Mitochondrial superoxide anions induced by exogenous oxidative stress determine tumor cell fate: an individual cell-based study

Abstract

Objective

Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H₂O₂ stimuli.

Methods

Modified microfluidics and imaging techniques were used to determine O₂^•− levels and construct an O₂^•− reaction network. To elucidate the consequences of increased O₂^•− input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca²⁺ levels, mitochondrial Ca²⁺ uptake, auto-amplification of intra-cellular ROS and the intrinsic apoptotic pathway.

Results and conclusions

Results from a modified microchip demonstrated that 1 mmol/L H₂O₂ induced a rapid increase in cellular O₂^•− levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca²⁺ and mitochondrial Ca²⁺ by confocal laser scanning microscopy and confirmed that Ca²⁺ stores in the endoplasmic reticulum were the primary source of H₂O₂-induced cytosolic Ca²⁺ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca²⁺ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.

概 要

目 的

通过细胞外过氧化氢 (H₂O₂) 的刺激建立单个人 肺癌SPC-A-1 细胞的氧化压力模型。

创新点

氧自由基 (ROS) 涉及多种生物现象, 包括有益 和有害两个方面。ROS 的定量检测和反应网络的 评估结果令人期待。但ROS 半衰期很短且反应过 程很快, 因此, 我们通过多种手段克服了检测和 评估的困难。

方 法

利用改进的微流控和成像技术测定 ROS 水平, 构 建氧反应网络。通过调控线粒体胞浆Ca²⁺水平、 线粒体 Ca²⁺摄取、细胞内ROS 自扩增以及内在 凋亡途径, 确定线粒体在外源氧化压力模式中扮 演的角色。

结论

研究结果表明1 mmol/L H₂O₂ 引起细胞O₂^•−水平 的快速增加, 从而导致细胞氧化能力增加和还原 能力降低。此外, 研究还证实了内质网中储存的 Ca²⁺是H₂O₂ 诱导的线粒体Ca²⁺爆发的主要来源。 外源氧化压力反应涉及细胞器间Ca²⁺信号的传 递、ROS 自身扩增、线粒体功能紊乱和半胱天冬 酶依赖性凋亡途径。线粒体在外源性氧化应激影 响细胞命运方面发挥着关键作用。