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The Role of Thymidine Phosphorylase in the Induction of Early Growth Response Protein-1 and Thrombospondin-1 by 5-Fluorouracil in Human Cancer Carcinoma Cells

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The Role of Thymidine Phosphorylase in the Induction of Early Growth Response Protein-1 and Thrombospondin-1 by 5-Fluorouracil in Human Cancer Carcinoma Cells 1193-1200.qxd 18/3/2010 09:17 Ì ™ÂÏ›‰·1193 INTERNATIONAL JOURNAL OF ONCOLOGY 36: 1193-1200, 2010 The role of thymidine phosphorylase in the induction of early growth response protein-1 and thrombospondin-1 by 5-fluorouracil in human cancer carcinoma cells SHIGETO MATSUSHITA1,2*, RYUJI IKEDA3*, YUKIHIKO NISHIZAWA3, XIAO-FANG CHE1, TATSUHIKO FURUKAWA1, KAZUTAKA MIYADERA4, SHO TABATA3, MINA USHIYAMA3, YUSUKE TAJITSU3, MASATATSU YAMAMOTO1, YASUO TAKEDA3, KENTARO MINAMI3, HIRONORI MATAKI3, TAMOTSU KANZAKI2, KATSUSHI YAMADA3, TAKURO KANEKURA2 and SHIN-ICHI AKIYAMA1 1Department of Molecular Oncology, 2Department of Dermatology, Field of Sensory Organology, 3Clinical Pharmacy and Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520; 4Taiho Pharmaceutical Co. Ltd., Misugidai 1-27 Hanno 357, Japan Received October 19, 2009; Accepted December 18, 2009 DOI: 10.3892/ijo_00000602 Abstract. Thymidine phosphorylase (TP) is an enzyme tetrahydrofolate (leucovorin; LV) stabilized the complex of involved in reversible conversion of thymidine to thymine. thymidylate synthase (TS) and 5-fluoro-deoxyuridine- TP is identical to an angiogenic factor, pletelet-derived monophosphate (FdUMP), increased the sensitivity to 5-FU endothelial cell growth factor (PD-ECGF) and the expression of TP expressing AZ521 cells, but not of the parental cells. levels of TP in a variety of malignant tumors were higher The levels of FdUMP in TP expressing cells were signifi- than the adjacent non-neoplastic tissues. To investigate the cantly higher than in parental cells and TPI considerably molecular basis for the effect of TP on the metabolic process decreased FdUMP to the level comparable to that in the and the anticancer effect of 5-fluorouracil (5-FU), human parental cells. 5-FU increased the expression of early growth gastric carcinoma AZ521 cells and epidermoid carcinoma KB response protein-1 (Egr-1) and an angiogenesis inhibitor, cells were transfected with TP cDNA, and AZ521/TP and KB/ thrombospondin-1 (TSP-1), in KB/TP cells but only slightly in TP were cloned. AZ521/TP and KB/TP cells overexpressed KB/CV cells, if any. TPI attenuated the induction of Egr-1 and TP and were more sensitive to 5-FU than the counterpart TSP-1 mRNA by 5-FU, while LV increased the expression parental cells. TPI, a newly synthesized inhibitor for TP of Egr-1 and TSP-1 mRNA in KB/TP cells. These findings (Ki=2.36x10-9 M), decreased the sensitivity to 5-FU of the demonstrate that the TP has a principal role in the production of TP expressing cells but not of the parental cells. 5-Formyl- FdUMP and the enhanced responses to 5-FU by leucovorin in TP-overexpressing KB and AZ521 cells, and FdUMP but not _________________________________________ FUTP is implicated in the induction of Egr-1 and TSP-1 in KB cells. Correspondence to: Dr Shin-ichi Akiyama, Department of Molecular Oncology, Graduate School of Medical and Dental Introduction Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan Thymidine phosphorylase (TP) catalyzes the reversible E-mail: [email protected] phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective bases and 2-deoxyribose-1- *Contributed equally phosphate (1-3). It also catalyzes deoxyribosyltransfer from one deoxynucleotide to another base, to form a second Abbreviations: TP, thymidine phosphorylase; PD-ECGF, platelet- deoxyuridine (4-6). Recent studies showed that TP is expressed derived endothelial cell growth factor; 5-FU, 5-fluorouracil; TPI, in a wide variety of solid tumors (7-13), but the role of TP in thymidine phosphorylase inhibitor; LV, leucovorin; PBS, phosphate- tumor proliferation was unknown. We have found that TP is buffered saline; DAB, diaminobenzidine; TS, thymidylate synthetase; identical to an angiogenic factor, platelet-derived endothelial FdUMP, 5-fluoro-deoxyuridine-monophosphate; MTT, 3-(4,5- cell growth factor (PD-ECGF), and the enzymatic activity of Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HPLC, high TP is need for the angiogenesis by TP (14-16). TP is also performance liquid chromatography; PARP, poly (ADP-ribose) poly- merase; TSP-1, thrombospondin-1; Egr-1, early growth response considered to activate some pyrimidine antimetabolites protein-1; aPAI-1, plasminogen activator inhibitor; 5'-DFUR, (17-19). 5-Fluorouracil (5-FU) was developed as a potential 5'-deoxy-5-fluorouridine anti-tumor drug (20), and reportedly activated through three alternative routes. It may be converted to FUMP either Key words: thymidine phosphorylase, 5-fluorouracil, leucovorin, directly by orotate phosphoribosyltranferase or by the FdUMP, thymidylate synthase, ternary complex, TSP-1 sequential actions of uridine phosphorylase and uridine kinase. FUMP may be converted to FUDP and then to FUTP, 1193-1200.qxd 18/3/2010 09:17 Ì ™ÂÏ›‰·1194 1194 MATSUSHITA et al: THE EFFECT OF 5-FU AND LV IN TP EXPRESSING CELL LINES which is incorporated into RNA, thus interfering with its RSV/TP (AZ521/TP and KB/TP cells) and TP-negative synthesis and nuclear processing. Alternatively, FUMP is clones (AZ521 and KB cells) were further analyzed. converted to FUDP by pyrimidine kinase and converted to FdUDP by ribonucleotide reductase. FdUDP is then converted Preparation of cell lysate. Cells were lysed in 10 mM Tris-HCl to 5-fluoro-deoxyuridine-monophosphate (FdUMP) which (pH 7.5) by rapid freeze-thawing in liquid nitrogen. The lysates inhibits thymidylate synthetase and hence DNA synthesis. were centrifuged at 15,000 x g for 20 min at 4˚C and the super- Another route of 5-FU activation to FdUMP is catalyzed by natants were resolved by electrophoresis (25). Protein concen- TP and thymidine kinase. Thymidylate synthase (TS) (EC trations were determined by the method of Bradford (26). 2.1.1.45), which converts deoxyuridine monophosphate (dUMP) to thymydilate, plays an essential role in the Immunoblot analysis. The proteins in the supernatants were synthesis of DNA. Inhibition of TS by 5-FU is mediated by separated using sodium dodecyl sulfate-polyacrylamide gel the formation of a stable ternary complex between the active electrophoresis (SDS-PAGE) according to the method of anabolite FdUMP, the target enzyme TS, and the cofactor Laemmli (27). Proteins in the gel were electrophoretically 5,10-methylenetetrahydrofolates (5,10-CH2H4PteGlun) transferred to a sheet of polyvinylidene difluoride (PVDF) (21,22). Recently, we have indicated that 5-FU activated p38 membrane (Immobilon-P transfer membrane; Millipore, MAPK and then upregulated Egr-1 expression, resulting in Bedford, MA) with Bio-Rad Transblot SD as described (28). the expression of an endogenous antiangiogenic factor, The membranes were then reacted with primary antibodies TSP-1. The induction of TSP-1 was suggested to be implicated against TP, Egr-1, TSP-1 and poly (ADP-ribose) polymerase in the antitumor effect 5-FU (23). However, the molecular (PARP). After incubation, membranes were washed and basis for the induction by 5-FU is not fully understood. In incubated with anti-mouse or anti-rabbit secondary antibodies. this study, we examined whether TP is needed for the induction The membranes were developed using the enhanced chemilu- of TSP-1 by 5-FU, and which of the two cytotoxic metabolites minescence Western blotting detection system (Amersham, of 5-FU, FUTP and FdUMP, is involved in the induction of Buckinghamshire, UK). Egr-1 and TSP-1. Quantitation of drug sensitivity. The sensitivity of parental Materials and methods and transfected cells to agents were determined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Chemicals. 5-FU and 3-(4,5-dimethylthiazol-2-yl)-2,5- colorimetric assay as described (29). diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO), leucovorin was from Assay of intracellular FdUMP level. Parental and transfected Lederle Co. (Tokyo) and [3H] 5-FU (15.3 mCi/mmol) was cells were suspended at 2x105 cells and incubated with or from Moravek Biochemicals, Inc. (Brea, CA). TPI, an inhibitor without 100 μM TPI overnight. After a 24-h incubation, cells of thymidine phosphorylase and an anti-TS polyclonal anti- were exposed to 400 nM [3H]5-FU for 3 h. Intracellular body were obtained from Taiho Pharmaceutical Co. Ltd. FdUMP level was analysed by high performance liquid Antibodies against Egr-1, TSP-1 and tubulin were from Santa chromatography (HPLC) method as previously descrived Cruz Biotechnology and NeoMarker, respectively. (30). Cell lines. Human gastric carcinoma AZ521 cells, AZ521/ Incorporation of fluoropyrimidines into RNA. Parental and TP, AZ521 cells transfected with PD-ECGF cDNA, human transfected cells were suspended at 5x105 cells and incubated KB epidermoid carcinoma cells and KB/TP, KB cells trans- with or without 100 μM TPI overnight. After a 24-h incu- fected with PD-ECGF cDNA, were maintained in minimal bation, cells were exposed to 80 nM [3H]5-FU for 12 h. Total essential medium (Life Technologies, Rockville, MD) cellular RNA was isolated and purified with the TRIzol reagent containing 10% fetal calf serum. These cells were mycoplasma- (Life Technologies, Rockville, MD) using phenol/ chloroform free. followed by isopropanol precipitation as recommended by the manufacturer. The RNA pellet was dissolved with 22 ml Transfection of PD-ECGF cDNA into AZ521 and KB cells. TE and quantitated by UV absorbance at 260/280 nm. Radio- PD-ECGF full length cDNA was kindly provided by Dr K. activity
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