A 5-fluorodeoxyuridine prodrug as targeted therapy for prostate cancer

A method for targeted delivery of the cytotoxic agent 5-fluorodeoxyuridine (FudR) (1) to sites of metastatic prostate cancer is described. The prodrug was synthesized by coupling the active drug (FudR) to the PSA-peptide via a self-cleaving diamino acid linker to produce HSSKLQ-Leu-Aib-FudR. This prodrug serves as a substrate for prostate specific antigen (PSA). This approach permitted efficient conversion of the inactive prodrug back to the active cytotoxic state by the enzymatic activity of PSA which is highly expressed by prostate cells.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Reductive activation of 5-fluorodeoxyuridine prodrug possessing azide methyl group by hypoxic X-irradiation

We prepared a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing azide methyl group (N(3)-FdUrd) as a novel radiation-activated prodrug. The parent antitumor agent, 5-FdUrd, was released efficiently from N(3)-FdUrd by hypoxic X-irradiation. On the other hand, the activation of N(3)-FdUrd was suppressed upon X-irradiation under aerobic conditions. A biological assay using A549 cells revealed that the cytotoxicity of N(3)-FdUrd was significantly enhanced by hypoxic X-irradiation.     

Checkpoint signaling, base excision repair, and PARP promote survival of colon cancer cells treated with 5-fluorodeoxyuridine but not 5-fluorouracil

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor.     

Hypoxia-selective activation of 5-fluorodeoxyuridine prodrug possessing indolequinone structure: radiolytic reduction and cytotoxicity characteristics

We synthesized a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing an indolequinone structure (IQ-FdUrd) to characterize the radiolytic reduction in aqueous solution and the radiation-activated cytotoxicity against EMT6/KU cells under hypoxic conditions. IQ-FdUrd released antitumor agent 5-FdUrd upon hypoxic, but not aerobic, irradiation with the G value of 0.38 x 10(-7) mol J(-1). Laser flash photolysis of IQ-FdUrd in Ar-purged aqueous solution with dimethylaniline as an electron donor gave rise to a transient absorption spectrum characteristic of semiquinone radical anion, which decayed via second order kinetics. It is most likely that bimolecular disproportionation of intermediate semiquinone radicals occurs to release 5-FdUrd. IQ-FdUrd showed enhanced cytotoxicity against EMT6/KU cells in a radiation dose-dependent manner upon hypoxic irradiation. IQ-FdUrd is potentially a prototype compound for new class of radiation-activated antitumor prodrugs that are useful for radiation treatment of hypoxic tumors.     

Synthesis of a fluorescent analogue of methotrexate lipophilic prodrug

A fluorescent analogue of the lipophilic prodrug of antitumor agent methotrexate has been synthesized. The conjugate consists of a residue of rac-1-[13-(Me₄-BODIPY-8)tridecanoyl]-2-oleoylglycerol connected to methotrexate by an ester bond via β-Ala-N-carbonylmethylene linker (Me₄-BODIPY-8 stands for 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl). The probe is designed for incorporation in the membrane of the liposomal vehicle to study a mechanism of interaction with tumor cells and intracellular traffic.     

Mechanism of resistance of Pediococcus cerevisiae strains to 5-fluorodeoxyuridine.

The kinetics of the reaction of OH radicals with ferricytochrome c was studied in the time range 1 microsecond to 1 s by means of pulse radiolysis. The OH radicals reduce ferricytochrome c by 40% +/- 10%. The time course of the reduction is explained by a mechanism whereby a radical formed after hydrogen has been abstracted from the outer surface of the protein reduces the iron by electron tunnelling. We have calculated that the reducing electron in the radical is bound with an energy of at least 1.75 eV and that the frequency factor of the tunnelling process is v=10(11.5)s-1. This model accounts for the observed absorbance change in time range 5 . 10(-6)--10(-1)s. The time course of the reduction of ferricytochrome c by H radicals (Lichtin, N.N., Shafferman A. and Stein, G. (1974) Biochim. Biophys. Acta 357, 386--398) is explained by the same model.     

Selective transfer of individual human chromosomes to recipient cells.

Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt). Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca. 10(-6) and contained mostly single, but occasionally multiple, copies of the plasmid sequences. These transformants actively express the Eco gpt marker. Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells. The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization. This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background. The complete library will represent the entire human karyotype.     

Toxoplasma gondii: the enzymic defect of a mutant resistant to 5-fluorodeoxyuridine.

We have previously described a mutant of Toxoplasma gondii that was 100-fold more resistant to 5-fluorodeoxyuridine, as measured by growth in human fibroblast cultures. Various pyrimidine salvage enzymes were measured in the wild type and the mutant parasites to determine the biochemical basis for resistance to fluorodeoxyuridine. Both the resistant mutant and the wild type parasite had little or no uridine kinase, an enzyme readily detectable in the human fibroblast host cells. Uridine and deoxyuridine phosphorylases were found in both parasites while human fibroblasts had much less of these enzymes. The critical difference between the mutant and the wild type parasites proved to be a 100-fold lower concentration of uracil phosphoribosyltransferase in the fluorodeoxyuridine-resistant mutant. A back mutant of the resistant strain, selected for its ability to use uracil, simultaneously regained uracil phosphoribosyltransferase and sensitivity to fluorodeoxyuridine. This enzymic evidence together with previously published data show that in wild type T. gondii, deoxyuridine is incorporated into nucleic acids through a phosphorolysis to produce uracil which is then converted to uridylic acid by uracil phosphoribosyltransferase.     

5-fluorodeoxyuridine induces radioresistant synthesis of DNA and sensitizes HeLa cells to gamma radiation

It was shown that preincubation of HeLa cells with 5-fluorodeoxyuridine (10(-6) M) induced DNA synthesis resistant to gamma-radiation (6 Gy). At the same time, the death rate of exposed cells increased and nucleoid relaxation decreased. The role of DNA synthesis inhibitors in the reproductive death of exposed cells is discussed.     

Short oligonucleotide prodrug having 5-fluoro and 5-iodouracil inhibits the proliferation of cancer cells in a photo-responsive manner

Photo-induced C1′ hydrogen abstraction of 5-fluoro-2′-deoxyuridine was adopted as the key reaction for releasing 5-fluorouracil (5-FU) anticancer drug from oligonucleotide strands. After photoirradiation following 5-FU release, anticancer activity was expected. We demonstrated that oligonucleotide tetramer, d(A^FU^IUA), can release 5-FU under physiological conditions in a photo-responsive manner thorough photo-induced C1′ hydrogen abstraction, and that the 5-FU released from d(A^FU^IUA) having a phosphorothioate backbone clearly suppresses the proliferation of HeLa cells in a photo-responsive manner.     

Suppression of TWIST1 enhances the sensitivity of colon cancer cells to 5-fluorouracil

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU.     

2'-deoxyribosyl analogues of UDP-N-acetylglucosamine in cells treated with methotrexate or 5-fluorodeoxyuridine

dUDP-GlcNAc, the 2'-deoxyribosyl analogue of UDP-GlcNAc, has been identified in human lymphoid cells treated with the dihydrofolate reductase inhibitor, methotrexate. It was shown previously that elevation of dUTP accompanies the gross expansion in intracellular deoxyuridylate pools that results from the methotrexate-induced block in thymidylate synthetase activity (1). dUDP-GlcNAc presumably is formed from dUTP acting in place of UTP in the normal pathway for formation of UDP-GlcNAc. Neither dUTP nor dUDP-GlcNAc has been detected in untreated cells. Inhibition of thymidylate synthetase by treatment of cells with 5-fluorodeoxyuridine (5-FdUrd) also causes the appearance of dUDP-GlcNAc, and, in addition, 5-FdUDP-GlcNAc, synthesized from 5-FdUTP. The metabolic effects, if any, of these analogues are not known. Synthesis of the analogues may help to limit accumulation of dUTP and 5-FdUTP under circumstances in which the deoxyuridine triphosphatase mechanism is insufficient.     

DNA synthesis in L-cells in the presence of 5-fluorodeoxyuridine.

After 16 h of incubation with 10-minus6 M FdUrd, the rate of (32P) orthophosphate uptake into DNA isolated from L-cells amounted to 15% of that of an untreated culture, although cell division had stopped several hours earlier. All 4 deoxynucleotides were present in this DNA but its nucleotide composition, as measured by enzymatic digestion and chromatography, reflected a decreased thymidine precursor pool in FdUrd-treated cells. Sedimentation analysis in alkaline sucrose gradients revealed that the DNA formed in the presence of FdUrd had a sedimentation coefficient of 10 S which corresponded to a single-stranded molecular weight of 5.5.105. This DNA could be "chased" into a high molecular weight DNA if the FdUrd block was bypassed with added dThd or BrdUrd. Other analyses failed to detect RNA covalently linked to the DNA fragments at a level of more than 5% RNA or about 90 ribonucleotides. The accumulation of these DNA fragments could be explained by assuming that in the presence of limiting precursor pool the rate of DNA chain initiation is greater than the rate of chain elongation.     

Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.

Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.     

Characterization of an immunosuppressive factor derived from colon cancer cells

The colon cancer cell line, HT29, produces a soluble substance (HT29 factor) that blocks mitogen-induced T cell proliferation and the production of interleukin 2 (IL 2). Inhibition of T cell proliferation by the HT29 factor is reversible and is not due to a decline in cell viability or an alteration in the kinetics of T cell proliferation. It occurs even when the HT29 factor is added only 24 hr before terminating the T cell cultures, indicating that the factor affects cell division after activation of T cells has already occurred. No inhibitory activity was found in medium conditioned by human colonic epithelial cells or fibroblasts. The factor has an apparent m.w. of 56,000 and an isoelectric point of 7.9. It is sensitive to endopeptidases, heating to 56 degrees C, and extremes of pH. The HT29 factor also suppresses IL 2 production by T cells. However, low IL 2 availability alone cannot account for the suppressive effect of the factor on T cell proliferation, because the addition of exogenous IL 2 does not reverse the inhibition. This block in IL 2 responsiveness is not primarily due to a decrease in IL 2 receptors because Tac expression on activated T cells is minimally decreased during a 24-hr exposure to the HT29 factor. In addition, IL 2-induced proliferation of mitogen-activated T cells is inhibited only slightly by the HT29 factor, indicating that a block in the interaction of IL 2 with its receptor is not its main mechanism of action. Thus the inhibition of T cell proliferation is likely to be due primarily to a mechanism independent of IL 2.     

Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine

Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeutic agents (e.g. 5-fluorouracil). Here, we used error-prone PCR to mutagenize the full-length human TS cDNA and then selected mutants resistant to 5-fluorodeoxyuridine in a bacterial complementation system. We found that resistant mutants contained 1-5 amino acid substitutions and that these substitutions were located along the entire length of the polypeptide. Mutations were frequent near the active site Cys(195) and in the catalytically important Arg(50) loop; however, many mutations were also distributed throughout the remainder of the cDNA. Mutants containing a single amino acid replacement identified the following 14 residues as unreported sites of resistance: Glu(23), Thr(51), Thr(53), Val(84), Lys(93), Asp(110), Asp(116), Pro(194), Ser(206), Met(219), His(250), Asp(254), Tyr(258), and Lys(284). Many of these residues are distant from the active site and/or have no documented function in catalysis or resistance. We conclude that mutations distributed throughout the linear sequence and three-dimensional structure of human TS can confer resistance to 5-fluorodeoxyuridine. Our findings imply that long range interactions within proteins affect catalysis at the active site and that mutations at a distance can yield variant proteins with desired properties.