Machover D, Zittoun J, Broët P, Metzger G, Orrico M, Goldschmidt E, Schilf A, Tonetti C, Tan Y, Delmas-Marsalet B, Luccioni C, Falissard B, Hoffman R M
Hematology and Oncology Department, Hospital Paul-Brousse, 12-14 Avenue Paul Vaillant-Couturier, F-94804, Villejuif, France.
Biochem Pharmacol. 2001 Apr 1;61(7):867-76. doi: 10.1016/s0006-2952(01)00560-3.
Potentiation of the cytotoxic activity of 5-fluorouracil (FUra) by folinic acid (5-HCO-H4folate) is due to elevation of the methylene tetrahydrofolate (CH2-H4folate) level, which increases the stability of the ternary complex of thymidylate synthase (TS), fluorodeoxyuridine monophosphate, and CH2-H4folate that inactivates the TS. Methionine deprivation results in the production of tetrahydrofolate (H4folate) and, subsequently, CH2-H4folate from methyl tetrahydrofolate, as a consequence of the induction of methionine synthesis. We hypothesized that the efficacy of FUra could be augmented by the combination of high-concentration 5-HCO-H4folate and recombinant methioninase (rMETase), a methionine-cleaving enzyme. Studies in vitro were performed with the cell line CCRF-CEM. Cytotoxic synergism of FUra + rMETase and FUra + 5-HCO-H4folate + rMETase was demonstrated with the combination index throughout a broad concentration range of FUra and rMETase. A subcytotoxic concentration of rMETase reduced the IC50 of FUra by a factor of 3.6, and by a factor of 7.5, in the absence and in the presence of 5-HCO-H4folate, respectively. 5-HCO-H4folate increased the intracellular concentrations of CH2-H4folate and H4folate from their baseline levels. Concentrations of folates were not changed by exposure to rMETase. Levels of free TS in cells treated with FUra + 5-HCO-H4folate and with FUra + rMETase were lower than those in cells exposed to FUra alone. The decrease of TS was still more pronounced in cells treated with FUra + 5-HCO-H4folate + rMETase. The synergism described in this study will be a basis for further exploration of combinations of fluoropyrimidines, folates, and rMETase.
亚叶酸（5-HCO-H4叶酸）增强5-氟尿嘧啶（FUra）的细胞毒活性是由于亚甲基四氢叶酸（CH2-H4叶酸）水平升高，这增加了胸苷酸合成酶（TS）、氟脱氧尿苷单磷酸和CH2-H4叶酸三元复合物的稳定性，从而使TS失活。蛋氨酸缺乏会导致四氢叶酸（H4叶酸）生成，随后从甲基四氢叶酸生成CH2-H4叶酸，这是蛋氨酸合成诱导的结果。我们假设高浓度5-HCO-H4叶酸与重组蛋氨酸酶（rMETase，一种裂解蛋氨酸的酶）联合使用可以增强FUra的疗效。使用CCRF-CEM细胞系进行了体外研究。在FUra和rMETase的广泛浓度范围内，通过联合指数证明了FUra + rMETase以及FUra + 5-HCO-H4叶酸 + rMETase具有细胞毒协同作用。在不存在和存在5-HCO-H4叶酸的情况下，亚细胞毒性浓度的rMETase分别使FUra的IC50降低了3.6倍和7.5倍。5-HCO-H4叶酸使CH2-H4叶酸和H4叶酸的细胞内浓度从基线水平升高。暴露于rMETase后叶酸浓度未发生变化。用FUra + 5-HCO-H4叶酸和FUra + rMETase处理的细胞中游离TS水平低于单独暴露于FUra的细胞。在用FUra + 5-HCO-H4叶酸 + rMETase处理的细胞中，TS的降低更为明显。本研究中描述的协同作用将为进一步探索氟嘧啶、叶酸和rMETase联合用药奠定基础。
