Renner S, Kuçi Z, d'Cruze H, Niethammer D, Bruchelt G
Children's University Hospital, Tübingen, Germany.
Electrophoresis. 2000 Aug;21(14):2828-33. doi: 10.1002/1522-2683(20000801)21:14<2828::AID-ELPS2828>3.0.CO;2-R.
The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu1) and MTX-polyglutamates (MTX-Glu2-5) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glun in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX-Glun derivatives including MTX-Glu1 could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP+ and ascorbate was achieved with another system (L: 0.01 M HCI/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX-Glu1-5 inhibited consumption of NADPH and production of NADP+. Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.
抗叶酸药物甲氨蝶呤（MTX）广泛应用于癌症化疗。在本研究中，我们发现MTX（MTX-Glu1）和MTX多聚谷氨酸盐（MTX-Glu2-5）强烈抑制白血病细胞系MOLT-4的生长。然而，这种作用可被抗坏血酸减弱。我们研究了抗坏血酸是否能够直接将二氢叶酸（DHF）还原为四氢叶酸（THF），或者通过规避MTX对二氢叶酸还原酶（DHFR）的抑制作用来实现。通过分析等速电泳测定了在不存在或存在抗坏血酸的情况下，MTX-Glun对这种依赖NADPH的DHF还原反应的抑制作用。使用0.01 M HCl/组氨酸，pH 6.0作为前导电解质（L），0.005 M 2-（N-吗啉代）乙磺酸（MES）/组氨酸，pH 6.0作为终止电解质（T），包括MTX-Glu1在内的MTX-Glun衍生物能够很容易地分离，然而对THF进行定量估计是不可能的。通过另一个系统（L：0.01 M HCl/β-丙氨酸，pH 3.73；T：0.01 M己酸，pH 3.27）实现了通过测量NADPH、NADP+和抗坏血酸对DHFR反应的定量表征。纳摩尔浓度的MTX-Glu1-5抑制了NADPH的消耗和NADP+的产生。抗坏血酸既不能直接还原DHF，在MTX抑制DHFR后也不能还原DHF。然而，抗坏血酸似乎减少了THF的氧化，这可能解释了其降低MTX对MOLT-4细胞抑制作用的能力。
