Berberine Exerts Anticancer Action On Lung Adenocarcinoma Via Activating FOXO1/3

Background:Lung cancer,the most common malignancy,now is a cancer with the highest morbidity and mortality worldwide.In recent years,lung adenocarcinoma has surpassed lung squamous cell carcinoma as the main pathological type of lung cancer.It is of great significance to explore the effect and mechanism of small molecule extract of Chinese medicine on lung adenocarcinoma.Berberine(BBR),an important alkaloid,is extracted from the rhizomes of Ranunculaceae,such as three needles and Coptis chinensis.BBR is widely used in domestic clinical treatment of gastroenteritis and other digestive diseases.Numerous studies reported BBR has significant anticancer actions,including the induction of apoptosis and inhibition of tumor proliferation,invasion and angiogenesis.Moreover,BBR has the role of lung adenocarcinoma inhibition,but its specific molecular mechanisms against lung adenocarcinoma are not yet fully understood.Forkhead box O 1(FOXO1)and FOXO3 are transcription factors and involve in the regulation of tumor progression.Studies documented activation of FOXO1 and FOXO3 can induce lung adenocarcinoma cell apoptosis and inhibit its proliferation,invasion and distant metastasis.The purpose of this study was to investigate the actions of BBR on A549 cells or tumor-bearing nude mice via analyzing cell viability,apoptosis and other hallmarks of tumor-related parameters,and understand the changes of upstream and downstream signaling of FOXO1/3 pathway.Then,the mechanism of FOXO1/3 in mediating BBR against lung adenocarcinoma was further clarified.Objective:To investigate the action of BBR treatment on cell viability,apoptosis,migration,adhesion and oxidative stress-related parameters of A549 cells,and understand the role of BBR treatment on the tumor volume of A549 cell tumor-bearing mice.Then,to further investigate whether the Akt-FOXO1 and JNK-FOXO3 signaling pathway involves in the regulation of BBR-induced anti-lung adenocarcinoma activities.Methods:1.Given the gradient concentration of BBR on A549 cells,then cells were divided into Control group,25 ?mol/L BBR group,50 ?mol/L BBR group and 75?mol/L BBR group,and the cell viability,apoptosis and oxidative stress were respectively investigated.In order to eliminate the effect of high concentration of BBR on cell viability,the effects of three concentrations of BBR(5 ?mol/L,10 ?mol/L and 15 ?mol/L)on cell migration and adhesion were determined.A549 cell viability was detected by CCK8 kit;apoptosis rate and membrane potential were respectively measured by TUNEL and JC-1 assay;the levels of ROS and Caspase3/9 were respectively analyzed by the DCFH-DA and Caspase kits.Later,we built the A549 cell tumor-bearing nude mice model to investigate the action of BBR treatment on tumor growth in vivo,and the mice were divided in to Control group,10 mg/kg BBR group and 20 mg/kg BBR group.2.Next,the effects of gradient concentration of BBR(25 ?mol/L,50 ?mol/L and 75 ?mol/L)on the expression of Akt-FOXO1 pathway and apoptosis-related signaling(Bax and Bcl2)in A549 cells were observed.To detect the effect of Akt agonist,1,3-Dicaffeoylquinic acid(DA),cotreated with BBR on A549 cells,the cells were divided into Control group,1 ?mol/L DA group,and 50?mol/L BBR group,and BBR + DA group,then repeating above mentioned experiments in section 1.Next,we built the tumor-bearing nude mice model to detect the role of DA and BBR cotreatment on tumor growth,Akt-FOXO1 pathway,and apoptotic signaling in vivo,and the mice were divided in to Control group,20 mg/kg BBR group and 20 mg/kg BBR+1 mg/kg DA group.3.Moreover,the effects of gradient concentration of BBR(25 ?mol/L,50 ?mol/L and 75 ?mol/L)on the expression of JNK-FOXO3 pathway in A549 cells were detected.To confirm the action of JNK antagonist,SP600125,cotreated with BBR on A549 cells,the cells were divided into Control group,20 ?mol/L SP600125 group,and 50?mol/L BBR group,and BBR + SP600125 group,then repeating above mentioned experiments in section 1.Later,we use the tumor-bearing nude mice to study the effect of SP600125 and BBR cotreatment on tumor growth,JNK-FOXO3 pathway,and apoptotic signaling in vivo,and the mice were divided in to Control group,20 mg/kg BBR group and 20 mg/kg BBR+5 mg/kg SP600125 group.Results:1.The A549 cell viabilities treated with the gradient concentration of BBR(25 ?mol/L,50 ?mol/L and 75 ?mol/L)were respectively decreased to 74.2±6.1%,59.4±5.4%,42.1±5.1%,compared with the control group.And the apoptotic rates were upregulated to 13.4±1.7%,9.8±2.9%,32.4±3.4%,and the rates of cell with low membrane potential were upregulated to 12.1±1.8%,28.4±2.1%,39.6±2.8%,compared with the control group.The levels of ROS,Caspase3 and Caspase9 were also increased by BBR treatment in a dose-dependent manner.Moreover,treatment of BBR(5 ?mol/L,10 ?mol/L and 15 ?mol/L)respectively increased the cell scratch distances to 125.4±7.9%,146.8±9.1%,183.2±10.3%,and decreased the cell adhesion abilities to 86.2±8.1%,65.3±5.4%,44.1±4.3%.In addition,intraperitoneal injection of BBR significantly decreased the tumor proliferation rate and tumor size in nude mice.2.After treatment of BBR with gradient concentration on A549 cells,BBR decreased the pAkt,pFOXO1 and Bcl2 levels,but upregulated the Bax level in a dose-dependent manner.Next,activation of Akt by DA cotreatment abolished the BBR-induced decrease of A549 cell viability and membrane potential,increase of apoptosis,and upregulation of ROS,Caspase3 and Caspase9 levels.Moreover,DA cotreatment inhibited the BBR-exerted decrease of pFOXO1 and Bcl2,and increase of Bax.Furthermore,intraperitoneal injection of DA blocked the BBR-induced prohibition of Akt and FOXO1 phosphorylation,apoptosis and tumor growth inhibition.3.After treatment of BBR with gradient concentration on A549 cells,BBR decreased the pFOXO3 level,but upregulated the pJNK level in a dose-dependent manner.Next,inhibition of JNK by SP600125 cotreatment abolished the BBR-induced decrease of A549 cell viability and membrane potential,increase of apoptosis,and upregulation of ROS,Caspase3 and Caspase9 levels.Moreover,SP600125 cotreatment significantly inhibited the BBR-exerted decrease of pFOXO3 and Bcl2,and increase of Bax levels.Furthermore,intraperitoneal injection of SP600125 blocked the BBR-induced activation of JNK and FOXO3,apoptosis and tumor growth inhibition.Conclusion:1.BBR can significantly inhibit the proliferation,migration and adhesion of lung adenocarcinoma A549 cells,and induce oxidative stress and apoptosis in A549 cells.2.BBR treatment decreased the phosphorylation levels of Akt and FOXO1 in A549 cells.Increase of FOXO1 phosphorylation by Akt activation abolished BBR-exerted anti-lung adenocarcinoma actions,suggesting BBR inhibits lung adenocarcinoma via Akt inhibition thus activating FOXO1.3.BBR treatment activated the JNK-FOXO3 signaling in A549 cells.Enhanced FOXO3 phosphorylation by JNK inhibition abolished BBR-exerted anti-lung adenocarcinoma actions,suggesting BBR inhibits lung adenocarcinoma via activation of JNK thus enhancing FOXO3 activities.