Study On Inhibition And Mechanism Of Antimicrobial Peptide 17BIPHE2 On Lung Adenocarcinoma A549 Cells

Objective To investigate the inhibition of antimicrobial peptide 17BIPHE2 on lung adenocarcinoma A549 cells and the related mechanism.Methods1.MTT assay was used to detect the cytotoxicity of 17BIPHE2,RL-10(0 to 40 o mol· L-1,)to lung adenocarcinoma A549 cells and pulmonary epithelial BEAS-2B cells.The IC50 was detected by the MTT method;.2.The cell cycle and invasion,migration ability of A549,treated by 17BIPHE2,were examined by flow cytometry and transwell experiment,.3.The apoptosis rate and cell morphology of A549 cells were detected by flow cytometry,TUNEL staining and transmission electron microscopy respectively.4.The mitochondria in cells die potential(delta ? m),calcium ion and reactive oxygen species(ROS)of A549 cell,treated by antibacterial peptide 17 biphe2,wereed detected by flow cytometry.5.The expression of ERK,p-erk,Bax and bcl-2 was detected by Western blot.6.Using siERK to transfect the A549 cells,detect the apoptosis of the A549 and the expression of ERK,p-ERK,Bax,and Bcl-2.7.Build lung adenocarcinoma nude mice transplantation tumor model,observe effect of antimicrobial peptide 17BIPHE2 treated on transplanted tumor in mice,and TUNEL staining and immunohistochemical technique were used to detect transplanted tumor cell apoptosis and the expression of Ki67.Result1.The antibacterial peptide 17BIPHE2 inhibited the proliferation of A549,and its inhibition concentration(IC50)was 34.33.The proliferation of normal pulmonary epithelial BEAS-2B cells cannot be inhibited by the antimicrobial peptide 17BIPHE2.2.The A549 cell cycle was delayed in S phase(P < 0.05).The ability of migration and invasion is effectively suppressed by antibacterial peptide 17BIPHE2.3.The A549 cell were treated with antibacterial peptide 17BIPHE2(0,25,35,mol· l-1)for 24 hours,and the TUNEL staining showed that the nuclei were lumpy and dense with dense chromatin.With the concentration of 17BIPHE2 increasing,the proportion of apoptosis was significantly increased in the treatment group(P < 0.05).Under transmission electron microscope,the control of A549 cells,organelles in cytoplasm structure is clear,there is a lot of endoplasmic reticulum,free ribosome and mitochondria,chromatin evenly distributed in the cell nucleus,nucleolus structure is clear;However,the A549 cells in the treatment group showed an increase in vacuoles in the cytoplasm,the cell nucleus was wrinkled,the organelle structure was destroyed,and the mitochondrial crest fracture and the swelling of the endoplasmic reticulum became empty.Flow cytometry showed that the apoptosis rate of A549 cells was significantly increased in the treatment group(P < 0.05).4.Treated with 17BIPHE2(0,25,35 umol · L-1)for 24 hours,the mitochondrial membrane potential(delta ? m)in A549 cell decreased obviously,and the intracellular calcium ions,intracellular reactive oxygen species(ROS)in A549 cell increased significantly(P < 0.05).5.Treated with 17BIPHE2(0,25,35 umol · L-1)for 24 hours,the expression of ERK and bcl-2 was down-regulated,and the expression of p-erk and Bax was up-regulated,and the ratio of Bax/ bcl-2 was increased(p < 0.05).6.After transfection of A549 with siERK,the apoptosis rate of A549 cells was significantly decreased,the expression of ERK,p-erk and Bax was down-regulated.Bcl-2 expression was up-regulated,and the ratio of Bax/ bcl-2 was decreased(p < 0.05).7.The antibacterial peptide 17BIPHE2(0,10 and 20mg/kg,)was injected in the tumor every two days,for 2 weeks.Compared with the untreated group,the tumor volume was significantly decreased,and the apoptosis rate of tumor cells increased significantly in the tumor tissue,and the expression of ki67 was down-regulated.Conclusion1.Antimicrobial peptide 17BIPHE2 can promote apoptosis of A549 and plays an anti-tumor role in vivo and vitro by inhibiting the cell cycle,migration and invasion of A549 cell.2.Antibacterial peptides 17 BIPHE2 can decrease the mitochondrial membrane potential(delta ? m)of lung adenocarcinoma A549 cell,reactive oxygen species(ROS),calcium(Ca2 +)to activate the ERK.Regulating the expression of apoptosis related proteins Bax,Bcl-2 through ERK,so as to regulate apoptosis of A549 cell.