Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow

• Published in: Integrative biology (Cambridge) 2011-03, Vol.3 (3), p.208-217
• Main Authors: Lin, Chunchen, Kolossov, Vladimir L, Tsvid, Gene, Trump, Lisa, Henry, Jennifer Jo, Henderson, Jerrod L, Rund, Laurie A, Kenis, Paul J A, Schook, Lawrence B, Gaskins, H Rex, Timp, Gregory
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Biosensing Techniques - methods; Buthionine Sulfoximine - pharmacology; Carmustine - pharmacology; Cell Line, Transformed; Cell Tracking - methods; CHO Cells; Cricetinae; Cricetulus; Diamide - metabolism; Diamide - pharmacology; Fibroblasts - drug effects; Fibroblasts - metabolism; Fluorescence Resonance Energy Transfer - methods; Glutathione - antagonists & inhibitors; Glutathione - metabolism; Glutathione Disulfide - metabolism; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Kinetics; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Microfluidic Analytical Techniques - methods; Microscopy, Confocal; Microscopy, Fluorescence - methods; Oxidation-Reduction; Oxidative Stress - drug effects; Oxidative Stress - physiology; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Swine; Transfection
• ISSN: 1757-9694; 1757-9708
• DOI: 10.1039/c0ib00071j

Despite the potential benefits of selective redox-modulating strategies for cancer therapy, an efficacious methodology for testing therapies remains elusive because of the difficulty in measuring intracellular redox potentials over time. In this report, we have incorporated a new FRET-based biosensor to follow in real time redox-sensitive processes in cells transformed to be tumorigenic and cultured in a microfluidic channel. A microfluidic network was used to control micro-scale flow near the cells and at the same time deliver drugs exogenously. Subsequently, the response of a redox homeostasis circuit was tested, namely reduced glutathione (GSH)/oxidized glutathione(GSSG), to diamide, a thiol oxidant, and two drugs used for cancer therapies: BSO (L-buthionine-[SR]-sulfoximine) and BCNU (carmustine). The main outcome from these experiments is a comparison of the temporal depletion and recovery of GSH in single living cells in real-time. These data demonstrate that mammalian cells are capable of restoring a reduced intracellular redox environment in minutes after an acute oxidative insult is removed. This recovery is significantly delayed by (i) the inhibition of GSH biosynthesis by BSO; (ii) the inactivation of glutathione reductase by BCNU; and (iii) in tumorigenic cells relative to an isogenic non-tumorigenic control cell line.