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

Abstract

Objective: To explore reactive oxygen species (ROS) generation and its role on A549 cell migration under hypoxic condition. Methods: Human non-small cell lung cancer (NSCLC) cell line A549 was incubated in a hypoxic environment (1%O(2), hypoxia group) or in a normoxic environment (21%O(2), 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. Results: After 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]. Conclusions: Hypoxia 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.