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.
ABSTRACT
Objective To investigate the killing effect of ethacrynic acid (EA) on lung cancer A549 cells derived spheres and explore the underlying mechanism.Methods A549 spheres were cultured in serum-free medium,and the protein expression of CD133,SOX2,EpCAM and ABCG2 was detected by Western blotting.MTT assay was used to evaluate the cell viability of A549 spheres and A549 cells after treated by 1,2,5,10 and 20 mg/mL cisplatin (DDP) for 48 h.The activity of glutathione S-transferase (GST) was measured by colorimetric method after A549 spheres were treated with 10,50,100 and 200 μmol/L EA,respectively.Flow cytometry,Western blotting,real-time PCR and luciferase assay were used to analyze the levels of cellular reactive oxygen species (ROS),formation of A549 spheres,mRNA and protein expression levels of β-catenin,Sox2 and ABCG2,and promoter activity of β-catenin upon 200 μmol/L EA treated cells for 48 h.A549 sphere was infected with β-catenin adenovirus for 24 h,followed by 200 μmol/L EA treatment (in presence or absence of 5 mg/mL DDP) for 24 h.The expression of β-catenin,Sox2 and ABCG2 at mRNA and protein levels was detected by real-time PCR and Western blotting,and cell growth of A549 spheres was evaluated by MTT assay.Results The A549 spheres,with high expression of tumor stem cells markers CD133,SOX2,EpCAM and drug resistance related molecule ABCG2,and resistance to DDP at different doses,were successfully derived.After 200 μmol/L EA had treated A549 sphere for 48 h,the levels of ROS were significantly increased (P ＜ 0.05),and the mRNA and protein levels of β-catenin,Sox2 and ABCG2,and promoter activity of β-catenin were notably decreased (P ＜ 0.05).The treatment of 200 μmol/L EA enhanced the inhibitory effect on proliferation and the promoting effect on apoptosis in A549 spheres induced by 5 mg/mL DDP (P ＜ 0.05).Up-regulation of β-catenin by adenoviral infection partly reversed the effects of 200 μmol/L EA on suppressing the expression levels of β-catenin,Sox2 and ABCG2,compared to the spheres infected with blank adenovirus.Additionally,β-catenin over-expression significantly remitted the inhibitory effect of 200 μmol/L EA and 5 mg/mL DDP on the proliferation in A549 spheres.Conclusion EA exerts inhibitory effect on the proliferation and stemness of A549 spheres through suppressing GST activity and β-catenin expression,and then promotes cell apoptosis.EA might be a novel drug in treatment of lung cancer and cancer stem cells.