Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine |
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| Institution: | 1. Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands;2. Advisory Council on Health Research, PO Box 16052, 2500 BB The Hague, The Netherlands;3. INC research, Amsterdam, The Netherlands;1. Department of Chemistry, Faculty of Science, University of Kragujevac, Radoja Domanovi?a 12, 34000 Kragujevac, Serbia;2. Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovi?a 69, 34000 Kragujevac, Serbia;3. Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovi?a 12, 34000 Kragujevac, Serbia;4. Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia;2. The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD;1. Charité – Universitätsmedizin Berlin, Department of Medical Oncology and Haematology, Augustenburger Platz 1, 13353 Berlin, Germany;2. Charité – Universitätsmedizin Berlin, Institute of Pathology, Charitéplatz 1, 10117 Berlin, Germany;3. Outpatient Department Hematology/Oncology, Friedrichstr. 53, 88045 Friedrichshafen, Germany;4. Charité – Universitätsmedizin Berlin, Department of General, Visceral and Transplantation Surgery, Augustenburger Platz 1, 13353 Berlin, Germany;1. Department of Medicine, Graduate Institute of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan;2. Department of Medicine, Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan;3. Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung Medical University, Kaohsiung, Taiwan;4. Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan;5. Department of Urology, College of Medicine, National Taiwan University, Taipei, Taiwan |
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| Abstract: | 2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of 5-3H]-2′-deoxyuridine or 5-3H]-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.Inhibition of the enzyme resulted in a relative increase of mis-incorporation of 5-3H]-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase. |
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| Keywords: | 2′ 2′-Difluoro-2′-deoxyuridine Thymidylate synthase 2′ 2′-Difluoro-2′-deoxycytidine Cytidine analogues |
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