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1852.Full.Pdf [CANCER RESEARCH 44, 1852-1856, May 1984] Potentiation of 5-Fluoro-2'-deoxyuridine Antineoplastic Activity by the Uridine Phosphorylase Inhibitors Benzylacyclouridine and Benzyloxybenzylacyclouridine1 Ming Yu W. Chu,2 Fardos N. M. Naguib, Max H. lltzsch,3 Mahmoud H. el Kouni, Shih Hsi Chu, Sungman Cha, and Paul Calabresi Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912 [M. Y. W. C., F. N. M. N., M. H. I., M. H. e. K., S. H. C., S. C., P. C.], and the Roger Williams General Hospital, Providence, Rhode Island 02908 [M. Y. W. C., P. C.] ABSTRACT in contrast to normal tissues, many types of neoplasia are deficient or have a low activity of thymidine phosphorylase when At a nontoxic dose (50 ^M), the two potent uridine phospho- compared to that of uridine phosphorylase (13, 18, 21, 24, 28, rylase inhibitors, benzylacyclouridine and benzyloxybenzylacy- 30, 34-37). Therefore, it was proposed (27, 28) that uridine clouridine (BBAU), potentiated 5-fluoro-2'-deoxyuridine (FdUrd) phosphorylase inhibitors, when coadministered with FdUrd, growth inhibition of human pancreatic carcinoma (DAN) and, to might cause selective toxicity against the tumor but not the host a lesser extent, human lung carcinoma (LX-1) cells in culture. tissues, because the host tissues would retain the capacity to BBAU was more effective than benzylacyclouridine. BBAU (50 cleave FdUrd to FUra by thymidine phosphorylase. A series of UM)enhanced the cytocidal effect of FdUrd (1 t¿M,3hr) on DAN acylouridines was developed as specific inhibitors of uridine grown on soft agar from 75 to 88%. In antithymocyte serum- phosphorylase (27, 28). It was shown that these compounds immunosuppressed mice bearing DAN, the mean tumor weight significantly inhibited the cleavage of FdUrd in extracts of tumors in animals treated with FdUrd (50 mg/kg/day for 2 days) was which lack or have low activity of thymidine phosphorylase (28). 11% less than that of untreated controls. When BBAU (10 mg/ Among these compounds, BAU and BBAU are the most potent kg/day for 2 days) was coadministered, the mean tumor weight (27, 29). at Day 10 was 78% less than untreated controls, with no The present study demonstrates the enhancement of FdUrd apparent host toxicity, clearly demonstrating the potentiation of antineoplastic efficacy by BAU and BBAU on the human pan the antitumor effects of FdUrd by BBAU. The fact that DAN creatic carcinoma (DAN) and the human lung carcinoma (LX-1 ) responded better than LX-1 to benzylacyclouridine and BBAU cell lines in vitro and in vivo. It also indicates that these BAUs could be due, in part, to the lower relative activity of thymidine are more effective in cell lines having a lower ratio of thymidine phosphorylase to uridine phosphorylase in DAN compared to phosphorylase to uridine phosphorylase. A preliminary report LX-1. The activities of other enzymes involved in FdUrd metab has been presented (10). olism, thymidine kinase, uridine kinase, orotate phosphoribosyl- transferase, 5'-nucleotidase, and dihydrouracil dehydrogenase, MATERIALS AND METHODS did not differ between the two cell lines. Chemicals. BAD and BBAU were synthesized as described previously (27). RPMI-1640 medium, FBS, A/-2-hydroxyethyl-1-piperazine-N'-2- INTRODUCTION ethanesulfonic acid buffer, trypsin:EDTA, Hanks' balanced salt solution, The efficacy of the cancer chemotherapeutic agent FdUrd4 is and trypan blue stain (4%) were obtained from Grand Island Biological limited by its cleavage to the less effective base FUra (15, 26). Co. (Grand Island, NY); rabbit ATS was from M. A. Bioproducts, (Walk- For many years, there has been a great deal of interest in ersville, MD); and FdUrd was from Calbiochem-Behring (La Jolla, CA). developing inhibitors of FdUrd degradation (1-3,16, 19, 20, 27- All unlabeled pyrimidine compounds, EDTA, DTT, carbamyl-DL-alanine, - 0-alanine, ninhydrin, 5-phosphoribosyl-1 -pyrophosphate, and NADPH 29,33) which might enhance the cytotoxicity and/or the selective were obtained from Sigma Chemical Co. (St. Louis, MO); dimethylamino- toxicity of this drug. As reviewed recently (29), 2 enzymes are benzaldehyde was from Aldrich Chemicals (Milwaukee, Wl); [2-"C]thymi- responsible for the cleavage of FdUrd: thymidine phosphorylase dine, [2-14C]uridine, [6-14C]uracil, [6-14C]orotate, 5-[2-uC]FUra, and [2- (EC 2.4.2.4) and uridine phosphorylase (EC 2.4.2.3). However, 14C]FdUrd were from Moravek Biochemicals, Inc. (Brea, CA); Silica Gel G/UV2M polygram, CEL 300/UV254 cellulose, and 300 PEI/UV254 poly- 1This investigation was supported by USPHS Grants CA 25631, CA 34228, CA ethyleneimine-cellulose polygram TLC plates were from Brinkmann In 31650, CA 13943, and CA 20892 awarded by the National Cancer Institute, struments, Inc. (Westbury, NJ); ACS scintillant was from Amersham/ Department of Health and Human Services, and Grant CH 136 awarded by the Searle Corp. (Arlington Heights, IL); Omnifluor scintillant was from New American Cancer Society. 2To whom requests for reprints should be addressed, at the Roger Williams England Nuclear (Boston, MA); Grade 1 neutral aluminum oxide was Hospital. from Woelm (Waters and Associates, Framingham, MA); and bovine y- 3 Recipient of support from the Training Program in Cancer Research, USPHS globulin and dye reagent for protein estimation were from Bio-Rad Grant CA 02904, awarded by the National Cancer Institute, Department of Health Laboratories (Richmond, CA). and Human Services. ' The abbreviations used are: FdUrd, 5-fluoro-2'-deoxyuridine; ATS, antithymo Cell Culture. The in vitro experiments to test the effect of BAU and cyte serum; BAU, benzylacyclouridine or 5-benzyl-1-{2'-hydroxyethoxymethyl)- BBAU on the cytotoxic effect of FdUrd were performed using human uracil; BBAU, benzylacyclouridine or 5-(m-benzyloxybenzyl)-1-(2'-hydroxyethoxy- pancreatic (DAN) and lung (LX-1) carcinoma cells, established in this methylXiracil; FBS, fetal bovine serum; FUrd, 5-fluouridine; FUra, 5-fluorouracil; TLC, thin-layer chromatography; DTT, dithiothreitol; OMP, orotidine 5'-monophos- laboratory (7,9). Cells at early passages (passages 10 to 20) were stored phate; FUMP, 5-fluorouridine 5'-monophosphate. at -120°C in growth medium (RPMI-1640 medium:10% FBS:0.02 M N- 2-hydroxyethyl-1-piperazine-N'-2-ethanesulfonic acid buffer) containing Received November 7, 1983; accepted February 2, 1984. 1852 CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1984 American Association for Cancer Research. Potentiation of FdUrd Effects by BAU and BBAU 10% dimethyl sulfoxide. For the present experiments, cells were main Ci (pH 7.5):10% glycerol:2 rnw DTT] was used for preparations to tained at 37°in growth medium in plastic tissue culture flasks (25 sq estimate the activity of thymidine kinase (EC 2.7.1.75), undine kinase (EC 2.7.1.48), orotate phosphoribosyltransferase (EC 2.4.2.10), and 5'- cm). Cells were recovered from exponentially growing cultures by using a solution of trypsin (0.05 g/liter) and EDTA (0.02 mg/liter) in Hanks' nucleotidase (EC 3.1.3.5). Buffer B [20 HIM potassium phosphate (pH balanced salt solution (free of calcium, magnesium, and bicarbonate). 8):1 mM EDTA:1 mw mercaptoethanol] was used for preparations to Tightly capped flasks were incubated at 37°in a nonhumidified incubator. estimate the activity of uridine phosphorylase, thymidine phosphorylase, Each cell line was analyzed routinely and found to be free of Mycoplasma and dihydrouracil dehydrogenase (EC 1.2.99.1). The homogenate was first centrifuged at 30,000 x g for 30 min at 4°, contamination (6). Determination of the Concentration of Drug Giving 50% Inhibition and the supernatant fluid was recentrifuged at 105,000 x g for 1 hr at of Growth Compared to Non-Drug-treated Control Cells. Plastic tissue 4°.The 105,000 x g supernatant fluid (cytosol) was used for all enzyme culture tubes (Corning 25200) were seeded with 4.8-ml suspensions of assays except 5'-nucleotidase. The 105,000 x g pellet (microsomes) 2x10* cells/ml in growth medium and incubated at 37°.After a 24-hr derived from the homogenate in Buffer A was resuspended in the same incubation, 0.1 ml of BAD or BBAU (final concentration, 50 /JM) was buffer and used to determine the activity of 5'-nucleotidase. added to the cell cultures. FdUrd (0.1 ml) was added 5 min later for a Enzyme Assays. All assays were run under conditions where the final concentration of 0.1 or 1 UM. Sterile 0.9% NaCI solution (saline) (0.2 activity was linear with time and enzyme concentration. ml) was added to controls. All experiments were carried out in duplicate. Pyrimidine Nucleoside Phosphorylases. The activity was measured Control and drug-treated cells were counted by the trypan blue exclusion by following the formation of base from nucleoside. The assay mixture method, and the number of doublings was calculated after 72-hr incu contained 20 mw potassium phosphate (pH 8.0), 1 mM EDTA, 1 mM bation at 37°.The dose that produced 50% inhibition of cell growth was mercaptoethanol, substrate (1 mw uridine or FdUrd, 9 mCi/mmol, or 2 estimated from the plot of the number of cell doublings versus the HIM thymidine, 4.5 mCi/mmol), and 50 pi of enzyme preparation in a final logarithm of drug concentration. volume of a 100 n\. The incubation was carried out at 37°.At 40 min, Determination of Cell Survival in Soft Agar. Cell survival was deter 50-jil aliquots were withdrawn from the assay mixture and placed into 5 mined by the soft agar cloning technique (8). A solution of 0.11 g of ¡Aof 40% perchloric acid to stop the reaction. Proteins were removed Noble agar in 5 ml of double-distilled water was autoclaved for 15 min, by centrifugation, and 25 n\ of the supernatant fluid were neutralized cooled down to approximately 60°,and transferred to a 44°water bath with 50 n\ of 0.1 N KOH.
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