2',2'-difluorodeoxyuridine (dFdU), competes with the parent compound for cellular entry via hENTs, but over time dFdU increases the net intracellular accumulation of dFdC by a currently unknown mechanism. In this study, we investigated whether dFdU affects intracellular phosphorylation of gemcitabine by modulating the activity of deoxycytidine kinase (dCK). We report here that coincubation of dFdU with dFdC significantly increases intracellular levels of dFdCTP. dFdCTP was not identified as a substrate for hENTs, suggesting that dFdU affects the formation rather than elimination of the triphosphate. To further characterize the disposition of dFdC in the presence of dFdU, the net intracellular radioactivity of [5-(3)H]dFdC and corresponding metabolic profile were evaluated in HeLa cells transfected with dCK-targeting small interfering RNA. Intracellular radioactivity significantly decreased in cells with compromised intracellular phosphorylation, which was mainly due to a loss in dFdCTP. Although dFdU increased the net intracellular radioactivity of [5-(3)H]dFdC at 24 h in control cells, this increase was abolished in the absence of dCK activity, strongly suggesting that the interaction between dFdU and dFdC occurs via modulation of both transport and metabolism. In conclusion, we have demonstrated that the intracellular distribution of dFdC is dependent on both transport and metabolic processes, and that by affecting the rate at which dFdC enters the cell, the presence of dFdU may be altering the metabolic fate of the parent compound (dFdC).

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil-DNA glycosylase UNG is the major route for FU-DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil-DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU-DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.

A novel phosphoramidate nucleotide prodrug of the anticancer nucleoside analogue 5-fluoro-2'-deoxyuridine (5-FdUrd) was synthesized and evaluated for its cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, NUC-3073 markedly kept its antiproliferative activity in TK-deficient tumour cells, and thus is largely independent of intracellular TK activity to exert its cytostatic action. NUC-3073 was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. NUC-3073 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with our observations that 5-FdUrd, but not NUC-3073, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, we propose NUC-3073 as a novel 5-FdUrd phosphoramidate prodrug that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP which is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Homodimeric thymidine phosphorylase from Escherichia coli (TP, E.C. 2.4.2.4) was immobilized on solid support with the aim to have a stable and recyclable biocatalyst for nucleoside synthesis. Immobilization by ionic adsorption on amine-functionalized agarose and Sepabeads(®) resulted in a very high activity recovery (>85%). To prevent undesirable leakage of immobilized enzyme away from the support, the ionic preparations were cross-linked with aldehyde dextran (MW 20 kDa) and the influence of the dextran oxidation degree on the resulting biocatalyst activity was evaluated. Although in all cases the percentage of expressed activity after immobilization drastically decreased (≤ 25%), this procedure allowed to obtain an active catalyst which resulted up to 6-fold and 3-fold more stable than the soluble (non immobilized) enzyme and the just adsorbed (non cross-linked) counterpart, respectively, at pH 10 and 37°C. No release of the enzyme from the support could be observed. Covalent immobilization on aldehyde or epoxy supports was generally detrimental for enzyme activity. Optimal TP preparation, achieved by immobilization onto Sepabeads(®) coated with polyethyleneimine and cross-linked, was successfully used for the one-pot synthesis of 5-fluoro-2'-deoxyuridine starting from 2'-deoxyuridine or thymidine (20mM) and 5-fluorouracil (10mM). In both cases, the reaction proceeded at the same rate (3 μmol min(-1)) affording 62% conversion in 1h., (Copyright © 2011 Elsevier Inc. All rights reserved.)

dUTPase is involved in preserving DNA integrity in cells. We report an efficient dUTPase silencing by RNAi-based system in stable human cell line. Repression of dUTPase induced specific expression level increments for thymidylate kinase and thymidine kinase, and also an increased sensitization to 5-fluoro-2'-deoxyuridine and 5-fluoro-uracil. The catalytic mechanism of dUTPase was investigated for 5-fluoro-dUTP. The 5F-substitution on the uracil ring of the substrate did not change the kinetic mechanism of dUTP hydrolysis by dUTPase. Results indicate that RNAi silencing of dUTPase induces a complex cellular response wherein sensitivity towards fluoropyrimidines and gene expression levels of related enzymes are both modulated.

5-Fluoro-2'-deoxyuridine (floxuridine, 5-FdUrd) and 5-fluorouracil (5-FU) are widely used for the treatment of colorectal cancers. The mechanisms of action of 5-FdUrd and 5-FU, as well as the biochemical pathway responsible for their metabolism, are well understood. Identification of every metabolite and achieving mass balance by conventional UV absorption-based HPLC analysis are not feasible because the metabolites beyond 5-FU in the 5-FdUrd metabolic pathway are undetectable by UV light. We therefore established a mass spectrometry method, designed for fast and convenient analysis, for simultaneously measuring 5-FdUrd, 5-FU, and their metabolites. Linearity, precision and accuracy were validated in the concentration ranges studied for each compound. Hydrolysis studies of 5-FdUrd and amino acid mono ester prodrugs of 5-FdUrd in Capan-2 cell homogenates were carried out and the achievement of mass balance was established with this method (recovery of 5'-O-l-leucyl-FdUrd was 96.6-108.2% and that of 5-FdUrd was 79.4-117.4%). This simple LC-MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts., Methods: The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation., Results: FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis., Conclusion: We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

5'-O-beta-D-Galactosyl-floxuridine, a potential novel prodrug, was synthesized with a yield of 75% through beta-galactosidase-catalyzed transgalactosylation. This enzyme displayed absolute regioselectivity toward the 5'-position of floxuridine. For the reaction, the optimal conditions were pH 6.5 at 45 degrees C for 60 h with floxuridine to o-nitrophenyl-beta-D-galactoside at 2:1 (mol/mol). Under these conditions, the initial reaction rate and the maximum yield were 0.28 mM h(-1) and 75%, respectively.

The survival and proliferation of the obligate intracellular malaria parasite Plasmodium falciparum require salvage of essential purines from the host. Genetic studies have previously shown that the parasite plasma membrane purine permease, PfNT1, plays an essential function in the transport of all naturally occurring purine nucleosides and nucleobases across the parasite plasma membrane. Here, we describe an intracellular permease, PfNT2. PfNT2 is, like PfNT1, a member of the equilibrative nucleoside transporter family. Confocal and immunoelectron microscopic analyses of transgenic parasites harboring green fluorescent protein- or hemagglutinin-tagged PfNT2 demonstrated endoplasmic reticulum localization. This localization was confirmed by colocalization with the endoplasmic reticulum marker PfBiP. Using yeast as a surrogate system, we show that targeting PfNT2 to the plasma membrane of fui1Delta cells lacking the plasma membrane nucleoside transporter Fui1 confers sensitivity to the toxic nucleoside analog 5-fluorouridine. This study provides the first evidence of an intracellular purine permease in apicomplexan parasites and suggests a novel biological function for the parasite endoplasmic reticulum during malaria infection.

The goal of this investigation was to evaluate the effects of structural differences between FIAU and FIRU on their ability to serve as a potential tracer for reporter gene imaging. To examine the characteristics of different configurations of FIAU and FIRU, a series of evaluations were done on HSV1-TK gene-expressing cells and on mice with HSV1-TK gene-expressing tumor. The results showed that, as an imaging agent for HSV1-TK-expressing cells, radiolabeled FIAU was more efficient for in vivo imaging than FIRU., (Copyright 2009 Elsevier Ltd. All rights reserved.)

We have investigated the molecular mechanisms regulating the necrosis and apoptosis that occur on treatment of mouse mammary tumor FM3A cells with 5-fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, using the original clone F28-7 and its variant F28-7-A cells. Previously, we reported an interesting observation that FUdR induces a necrotic morphology in F28-7 but an apoptotic morphology in F28-7-A cells. We have now analyzed the protein expression profiles of these FUdR-induced necrosis and apoptosis. Thus, proteome analysis of these clones by two-dimensional gel electrophoresis and mass spectrometry showed that the cytoplasmic intermediate filament protein, cytokeratin-19, is expressed at a significantly higher level in F28-7 than in F28-7-A cells. This strong expression was detected both in untreated and FUdR-treated stages of F28-7 cells. We interpreted this phenomenon as suggesting that cytokeratin-19 possesses a function in leading the cell to apoptosis. We performed a knockdown of cytokeratin-19 expression in F28-7 cells by use of the small interfering RNA technique. Indeed, a lowering of the cytokeratin-19 expression down to the level in F28-7-A occurred, and the FUdR-induced death morphology of this knockdown F28-7 was apoptosis, instead of the necrosis usually observable in the FUdR-treated F28-7. It is known that the cytoskeletal protein cytokeratin-19 undergoes caspase-mediated degradation during apoptosis. Our present finding provides an interesting possibility that cytokeratin-19 may have a key role in regulating cell-death morphology.

Purpose: To examine the role of DNA repair and altered thymidine analogues in altering the response to radiation during thymidine deprivation., Methods and Materials: Mismatch repair-deficient and -proficient cell lines HEC59 and HC-2.4 were treated with fluorodeoxyuridine (FUdR), azidothymidine (AZT), and irradiation either alone or in combination, and outcomes of clonogenic survival and cell-cycle distributions were determined., Results: Survival outcomes for all treatments were similar for both cell lines, suggesting that hMSH2 does not significantly influence thymidine deprivation toxicity or radiosensitization. The chain-terminating thymidine analogue AZT increased the toxicity of FUdR and increased DNA fragmentation. The combination of FUdR and AZT afforded greater radiosensitization than either drug alone. Drug enhancement ratios, the degree of excess radiation-induced cell death in drug-treated cultures compared with radiation alone for HEC59, were 1.2, 1.4, and 1.8 for AZT, FUdR, and the combination, respectively. Enhancement ratios for HC-2.4 were 1.3, 1.5, and 1.8 for AZT, FUdR, and the combination, respectively., Conclusion: Azidothymidine, a chain-terminating thymidine analogue, can enhance the radiosensitizing affects of thymidine deprivation. Deoxyribonucleic acid strand breaks may play an important role in the mechanism of thymidine deprivation-induced radiosensitization., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2'-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2'-deoxyuridine-5'-monophosphate (dUMP)/thymidine-5'-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5microm, (100mmx2.1mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5-150ngmL(-1) according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at -20 degrees C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.

Unexpected inversion of the 3':5'-regioselectivity was observed in the enzymatic methacryloylation, crotonylation and cinnamoylation of floxuridine (1.5:1, 2.3:1 and 4.4:1, respectively), where Thermomyces lanuginosus lipase preferentially catalyzed the acylation of 3'-hydroxyl rather than that of 5'-hydroxyl group. The possible reason might be the presence of a remote interaction between the unsaturated bond in the acyl group and the aromatic ring of amino acid residue Trp89 in the lid of the lipase.

Among the five vinyl esters bearing phenyl ring in acyl moiety, the lipase from Burkholderia cepacia was most specific toward vinyl 4-phenylbutyrate. Additionally, 3'-regioselectivity of the enzyme was enhanced with the increment of acyl chain length. The lipophilic 3'-arylaliphatic acid esters of floxuridine, its potential prodrugs, were synthesized with high conversions (>98%) and good to excellent regioselectivities (85-99%) via the enzymatic approach.

Pseudomonas cepacia lipase-catalysed preferential acylation of the secondary hydroxy group of FUdR (floxuridine) with vinyl crotonate was carried out in spite of the presence of the primary hydroxy group, and 3'-O-crotonylfloxuridine was prepared successfully for the first time. The isomerization of the double bond of crotonate, which occurs in conventional organic synthesis, could be effectively avoided during the enzymatic acylation. The effects of some key factors such as reaction medium, initial a(w) (water activity), molar ratio of vinyl crotonate to FUdR, FUdR concentration and reaction temperature on the reaction were examined. Under the optimized reaction conditions, the initial reaction rate, substrate conversion and 3'-regioselectivity of the reaction were as high as 24 mM/h, 98% and 85% respectively.

Dipeptide monoester prodrugs of floxuridine were synthesized, and their chemical stability in buffers, resistance to glycosidic bond metabolism, affinity for PEPT1, enzymatic activation and permeability in cancer cells were determined and compared to those of mono amino acid monoester floxuridine prodrugs. Prodrugs containing glycyl moieties were the least stable in pH 7.4 buffer ( t 1/2 < 100 min). The activation of all floxuridine prodrugs was 2- to 30-fold faster in cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of dipeptide monoester prodrugs containing aromatic promoieties in cell homogenates was 5- to 20-fold slower than that of other dipeptide and most mono amino acid monoester prodrugs ( t 1/2 approximately 40 to 100 min). All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent floxuridine. In general, the 5'-O-dipeptide monoester floxuridine prodrugs exhibited higher affinity for PEPT1 than the corresponding 5'-O-mono amino acid ester prodrugs. The permeability of dipeptide monoester prodrugs across Caco-2 and Capan-2 monolayers was 2- to 4-fold higher than the corresponding mono amino acid ester prodrug. Cell proliferation assays in AsPC-1 and Capan-2 pancreatic ductal cell lines indicated that the dipeptide monoester prodrugs were equally as potent as mono amino acid prodrugs. The transport and enzymatic profiles of 5'- l-phenylalanyl- l-tyrosyl-floxuridine, 5'- l-phenylalanyl- l-glycyl-floxuridine, and 5'- l-isoleucyl- l-glycyl-floxuridine suggest their potential for increased oral uptake, delayed enzymatic bioconversion and enhanced resistance to metabolism to 5-fluorouracil, as well as enhanced uptake and cytotoxic activity in cancer cells, attributes that would facilitate prolonged systemic circulation for enhanced therapeutic action.

The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.145) has a high turnover rate and a wide substrate range that makes it a very good candidate for gene therapy. This concept is based on introducing a suicide gene into malignant cells in order to activate a prodrug that eventually may kill the cell. To be able to optimize the function of dNK, it is vital to have structural information of dNK complexes. In this study we present crystal structures of dNK complexed with four different nucleoside analogs (floxuridine, brivudine, zidovudine and zalcitabine) and relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside analogs investigated bound in a manner similar to that of the pyrimidine substrates, with many interactions in common. In contrast, the base of dGTP adopted a syn-conformation to adapt to the available space of the active site.

Background: The antipoxviral activities and phosphorylation of N-methanocarbathymidine ([N]-MCT) and four 5-halo-2'-deoxyuridines, namely 5-fluoro-(FdU), 5-chloro-(CldU), 5-bromo-(BrdU), and 5-iodo-(IdU) derivatives, were explored., Methods: Antiviral activities and nucleoside metabolism were determined in C127I mouse, LLC-MK2 monkey, and A549 human cells infected with thymidine-kinase-containing and -deficient (TK+ and TK-) vaccinia (WR strain) viruses., Results: The antiviral potencies of CldU, BrdU and IdU were increased 16-26-fold in LLC-MK2 cells infected with TK+ compared with TK- virus infections, but enhancement of activity was much less in the other cell lines. (N)-MCT was nearly equally active against TK+ and TK- viruses in the three cell lines. Antiviral activity of FdU was associated with cytotoxicity. Uninfected and infected cells metabolized compounds to mono-, di- and triphosphates. The thymidine, BrdU and IdU triphosphate levels were higher in C127I and LLC-MK2 cells infected with TK+ than with TK- virus. (N)-MCT monophosphate levels were much higher in TK+ virus-infected cells, but without corresponding increases in (N)-MCT triphosphate. Furthermore, TK+ virus infections did not appreciably alter (N)-MCT triphosphate levels in other mouse (L929), monkey (MA-104 and Vero) and human cell lines (A549). Antiviral potency of the compounds was greater in C127I than in LLC-MK2 cells, yet lower intracellular triphosphate levels were found in C127I cells., Conclusion: We conclude that viral TK plays an important role in increasing the antiviral potencies of these compounds in some cell lines, but minimally in others. These findings may have implications in treating infected animals with compounds that are dependent upon poxvirus TK for their activation, because viral TK activity may vary greatly due to cell type.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) are often upregulated in solid tumors and catalyze the phosphorolysis of natural (deoxy)nucleosides and a wide variety of fluorinated pyrimidine nucleosides. Because the relative contribution of each of the two enzymes to these reactions is still largely unknown, we investigated the substrate specificity of TP and UP in colon cancer cells for the (fluoro)pyrimidine nucleosides thymidine (TdR), uridine (Urd), 5'-deoxy-5-fluorouridine (5'DFUR), and 5FU. Specific inhibitors of TP (TPI) and UP (BAU) were used to determine the contribution of each enzyme in relation to their cytotoxic effect. The high TP expressing Colo320TP1 cells were most sensitive to 5'DFUR and 5FU, with IC50 values of 1.4 and 0.2 microM, respectively, while SW948 and SW1398 were insensitive to 5'DFUR (IC50>150 microM for 5'DFUR). TPI and BAU only moderately affected sensitivity of Colo320, SW948, and SW1398, whereas TPI significantly increased IC(50) for 5'DFUR (50-fold) and 5FU (11-fold) in Colo320TP1 and BAU that in C26A (9-fold for 5'DFUR; p<0.01). In the epithelial skin cell line HaCaT both inhibitors were able to decrease sensitivity to 5'DFUR and 5FU separately. HaCaT might be a model for 5'DFUR toxicity. In the colon cancer cells 5'DFUR degradation varied from 0.4 to 50 nmol 5FU/h/10(6)cells, that of TdR from 0.3 to 103 nmol thymine/h/10(6)cells, that of Urd from 0.8 to 79 nmol uracil/h/10(6)cells, while conversion of 5FU to FUrd was from 0.3 to 46 nmol/h/10(6)cells. SW948 and SW1398 were about equally sensitive to 5'DFUR and 5FU, but SW1398 had higher phosphorylase activity (>65-fold) compared to SW948. In SW948 and HaCaT TPI and BAU inhibited TdR and Urd phosphorolysis (>80%), respectively. Both TP and UP contributed to the phosphorolysis of 5'DFUR and 5FU. In the presence of both inhibitors, still phosphorolysis of 5FU (>40%) was detected in the tumor and HaCaT cell lines, and remarkably, that of all four substrates in SW1398 cells. 5'DFUR phosphorolysis was also measured in situ, where Colo320TP1, SW1398, and HaCaT cells produced significant amounts 5FU from 5'DFUR (>10 nmol/24h/10(6)cells). In Colo320TP1 and in HaCaT cells TPI completely prevented 5FU production, but not in SW1398 cells, where BAU decreased this by 67% (p<0.01). High uracil and dUrd levels were detected in the medium. Uracil accumulation was heavily reduced in the presence of TPI for Colo320TP1 and HaCaT cells, whereas 5FU-induced dUrd production by these cell lines increased (p<0.01). In contrast, for SW1398 cells only BAU was able to reduce uracil levels, and dUrd production remained unchanged. In conclusion, overlapping substrate specificity was found for TP and UP in the cell lines, in which both enzymes were responsible for converting TdR and Urd, and 5'DFUR. 5'DFUR and 5FU were converted to their products in both the colon cancer cells and keratinocytes.

Nucleoside analogues are broadly used in cancer treatment. Although nucleoside metabolism is a necessary step in the development of their cytotoxicity, mediated transport across the plasma membrane might be needed for nucleoside-derived drugs to exert their pharmacological action. In this study, we have addressed the question of whether particular plasma membrane transporters contribute to the transcriptomic response associated with nucleoside-derived drug therapy. Firstly, we have characterized the nucleoside transporters responsible for 5'-DFUR uptake into the breast cancer cell line MCF7. 5'-DFUR is the immediate precursor of 5-FU and a metabolite of the orally administered pro-drug capecitabine, currently used in the treatment of breast cancer and other solid tumors. Although 5'-DFUR is a substrate for both plasma membrane equilibrative nucleoside carriers, hENT1 shows higher affinity for this molecule than hENT2. Inhibition of hENT1 function partially protected MCF7 cells from 5'-DFUR-induced cytotoxicity. Secondly, we have used a pharmacogenomic approach to determine how inhibition of hENT1 function contributes to the transcriptomic response associated to 5'-DFUR treatment. Under hENT1 inhibition most of the transcriptional targets of 5'-DFUR action, which were genes associated with apoptosis and cell cycle progression were blocked. This study demonstrates that although 5'-DFUR is substrate for both equilibrative nucleoside carriers, hENT1 function is essential for the full transcriptional response to 5'-DFUR treatment.

Uridine phosphorylase (UPase) is an enzyme that converts the pyrimidine nucleoside uridine into uracil. Upon availability of ribose-1-phosphate, UPase can also catalyze the formation of nucleosides from uracil as well as from 5-fluorouracil, therefore involved in fluoropyrimidine metabolism. UPase gene expression is strictly controlled at the promoter level by oncogenes, tumor suppressor genes, and cytokines. UPase activity is usually elevated in various tumor tissues, including breast cancer, compared to matched normal tissues and this induction appears to contribute to the therapeutic efficacy of fluoropyrimidines in cancer patients. In this review, we will discuss in detail the role of UPase in the activation of fluoropyrimidines and its effect on the prognosis of breast cancer patients.

Chemoresistance is a biological response of cells to survive toxic stress. During cancer treatment the development of chemoresistance is a major problem. The mechanisms how cells become insensitive, and which downstream pathways are affected are not completely understood. Since it has not been well analysed which and how many regulative disorders are subsummised under the term "chemoresistance", we examined and measured arabinosylcytosine (AraC)-mediated desensitation of two mechanisms relevant for tissue homeostasis, cell cycle inhibition and apoptosis induction. MCF-7 cells harbouring ectopic mutated p53 were suitable for this investigation because they activated these mechanisms subsequently and became insensitive to AraC with regard to cell cycle inhibition and apoptosis induction. The major causal mechanism of acquired resistance against AraC was most likely through the inhibition of the first step of AraC phosphorylation within the cell, which is rate limiting for its activation. With regard to cell cycle inhibition AraC-resistant cells were also resistant against 5-fluorodeoxyuridine (5-FdUrd), but fully responsive to 5-FdUrd-induced apoptosis, evidencing that cell cycle and apoptosis are independent of each other. Apoptosis correlated with AIF-activation and was independent of Caspase 7, whereas cell cycle inhibition correlated with cyclinD1 expression but not with induction of p21 or p27. The phosphate conjugated 5-FdUrd-araC heterodimer (5-Fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine), which is a prodrug of AraC-monophosphate, reactivated AIF and down-regulated cyclin D1 in AraC-resistant cells and circumvented resistance to apoptosis and to cell cycle inhibition. Also, cells which were resistant to 5-FdUrd or doxorubicin were sensitive to 5-FdUrd-araC. This investigation demonstrates that chemoresistance affects apoptosis induction and cell cycle inhibition independently and that detailed knowledge about the affected downstream pathways would enable the design of targeted intervention with small molecules to restore chemosensitivity.

Amino acid ester prodrugs of antiviral and anticancer nucleoside drugs were developed to improve oral bioavailability or to reduce systemic toxicity. We studied the interaction of human concentrative nucleoside transporter (hCNT2) cloned from intestine with various amino acid ester prodrugs of floxuridine (FUdR) and 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB). Na(+)-dependent uptakes of [(3)H]-inosine and [(3)H]-adenosine were measured in U251 cells transiently expressing intestinal hCNT2. FUdR significantly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine (60-70% of control), while its amino acid ester prodrugs including Val, Phe, Pro, Asp, and Lys esters exhibited markedly decreased inhibition potency (10-30% of control). On the other hand, BDCRB and its amino acid prodrugs markedly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine. Val, Phe, and Pro ester prodrugs of BDCRB showed similar inhibition capacities as parent compound BDCRB (80-90% for adenosine and 60-80% for inosine). The amino acid site of attachment (3'- and 5'-monoesters) and stereochemistry (L- and D-amino acid esters), did not significantly affect the uptake of [(3)H]-inosine and [(3)H]-adenosine. These results demonstrate that the hCNT2 favorably interacts with BDCRB and its amino acid prodrugs, compared to those of FUdR, and that neutral amino acid esters of BDCRB have a high affinity toward this transporter. Therefore, the intestinal hCNT2 may be a target transporter as a factor for modulating oral pharmacokinetics of BDCRB prodrugs.