University of Notre Dame, 316 Stinson-Remick Hall, South Bend, IN 46556.
Integr Biol (Camb). 2011 Mar;3(3):208-17. doi: 10.1039/c0ib00071j. Epub 2010 Dec 23.
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.
尽管选择性氧化还原调节策略在癌症治疗方面具有潜在的益处，但由于难以随时间测量细胞内的氧化还原电位，因此仍然难以找到有效的治疗方法测试方法。在本报告中，我们已经结合了一种新的基于 FRET 的生物传感器，以实时跟踪转化为致瘤性并在微流控通道中培养的细胞中的氧化还原敏感过程。使用微流网络来控制细胞附近的微尺度流动，同时外源性地输送药物。随后，测试了氧化还原平衡回路的响应，即还原型谷胱甘肽（GSH）/氧化型谷胱甘肽（GSSG）对二酰胺，一种硫醇氧化剂，以及两种用于癌症治疗的药物：BSO（L-丁硫氨酸-[SR]-亚砜亚胺）和 BCNU（卡莫司汀）。这些实验的主要结果是比较单个活细胞中 GSH 的实时消耗和恢复。这些数据表明，哺乳动物细胞能够在急性氧化应激消除后的几分钟内恢复还原的细胞内氧化还原环境。BSO 抑制 GSH 生物合成会显著延迟（i）这种恢复；BCNU 使谷胱甘肽还原酶失活会显著延迟（ii）这种恢复；与同基因非致瘤性对照细胞系相比，致瘤细胞会显著延迟（iii）这种恢复。
