Effects Of Periplaneta Americana Extract On Apoptosis And PI3K/AKT Signaling Pathway Of Lung Adenocarcinoma A549 Cells

Objective:Lung cancer has a high incidence in China.Non-small cell lung cancer（NSCLC）accounts for about 80%of lung cancer,and Lung adenocarcinoma is the most common type of non-small cell lung cancer.At present,the main treatments for lung cancer include surgery,radiotherapy,chemotherapy and biotherapy,but surgical treatment has some limitations.Although radiotherapy and chemotherapy can improve the survival rate of patients,they often have metastasis and recurrence,and have some toxic and side effects,and the therapeutic effect is not ideal.Abnormal proliferation of tumor cells is an important cause of tumorigenesis and development,and most of them acquire infinite proliferation through apoptotic escape.Therefore,from the pathway of inducing apoptotic transmission,searching for safe and effective drugs targeting to regulate the apoptosis of cancer cells has always been the focus of cancer researchers.In recent years the influence of Chinese herbal medicine on tumor had attracted wide attention of scholars.Periplaneta Americana not only had important medicinal value in traditional Chinese medicine,but also had been shown to inhibit the growth of various human cancer cells by more and more studies.Although it had been proved that Periplaneta Americana extract could inhibit the growth of lung cancer H125 cells and induce cell cycle arrest and apoptosis,its regulatory mechanism was still unclear,and the experimental basis of anti-lung cancer of Periplaneta Americana extract was not sufficient.The information exchange and transmission of intracellular and extracellular environment can not be separated from the role of cell signaling pathways,and the abnormal signal transduction is an important cause of many diseases,including tumors.PI3K/AKT signaling pathway is one of the important pathways affecting various biological behaviors of cells.It can affect the biological processes of cell proliferation,cycle,invasion and apoptosis by regulating the expression level of downstream related genes.It plays an important role in the occurrence and development of a variety of cancer cells,including lung cancer.It has been proved that Periplaneta Americana extract can promote apoptosis of ovarian cancer cells and hepatic stellate cells by inhibiting PI3K/AKT signaling pathway,but whether it can induce apoptosis of lung cancer cells through this mechanism is not clear.In this study,we observed the apoptosis of lung adenocarcinoma A549 cells induced by Periplaneta americana extract and its effect on PI3K/AKT signaling pathway in vitro,and explored the molecular mechanism of Periplaneta americana extract against lung cancer,in order to provide a new theoretical basis for the clinical application of Periplaneta americana extract against lung cancer.Method:1.After treating lung adenocarcinoma A549 cells cultured in vitro with the extracts of Periplaneta Americana at different concentrations of 0,12.5,25,50,100and 200μg/ml for 24,48 and 72 hours,the inhibition rate of proliferation of A549cells was measured by MTT method,and the IC₅₀0 of the extracts of Periplaneta Americana against A549 cells at different time was calculated.In the latter experiment,the extracts of Periplaneta Americana at concentrations of 50,100 and 500 ug/ml were used to treat A549 cells for 48 hours.2.The changes of cell cycle was measured by flow cytometry.3.The apoptotic rate of A549 cells was detected by AnnexinV-FITC/PI double staining.4.The changes of mitochondrial membrane potential was measured by flow cytometry.5.The expression of PI3K/AKT signaling pathway related genes PI3K,AKT and apoptosis related genes p53,Caspase-3 and CytC mRNA and proteins were checked by Semi-quantitative RT-PCR and Western blot.6.The expression of PI3K/AKT signaling pathway related genes PI3K,AKT and apoptosis related genes p53,Caspase-3 and CytC proteins were checked by Western blot.Result:1.IC₅₀ of A549 cells treated with 12.5,25,50,100 and 200μg/ml of Periplaneta Americana extract for 24,48 and 72 hours were 249.20μg/ml,135.90μg/ml and95.87μg/ml,respectively.The inhibition rate of cell proliferation in Periplaneta americana extract treatment group was significantly higher than that in control group,and there was a concentration-time dependence（P<0.05）.2.Compared with control group[（62.20±1.12）%,（24.75±1.02）%],the percentage of A549 cells in G0/G1 phase[（59.83±1.02）%,（56.56±1.10）%]and S phase[（17.96±0.78）%,（15.35±0.54）%]treated with medium（M）concentration 100ug/ml and high（H）concentration 200 ug/ml Periplaneta Americana extract were all decreased significantly,however the percentage of G2/M phase[（22.18±1.06）%,（27.85±1.22）%]was significantly higher than that of the control group（13.05±0.36）%（P<0.05）.3.After 48 hours of treatment with 50（low concentration,L）,100 and 200ug/ml extracts of Periplaneta americana,the apoptotic rates of A549 cells were（9.26±0.25）%,（15.48±1.38）%and（20.85±1.46）%,respectively.Compared with the control group（3.38±1.06）%,the apoptotic rates of A549 cells were significantly increased and were concentration-dependent（P<0.05）.4.After 48 hours of treatment with 50,100 and 200 ug/ml extracts of Periplaneta americana,the percentage of green fluorescence in A549 cells was（15.06±0.75）%,（20.59±1.18）%,（26.25±1.26）%.Compared with the control group（1.23±0.25）%),the percentage of green fluorescence in A549 cells was significantly increased and was concentration-dependent（P<0.05）.5.The extracts of Periplaneta Americana at 50,100 and 200 ug/ml could up-regulate the expression of p53（1.67±0.15,2.43±0.18,4.16±0.35,and the control group was 0.96±0.08）Caspase-3（1.78±0.53,2.51±0.16,3.86±0.25,and the control group was 0.98±0.05）,CytC（1.51±0.12,2.16±0.12,3.75±0.26,and the control group was0.95±0.06）,anddown-regulationofexpression PI3K（0.84±0.06,0.72±0.05,0.35±0.03,and the control group was 1.00±0.06）,AKT（0.65±0.03,0.42±0.03,0.33±0.02,and the control group was 1.02±0.06）mRNA in a concentration-dependent manner.6.The extracts of Periplaneta Americana at 50,100 and 200 ug/ml could up-regulate the expression of p53（0.28±0.02,0.44±0.03,0.54±0.04,and the control group was 0.09±0.02）Caspase-3（0.33±0.02,0.40±0.03,0.49±0.03,and the control group was 0.12±0.02）,CytC（0.25±0.03,0.37±0.04,0.52±0.05,and the control group was 0.06±0.01）,and down-regulation of expression PI3K（0.57±0.04,0.43±0.04,0.11±0.02,and the control group was 0.66±0.04）,p-AKT（0.40±0.0 2,0.34±0.03,0.11±0.03,and the control group was 0.46±0.03）proteins in a concentration-dependent manner.Conclusion:1.Periplaneta Americana extract can inhibit the proliferation of lung adenocarcinoma A549 cells,block cell cycle at G2/M phase,and induce cell apoptosis.2.Periplaneta Americana extract can induce apoptosis of lung adenocarcinoma A549 cells through p53-mediated mitochondrial pathway.3.Periplaneta Americana extract can induce apoptosis of lung adenocarcinoma A549 cells by Upregulation of p53 expression through inhibiting PI3K/AKT signaling pathway.