Intracellular metabolite kinetics after 5-FU exposure by CE-TOFMS metabolome analysis
Authors: Akito Nishimuta, Yasuko Yamayoshi, Akiyoshi Hirayama, Junji Abe, Atsushi Nagashima, Hajime Sato, Sayo Suzuki, Mitsuhiro Watanabe, Tomoyoshi Soga, Yusuke Tanigawara
Journal: Cancer Research (2008)

2563  Purpose:5-Fluorouracil (5-FU) is thought to exert anticancer effects via conversion to several active metabolites: fluorodeoxyuridine monophosphate (FdUMP), fluorouridine triphosphate (FUTP), and fluorodeoxyuridine triphosphate (FdUTP). Most studies have addressed the correlation between the expression levels of metabolic enzymes that convert 5-FU to its active metabolites and chemosensitivity to 5-FU. There is no report focusing on changes in the total intracellular metabolite pool (metabolome) caused by 5-FU, which are thought to reflect its anticancer effects directly. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) is an ideal tool for quantitative metabolome analysis that can simultaneously detect over 1000 charged species. The purpose of this study is to elucidate the metabolic responsiveness of cancer cells to 5-FU exposure by CE-TOFMS metabolome analysis.  Experimental Procedures: Exponentially growing human colorectal carcinoma LS174T cells were exposed to high (100 µM) or low (2 µM) 5-FU concentration under reduced folate concentration close to physiological condition. After 0, 4, 8, and 12 h exposure, all intracellular metabolites were extracted by liquid-liquid extraction and analyzed by CE-TOFMS.  Results:Among many peaks detected by CE-TOFMS, approximately 90 metabolites including 5-FU derivatives and nucleotide metabolites were identified. The intracellular level of dUMP, a substrate for thymidylate synthase (TS), remarkably increased after 5-FU exposure, which subsequently led to strikingly increased dUDP and dUTP levels. In contrast, the level of dTMP, a product of TS, decreased after 5-FU exposure. Its depletion caused subsequent depletion of dTDP and dTTP required for DNA synthesis. We also observed the increase in the level of FdUMP. These results strongly supported that TS inhibition by FdUMP is one of the main anti-cancer mechanisms of 5-FU. In addition, the levels of fluorouridine monophosphate (FUMP), fluorouridine diphosphate (FUDP), and FUTP, which are extensively incorporated into RNA and disrupting normal RNA processing and function, remarkably increased after exposure to 100 µM 5-FU, and reached a plateau after 8 hr exposure to 5-FU. These results suggest that RNA inhibition by FUTP may contribute to cell death under the high 5-FU concentration.  Conclusions: We showed remarkable changes in intracellular metabolites after 5-FU exposure by the CE-TOFMS metabolome analysis; these changes appeared to correlate well with the anti-cancer mechanism of 5-FU. The concept of simultaneous evaluation of each of the metabolites over time (metabolomics) is novel. CE-TOFMS metabolome analysis can be a strong tool to understand comprehensively the unknown kinetics of intracellular metabolites, including drug derivatives, after drug exposure. Our approach can be applied to pharmacodynamic analysis, toxicity assessment, and biomarker analysis.