The role of GPx1 in proliferation and apoptosis of lung cancer A549 spheres and its possible mechanism / 肿瘤

ABSTRACT

Objective: To investigate the effects of glutathione peroxidase 1 (GPx1) on proliferation and apoptosis of lung adenocarcinoma A549 pheres in vitro, and to explore its possible mechanism. Methods: A549 spheres were cultured in serum-free medium. The proportion of CD133+CD44+ cells in A549 spheres was detected by FCM, and the expression levels of GPx1 protein and stem cell markers Sox2 and Nanog were detected by Western blotting. The A549 spheres and their parental A549 cells were treated with 5 mg/mL cisplatin (DDP) for 48 h, then the cell survival rate was detected by CCK-8 method, the glutathione (GSH) concentrations and GPx1 activities in A549 spheres and parental A549 cells were measured by colorimetric method, while the expression level of GPx1 protein and the level of reactive oxygen species (ROS) were detected by Western blotting and FCM, respectively. When GPx1-siRNA was transfected into A549 spheres, the expression levels of GPx1 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After silencing GPx 1 gene expression in A549 spheres, the level of ROS in A549 spheres was detected by FCM, the expression levels of Sox2 and Nanog proteins were analyzed by Western blotting, and the sphere formation ability was detected by sphere-forming experiment. When A549 spheres were transfected with GPx1-siRNA and treated with DDP (5 mg/mL), the survival rate and the apoptosis rate of A549 sphere cells were measured by CCK-8 and FCM, respectively. Additional, the expression levels of phospho-p38 (p-p38), phospho-activating transcription factor 2 (p-ATF2), p53 and Bax proteins were detected by Western blotting. Results: A549 spheres were obtained successfully. The proportion of CD133+CD44+ cells was (11.7±0.6) % in all A549 spheres, which was higher than (2.2±0.3)% of parental A549 cells. The protein levels of Sox2 and Nanog in A549 spheres were higher than those in parental A549 cells (both P 0.05). The GSH concentration, GPx1 activity and GPx1 protein expression level in A549 spheres were higher than those in parental A549 cells (all P < 0.01), but the ROS level in A549 spheres was lower than that in parental A549 cells (P < 0.05). After GPx1- siRNAs were transfected into A549 spheres, the expression levels of GPx1 and Sox2 were downregulated, and the sphere formation was suppressed (all P < 0.05). After GPx 1 gene-silencing and DDP (5 mg/mL) treatment, the survival rate of A549 spheres was significantly decreased, and the apoptosis rate was elevated (both P < 0.05), while the protein expression levels of p-p38, p-ATF2, p53 and Bax were significantly up-regulated (all P < 0.05). Conclusion: Down-regulation of GPx1 expression may suppress the expression of Sox2 and increase the level of ROS, so as to inhibit the proliferation and induce the apoptosis of A549 spheres via p38-p53 signal pathway. Thus GPx1 may be a novel potential target for lung cancer treatment.