Monitoring the Glutathione Redox Reaction in Living Human Cells by Combining Metabolic Labeling with Heteronuclear NMR

Angew Chem Int Ed Engl. 2016 Jul 4;55(28):7939-42. doi: 10.1002/anie.201601026. Epub 2016 May 13.

Abstract

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.

Keywords: cancer; drug resistance; glutathione; in-cell NMR; redox chemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosynthetic Pathways
  • Cell Line, Tumor
  • Cell Survival
  • Cysteine / analysis
  • Cysteine / metabolism
  • Drug Resistance, Neoplasm
  • Glioblastoma / drug therapy
  • Glioblastoma / metabolism
  • Glutathione / analysis
  • Glutathione / metabolism*
  • Humans
  • Isotope Labeling / methods
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Oxidation-Reduction
  • Oxidative Stress
  • Reactive Oxygen Species / metabolism

Substances

  • Reactive Oxygen Species
  • Glutathione
  • Cysteine