Effect Of Eurycomanone On Phenotype Of Lung Adenocarcinoma And Its Molecular Mechanism

Objective The drug action model was established to explore the effects of different concentration gradients on the proliferation,apoptosis and invasion of lung adenocarcinoma cells.At the same time,the action mechanism of the drug was predicted by the target of the interaction between the drug and intracellular protein.and the mechanism was verified at the protein level.Methods The 2D and 3D molecular structures of Eurycomanone were obtained by Pubchem(https://pubchem.ncbi.nlm.nih.gov/).Through the Pharm Mapper(http://www.lilab-ecust.cn/pharmmapper/)pharmacophore matching platform,the human protein targets are matched according to the drug 3D structure.The protein gene names were annotated by multiple databases.Lung cancer susceptibility genes were obtained from Gene Card(https://www.genecards.org/)human gene database and intersected with Eurycomanone protein targets by Venn diagram to construct PPI protein interaction network and drug-target-disease regulatory network.The action mechanism of Eurycomanone was predicted by GO enrichment and KEGG enrichment.Based on the enrichment of the pathway,the pathway prediction model under the action of Eurycomanone was constructed.The IC50 of A549 lung adenocarcinoma cell line was detected by CCK8(Cell Counting Kit-8,CCK-8)to determine the concentration of subsequent experiment.EDU cell proliferation assay was used to detect the cell proliferation ability.The apoptosis rate was detected by Annexin V-FITC/PI method.The invasive ability of the cells was detected by Transwell migration test and invasion test.Westernblot was used to detect the changes of intracellular expression of cell proliferation,apoptosis and invasion-related proteins under different drug concentration gradients.The phosphorylation levels of MAPK signal pathway and PI3K-AKT signal pathway were detected by Westernblot.Results Eurycomanone significantly inhibits the proliferation of lung adenocarcinoma cells.In Ed U assay,the number of Ed U positive cells in A549 lung adenocarcinoma cell line decreased gradually with the increase of Eurycomanone concentration(P < 0.0001).In clone formation test,the number of clones in A549 lung adenocarcinoma cell line decreased significantly(P <0.05).It is suggested that Eurycomanone inhibits DNA synthesis and cell proliferation in lung adenocarcinoma cells in a concentration-dependent manner.Eurycomanone significantly promotes apoptosis of lung adenocarcinoma cells.Annexin V/PI apoptosis was detected by flow cytometry.The results showed that with the increase of the concentration of Eurycomanone,the total apoptosis rate of lung adenocarcinoma cells increased,the phosphorylation level of pro-apoptotic protein p53 increased,and the level of apoptosis-related protein Bcl-2 decreased.This suggests that Eurycomanone promotes the apoptosis of lung adenocarcinoma cells.Eurycomanone inhibits the migration and invasion of lung adenocarcinoma cells.The effect of Eurycomanone on the migration ability of lung adenocarcinoma cells was verified by Transwell migration assay.The number of cells penetrating the carbonate membrane decreased with the increase of the concentration of Eurycomanone.The effect of Eurycomanone on the invasive ability of lung adenocarcinoma cells was verified by Transwell invasion assay.The degradation ability of extracellular matrix of the two cell lines decreased significantly with the increase of the concentration of Eurycomanone,indicating that Eurycomanone inhibited the invasive ability of lung adenocarcinoma cells.Eurycomanone inhibited the activation of MAPK pathway and PI3K-AKT pathway.Compared with the blank control,the AKT of lung adenocarcinoma cells decreased at different concentrations(P < 0.01).The results showed that the inhibition of MAPK signal pathway and PI3K-AKT signal pathway phosphorylation was concentration-dependent and promoted cell apoptosis.ConclusionsIt is predicted by network pharmacology that there is a direct interaction between Eurycomanone and many lung cancer-related proteins,and the target proteins are significantly enriched in MAPK signal pathway and PI3K-AKT signal pathway.In cytological experiments,Eurycomanone can effectively inhibit the activation of MAPK pathway and PI3K-AKT pathway in lung adenocarcinoma cells.At the same time,lung adenocarcinoma cells showed proliferation,invasion inhibition and increase of apoptosis rate under different drug concentration gradients.