Metabolism Of Pyrimidine Nucleosides Research Articles

X-ray structure ofSalmonella typhimuriumuridine phosphorylase complexed with 5-fluorouracil and molecular modelling of the complex of 5-fluorouracil with uridine phosphorylase fromVibrio cholerae

Uridine phosphorylase (UPh), which is a key enzyme in the reutilization pathway of pyrimidine nucleoside metabolism, is a validated target for the treatment of infectious diseases and cancer. A detailed analysis of the interactions of UPh with the therapeutic ligand 5-fluorouracil (5-FUra) is important for the rational design of pharmacological inhibitors of these enzymes in prokaryotes and eukaryotes. Expanding on the preliminary analysis of the spatial organization of the active centre of UPh from the pathogenic bacterium Salmonella typhimurium (StUPh) in complex with 5-FUra [Lashkov et al. (2009), Acta Cryst. F65, 601-603], the X-ray structure of the StUPh-5-FUra complex was analysed at atomic resolution and an in silico model of the complex formed by the drug with UPh from Vibrio cholerae (VchUPh) was generated. These results should be considered in the design of selective inhibitors of UPhs from various species.

Protection against DNA damage-induced apoptosis by the angiogenic factor thymidine phosphorylase

Thymidine phosphorylase (TP) is involved both in pyrimidine nucleoside metabolism and in angiogenesis. TP also conferred the resistance to hypoxia-induced apoptosis of the cancer cells. In U937 cells, DNA damage-inducing agents significantly enhanced the expression of TP. Cell lines stably transfected with TP cDNA were more resistant to the DNA damage-inducing agents than the mock-transfected cells and showed augmented activity of Akt. The cytoprotective function of TP against DNA damage was independent of its enzymatic activity. The resistance to apoptosis was partially abrogated by treatment with the phosphatidyl inositol 3-kinase (PI3K) inhibitors, suggesting that the cytoprotective function of TP is mediated, at least in part, by regulation of the PI3K/Akt pathway. These findings indicate that TP expression in increased by various stress including DNA damage and that TP molecules confer resistance to DNA damage-induced apoptosis in cancer cells.

Clinical impact of thymidine phosphorylase expression in gastric cancer.

Thymidine phosphorylase (dThdPase) is an enzyme that is involved in pyrimidine nucleoside metabolism and DNA synthesis and converts 5'-deoxy-5-fluorouridine (5'-DFUR) to 5-fluorouracil (5-FU). The aim of this study was to elucidate the relationship between dThdPase expression and biological malignancy, prognosis, and sensitivity to postoperative chemotherapy, using immunohistochemical staining. We studied 148 patients with gastric cancer who underwent surgery at Department of Surgery II in Oita Medical University between 1990 and 1999. Immunohistochemical expression of dThdPase was correlated to clinicopathological factors and postoperative survival. Tumor tissue was dThdPase-positive in 112 patients. The results suggested a relationship between the degree of histological differentiation and dThdPase expression (p=0.0697). Examination of dThdPase expression based on the site of the tumor revealed that the groups with upper or lower gastric cancer included a significantly greater number of dThdPase-positive patients (p=0.0011). Analysis of the patients as a whole showed no significant difference between the survival. In the chemotherapy group, the dThdPase-positive patients tended to have a more favorable prognosis than the dThdPase-negative patients (p=0.0578). The results suggest that postoperative adjuvant chemotherapy that makes use of FU metabolic pathways may improve prognosis in patients with dThdPase-positive gastric cancer.

The activity of thymidine phosphorylase correlates with tumor size and lymph nodes status in breast carcinoma.

The platelet-derived endothelial cell growth factor (PD-ECGF) is one of the potent angiogenic factors. Recently, its homology with thymidine phosphorylase (dThdPase), an enzyme involved in pyrimidine nucleoside metabolism, has been shown. In the present study, dThdPase activity was evaluated spectrophotometrically in 43 breast carcinomas and in 19 cases of non-neoplastic breast tissues. The mean dThdPase activity in breast cancer was almost six fold higher than in normal, non-neoplastic breast tissues (1.92 and 0.29 mumol thymine (T) x mg prot.-1 x h-1 respectively). The enzyme activity significantly correlated with axillary lymph node status (p = 0.0076) and with tumor size (p = 0.0099). Besides, the intratumoral microvessel density (MD) was evaluated using the CD 31 mouse anti-human monoclonal antibody, and there was no correlation between the level of enzymatic activity and a number of microvessels. The positive significant correlation of thymidine phosphorylase activity with prognostic factors in breast cancer patients with no relation to the number of microvessels needs further examination to confirm the prognostic significance of the level of dThdPase.

Expression of thymidine phosphorylase and angiogenesis in human ovarian tumor

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in various types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We measured the dThdPase activity and the level of thrombomodulin (TM), as a marker for endothelial cells, in ovarian carcinomas, benign tumors and normal ovarian tissues. The average dThdPase activity of ovarian carcinomas (10.86+/-6.98 nmol/100 mu g protein/h) was significantly higher than that of benign tumors (4.66+/-3.91 nmol/100 mu g protein/h) or normal tissues (2.52+/-1.90 nmol/100 mu g protein/h). The expression of dThdPase detected by immunoblot analysis was well correlated with dThdPase activity. In an immunohistochemical study, the expression of dThdPase was more frequently observed in ovarian carcinomas than in benign tumors or normal tissues. dThdPase activity in human ovarian carcinomas was significantly correlated with the expression of TM in human ovarian carcinomas. These findings suggest that dThdPase expression is significantly correlated with angiogenesis in ovarian tumors.

The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinomas.

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in several types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and that dThdPase has angiogenic activity. We measured dThdPase activity and the level of thrombomodulin (TM) as a marker for endothelial cells in colorectal carcinomas and adjacent normal tissues from 21 patients, and in adenomas from 13 patients. The average dThdPase activity of colorectal carcinomas (11.58 +/- 6.30 nmol/100 micrograms protein/h) was significantly higher than that of adenomas (8.57 +/- 4.14 nmol/100 micrograms protein/h) or normal tissues (4.89 +/- 3.16 nmol/100 micrograms protein/h). In immunohistochemical study, the expression of dThdPase was observed more frequently in colorectal carcinomas than in adenomas or normal mucosas. The amount of TM in colorectal carcinomas (8.32 +/- 5.07 ng/100 micrograms protein) was significantly higher than that of adenomas (4.51 +/- 4.49 ng/100 micrograms protein) or normal tissues (3.51 +/- 2.78 ng/100 micrograms protein). dThdPase activity in human colorectal carcinomas, adenomas and normal tissues was significantly correlated with the expression of TM in these tissues. These results indicate that the expression levels of both dThdPase and TM in colorectal carcinomas are higher than those in colorectal adenomas and normal tissues and suggest that dThdPase may be involved in angiogenesis in human colorectal carcinomas, adenomas and normal tissues.

Hepatic and pulmonary metabolism of pyrimidine nucleosides in rats

The water-soluble antineoplastic agent 5-fluoro-2′-deoxyuridine (FUdR) was encapsulated in the water phase of liposomes of different lipid compositions. The retention of this drug upon storage and during contact with plasma was assessed. It was found that, upon refrigeration, diffusion of FUdR across the liposome bilayer was considerably faster when the drug was encapsulated in fluid-type liposomes (egg PC/PS/CHOL) than in solid-type liposomes (DSPC/DPPG/CHOL). With either composition, leakage of the drug from the liposomes was accelerated upon contact with plasma. To achieve improved liposomal retention of the drug, FUdR was converted to a lipophilic prodrug by esterifying the free hydroxyl groups in the deoxyribose moiety with fatty acids of different chain lengths. Thus FUdR-dipalmitate (C-16) and FUdR-dioctanoate (C-8) were synthesized and incorporated in liposomes. The dipalmitoyl derivative could be incorporated upto 13 mol% in solid-type liposomes but to only 2 mol% in fluid-type liposomes. Freeze-fracture electron microscopy revealed no major differences between control liposomes and those containing the prodrug. FUdR-dipalmitate was found to be firmly associated with the liposomal bilayer in both liposome-types: no exchange of the pro-drug with blood constituents or hydrolysis by serum esterases could be registered when the liposomes were incubated with serum. On the other hand, liposome-incorporated FUdR-dioctanoate was found to be readily extracted from the liposomes by serum components (predominantly albumin) and was found to be degraded rapidly by serum esterase activity. The antitumor activity of FUdR-prodrugs was determined using C26 colon adenocarcinoma cells. This cell line was found to be highly sensitive to FUdR. Liposomal FUdR-dioctanoate inhibited cell growth in the same concentration range as unesterified FUdR. FUdR-dipalmitate, however, was more than two orders of magnitude less potent in inhibiting cell proliferation. Its antiproliferative activity was dependent on the liposome-type used: when incorporated in fluid-type liposomes, antiproliferative activity of FUdR-dipalmitate was several-fold higher than in solid-type liposomes. The difference in antitumor activity between FUdR-dipalmitate and FUdR-dioctanoate and between FUdR-dipalmitate in the fluid- and solid-type liposomes could be explained by differences in the rate of hydrolysis of the prodrugs to FUdR by esterase activity in the tumor cells or in the growth medium.

Purification and tissue distribution of human thymidine phosphorylase; high expression in lymphocytes, reticulocytes and tumors

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism, but little is known about its physiological functions. We purified dThdPase from human placenta and used it for antibody preparation. The purified material appears as a single band at 55 000 dalton on sodium dodecylsulfate-polyacrylamide gel electrophoresis. We obtained a specific antibody raised in rabbits that detected a single polypeptide with a molecular weight of 55 000 dalton in the post nuclear homogenates of several human tissues, on immunoblotting. Using the same technique, dThdPase was highly expressed in the liver, lung, spleen, lymph nodes and peripheral lymphocytes. Immunohistochemical staining revealed that macrophage-like cells contained a much higher amount of dThdPase than parenchymal cells in the liver and lung. dThdPase was found to be highly expressed in T- and B-cell-type malignant lymphoma cells, but low in lymphoblastic and myeloblastic leukemia cells. We also found that carcinomas in the stomach, colon and ovary contained higher amounts of this enzyme than non-neoplastic regions of the tissues. These data suggest that dThdPase plays a role in proliferation and/or differentiation of leukocytes and in cancer proliferation.

Nucleosides of 1,4-thiazin-3-one and derivatives as tetrahedral intermediate analogs of enzymes in pyrimidine nucleoside metabolism

Reaction of the trimethylsilylated derivative of 1,4-thiazin-3-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of SnCl4 gave, after deblocking, 4-beta-ribofuranosyl-1,4-thiazin-3-one (8). Treatment of 1,4-thiazin-3-one with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose in the presence of sodium hydride provided, after deblocking, the corresponding 2-deoxy-beta-D-ribofuranosyl derivatives (19). Oxidation of 4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,4-thiazin-3-one (7) with 1 equiv of m-chloroperbenzoic acid resulted in 4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,4-thiazine-2,3-dione (9) and 4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,4-thiazin-3-one 1-oxide (10). Evidence is presented that indicates that the oxidation of the thiazine at the 2-position is due to a Pummerere rearrangement. The new compounds failed to show significant activity against tumor cell lines in culture, L1210 cells in vivo, virus cytotoxicity in cell culture, or cytidine deaminase.

Enzymatic cleavage of various fluorinated pyrimidine nucleosides to 5-fluorouracil and their antiproliferative activities in human and murine tumor cells.

The antitumor agent 5'-deoxy-5-fluorouridine (5'-dFUrd) is a prodrug from which 5-fluorouracil (5-FUra) is generated mainly by uridine phosphorylase in mice and by thymidine phosphorylase in human tumors. In the present study varios derivatives related to 5'-dFUrd were examined to establish the relationship between their susceptibilities to these enzymes, and their in vitro antitumor activities against both human and murine tumor cells. These studies divided the compounds into four distinct groups. 1) Compounds (including 5'-dFUrd) which were phosphorolyzed to 5-FUra by extracts from both human tumors and murine sarcoma 180; these compounds inhibited the growth of both murine (sarcoma 180 and B16 melanoma) and human (HeLa and G361 melanoma) cells. 2) Compounds which were phosphorolyzed only by the extract from the murine tumor; these compounds inhibited the growth of only the murine tumor cells. 3)Compounds phosphorolyzed only by the human tumor extracts; these compounds showed activity against the tumor cells of both species, although the relative activity against the human tumor cells appeared to be higher than that of group 1 compounds. 4) Compounds which were not phosphorolyzed by the extracts from the human or murine tumors; these compounds showed no antitumor activity. These results suggest that phosphorolysis of the 5'-dFUrd analogs is essential for antitumor activity, and that the metabolism of pyrimidine nucleosides is different between human and mouse tumors. Future approaches towards the design of prodrugs of 5-FUra and 5'-dFUrd should be aimed at compounds which generate 5-FUra in human tumor cells, or which are substrates for the human enzyme.

Synergistic toxicity of pyrazofurin and cytidine in cytidine deaminase deficient lymphoid cells (Raji)

The intermediary metabolism of pyrimidine nucleosides was studied in a line of human B lymphoblasts (Raji) in which pyrimidine de novo synthesis deficiency was pharmacologically induced by pyrazofurin. It was found (a) that Raji cells are cytidine deaminase deficient (b) that cytidine has a synergistic effect on the toxicity of pyrazofurin towards these cytidine deaminase deficient cells, affecting both the proliferation and the viability of the cells. Indirect evidences suggest that this synergistic toxicity is not mediated by an effect on nucleoside diphosphate reductase nor on the first steps of pyrimidine de novo synthesis.

Salvage of circulating pyrimidine nucleosides by tissues of the mouse.

The metabolism of pyrimidine nucleosides present in the plasma of the mouse has been examined. Uridine and cytidine are rapidly cleared from the circulation with t1/2 of less than 5 min. Uracil, deoxycytidine, deoxyuridine, and thymidine are cleared more slowly with t1/2 of 9 to 13 min. Various tissues differed markedly in the extent of nucleotide formation from circulating nucleosides. Cytidine and uridine are predominantly converted to nucleotides (greater than 50%) rather than catabolized, whereas uracil is almost entirely degraded. Thymidine, deoxyuridine, and deoxycytidine are intermediate in the extent of their conversion to nucleotides: 8.9 to 21% of these nucleosides are salvaged in the mouse. Both anabolic and catabolic routes are important in the metabolism of pyrimidine nucleosides in vivo.

Pyrimidine metabolism in microplasmodia of Physarum polycephalum.

If microplasmodia of Physarum polycephalum are exposed to 14C-labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2-14C]pyrimidine nucleosides readily gives rise to [14C]O2, particularly in the case of [2-14C]thymidine. Due to this a significant part of the trichloroacetic-acid-insoluble radioactivity from exogenously supplied [2-14C]thymidine is not associated with DNA. The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell-free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases. Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic. The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.

Pyrimidine nucleoside metabolism in mammalian cells: An in vitro comparison of two rodent species

The incorporation of thymidine into DNA has been studied in splenic and cultured cells of the golden hamster ( Mesocricetus auratus) and the goldenmantled ground squirrel ( Citellas lateralis). Considerably less uptake was observed in cells of the latter species. The reduced incorporation of thymidine found in cells of the ground squirrel is not due to a defective intracellular permeability, an inability to synthesize thymidine kinase or to trapping in an abnormally large nucleotide pool. It appears to derive from an unusually active catabolic pathway for thymidine and other pyrimidine nucleosides including deoxyuridine, uridine, deoxycytidine and cytidine. At critical concentrations the breakdown of nucleosides is sufficient to deplete the medium of these substances. Little, if any, difference is observed in the degradation of thymidine by liver slices from the same animals.