College of Pharmacy, Natural Product Research Institute, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Korea.
Department of Pathology, Laboratory of Cancer Research, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan.
Angew Chem Int Ed Engl. 2016 Jul 4;55(28):7939-42. doi: 10.1002/anie.201601026. Epub 2016 May 13.
The glutathione (GSH) redox reaction is critical for defense against cellular reactive oxygen species (ROS). However, direct and real-time monitoring of this reaction in living mammalian cells has been hindered by the lack of a facile method. Herein, we describe a new approach that exploits the GSH biosynthetic pathway and heteronuclear NMR. [U-(13) C]-labeled cysteine was incorporated into GSH in U87 glioblastoma cells, and the oxidation of GSH to GSSG by a ROS-producing agent could be monitored in living cells. Further application of the approach to cells resistant to temozolomide (TMZ), an anti-glioblastoma drug, suggested a possible new resistance mechanism involving neutralization of ROS. This result was corroborated by the observation of up-regulation of glutathione peroxidase 3 (GPx3). This new approach could be easily applied to redox-dependent signaling pathways and drug resistance involving ROS.
谷胱甘肽（GSH）的氧化还原反应对于抵御细胞内的活性氧（ROS）至关重要。然而，由于缺乏一种简便的方法，直接和实时监测活哺乳动物细胞中的这种反应受到了阻碍。在此，我们描述了一种新的方法，该方法利用了 GSH 的生物合成途径和异核 NMR。[U-（13）C]标记的半胱氨酸被掺入 U87 神经胶质瘤细胞中的 GSH 中，并且可以在活细胞中监测 ROS 产生剂将 GSH 氧化为 GSSG 的过程。该方法在对替莫唑胺（TMZ）耐药的细胞中的进一步应用表明，一种可能的新耐药机制涉及 ROS 的中和。这一结果得到了谷胱甘肽过氧化物酶 3（GPx3）上调的观察结果的证实。这种新方法可以很容易地应用于涉及 ROS 的依赖于氧化还原的信号通路和耐药性。
