活性氧在低氧诱导非小细胞肺癌转移中的作用

Role of reactive oxygen species in hypoxia-induced non-small cell lung cancer migration

目的探讨活性氧（ROS）在低氧诱导非小细胞肺癌转移中的作用。方法人非小细胞肺癌A549细胞分为常氧组和低氧组，分别在常氧（21%O2）或低氧（1%O2）条件下培养；Transwell检测细胞迁移能力；采用流式细胞术检测ROS的生成；Western印迹检测磷酸化蛋白激酶B（AKT）、38 000促分裂素原活化蛋白激酶（p38）等信号分子表达。结果经16 h常氧或低氧培养后，低氧组细胞迁移数显著高于常氧组[（85±10）比（56±7）个/低倍视野，P〈0.001]；低氧时间越长细胞内ROS越多，在低氧24 h后，低氧组细胞内ROS相对含量显著高于常氧组[（273±4）%比（102±6）%，P〈0.001]；经2 mmol/L N－乙酰半胱氨酸（NAC）预处理1 h后培养16 h，低氧＋NAC组细胞迁移数显著低于低氧组[（47±13）比（105±14）个/低倍视野，P＝0.011]，NAC显著抑制低氧组细胞的迁移，同时NAC在低氧12 h时抑制低氧引起的AKT、p38活化；使用0.1 μmol/L AKT变构抑制剂MK－2206与低氧同时处理，低氧＋MK－2206组细胞迁移数显著低于低氧组[（155±21）比（249±32）个/低倍视野，P〈0.001]。结论低氧环境下，由于人非小细胞肺癌A549细胞氧化应激导致ROS生成增多，增多的ROS通过激活AKT进而促进细胞的转移。

ObjectiveTo explore reactive oxygen species （ROS） generation and its role on A549 cell migration under hypoxic condition.MethodsHuman non-small cell lung cancer （NSCLC） cell line A549 was incubated in a hypoxic environment （1%O2, hypoxia group） or in a normoxic environment （21%O2, normoxia group）. The generation of ROS was measured by flow cytometry. The cell motility of A549 cells was detected by Transwell assay. The protein levels of protein kinase B （AKT）, p38 mitogen-activated protein kinase （p38） were determined by Western blot analysis.ResultsAfter 16 h hypoxic treatment, the migration of A549 cells in hypoxia group was significantly more than that of normoxia group [（85±10） vs （56±7） per lower magnification, P〈0.001]. Besides, the generation of ROS was in a time-depended manner in hypoxia group. The ROS level was increased with the prolonged hypoxia time. It was significantly higher at 24 h than that in normoxia group [（273±4）% vs （102±6）%, P〈0.001]. The migrated cells in hypoxia group co-treated with 2 mmol/L NAC for 16 h were less than that with hypoxic treatment alone [（47±13） vs （105±14） per lower magnification, P=0.011]. Meanwhile, the phosphorylation of AKT and p38 increased after 12 h hypoxic treatment in hypoxia group, however, 2 mmol/L NAC co-treatment attenuated this effect. Furthermore, inhibition of phosphorylated AKT with 0.1 μmol/L allosteric AKT inhibitor （MK-2206） in hypoxia group for 16 h reversed the hypoxia-induced A549 cell migration. The migrated cells in hypoxia＋ MK-2206 group were significantly less than that in hypoxia group [（155±21） vs （249±32） per lower magnification, P〈0.001].ConclusionsHypoxia increases the generation of ROS in A549, resulting from oxidative stress under hypoxia. The increased ROS level promotes cell motility through the activation of AKT.