Mechanism Of Inhibition Of Growth,Migration,Invasion And Angiogenesis In Non-small Cell Lung Cancer By Huaier Extract

Objective: Lung Cancer is the leading cause of Cancer death in humans,and about 80%of patients are non-small Cell Lung Cancer(NSCLC).At present,there is an urgent need for the research on the treatment of lung cancer and the development of new drugs,so the search for more effective and safe treatment drugs has become a hot topic in the current NSCLC research.Traditional Chinese medicine(TCM)has a long history and a wide range of applications in the treatment of tumors with low side effects,high safety and improving quality of life.80% of Chinese traditional medicine is plant source,among which fungal Chinese traditional medicine is an important part.Huaier(Trametes robiniophila Murr.)extract as a kind of fungal traditional Chinese medicine,is a complex amino polysaccharide,the anti-tumor effect of sophobia in the treatment of liver cancer ascites was found,now has been widely used in the treatment of triple negative breast cancer and hepatocellular carcinoma.In a prospective study of more than 1000 patients,the efficacy of huaier extract granule as an adjunctive agent in the treatment of primary hepatocellular carcinoma after hepatectomy was confirmed.Patients who received the adjunctive treatment of huaier extract granule had longer RFS and OS than those who received placebo.However,the effect of Huaier extract on NSCLC and its mechanism are still unclear.Proliferation,metastasis and angiogenesis are the most basic characteristics of malignant tumors.This study intends to clarify the inhibitory effect of Huaier on proliferation,metastasis and angiogenesis of non-small cell lung cancer.In the research of Chinese herbal medicine,the use of bioinformatics to study the pharmacodynamics and the mechanism of action can provide new research directions and ideas for the research methods based on traditional experiments.Molecular docking is a process of mutual recognition by energy matching and space matching between receptor and ligand.In this study,we predicted the action target of Huaier extract and the way of Huaier extract and target through bioinformatics and molecular docking.This study provides more drug options for adjuvant treatment of NSCLC.Methods: 1.The inhibition of Huaier extract on the proliferation of lung adenocarcinoma cell lines was detected by MTT and colony formation test.2.Flow cytometry was used to detect the effect of Huaier extract on cell apoptosis and cell cycle arrest.3.The influence of Huaier extract on the migration and invasion of lung adenocarcinoma cell lines wasdetected by wound healing and Transwell test.4.In the human bronchial epithelial cell line,we tested whether Huaier extract has an effect on normal lung epithelial cells.5.To examine the effect of Huaier extract on angiogenesis,we tested the effects of Huaier extract on vascular endothelial cell proliferation,migration,and tube formation.6.The inhibitory effect of Huaier extract on angiogenesis was tested in vivo in a chicken choriallantoic membrane model(CAM).7.Through Pharmmapper database,combined with previous reports on the molecular structure of Huaier component,the possible targets of Huaier extract were predicted.8.We analyzed the GO and KEGG of Huaier's target to clarify the overall function of the target of Huaier extract.9.The intersection of Huaier target proteins and disease-related proteins is adopted to target the inhibition of Huaier extract on non-small cell lung cancer and angiogenesis.10.Western Blotting was used to verify the inhibition of Huaier extract on the target and its downstream related molecules.11.We predicted the binding of Huaier extract to target by molecular docking.12.The binding ability of Huaier extract and target was verified by the kinase activity assay.13.The effect of Huaier extract on proliferation,angiogenesis,and survival of nude mice,as well as its inhibitory effect on targets,was verified in vivo by tumor-forming model in nude mice.Results: 1.Huaier extract inhibits the proliferation of lung adenocarcinoma cell lines(A549 and H1299).2.Huaier extract promoted the apoptosis of the lung adenocarcinoma cell line,mainly in the late stage,and promoted the arresting of the cell cycle of the lung adenocarcinoma cell line in the S phase.3.Huaier extract inhibited the migration and invasion of lung adenocarcinoma cell lines.4.Huaier extract did not significantly inhibit the proliferation of human bronchial epithelial cell lines at normal doses.5.Huaier extract can inhibit angiogenesis in vitro by inhibiting the proliferation,DNA replication,migration,and tube formation of human umbilical vein endothelial cells(HUVECs),and inhibit VEGF-induced HUVECs migration and tube formation.6.In the chicken tomoallantoic membrane model,we found that Huaier extract can also inhibit angiogenesis in vivo.7.By Pharmmapper database,we predicted 575 possible targets of Huaier.8.GO analysis showed that the target protein was enriched in multiple tumor-related biological processes(BP),such as the metabolic process;In addition,the molecular function(MF)analysis showed that the target protein mainly plays a role inthe protein binding.The KEGG pathway revealed the accumulation of interacting proteins in metabolic pathways.9.The intersection of Huaier target and NSCLC and angiogenesis related targets is adopted to obtain 32 possible targets for Huaier inhibition of lung cancer and angiogenesis;Among them,EGFR,VEGFR2 is the classic lung cancer,angiogenesis driver genes,so we speculated that Huaier through EGFR,VEGFR2 inhibit lung cancer and angiogenesis.10.Western Blotting was used to verify the inhibitory effect of Huaier extract on the target.After treatment with Huaier extract,there was no significant change in the total expression of EGFR(t-EGFR),the expression of phosphorylated EGFR(p-EGFR)was down-regulated,the expression of GRB2 was decreased,and the phosphorylation and activation of downstream Akt and ERK were reduced.Similarly,the total expression of VEGFR2 remains unchanged,the phosphorylation activation level is reduced,and the activation of downstream Src is also inhibited.Meanwhile,Huaier extract inhibited EGF-induced EGFR activation and VEGF-induced VEGFR2 activation.11.The results of molecular docking indicate that the active components of Huaier extract may dock to EGFR and VEGFR2 to inhibit its activation: both the components of Huaier extract and the positive controls(geffitinib,sorafenib)can form the same chemical bond with EGFR and VEGFR2(EGFR: hydrogen bond;VEGFR2: disulfide and PI-PI bonds),and the release of similar free energy during the binding process,which predicted that Huaier extract could dock to EGFR and VEGFR2.12.The predicted result of molecular docking was verified by the kinase activity assay: Huaier extract inhibited activation by docking to EGFR and VEGFR2.13.In vivo experiments,we applied the lung adenocarcinoma cancer nude mouse tumor model,found Huaier extract inhibited the lung cancer subcutaneous transplantation tumor growth in mice,prolonged the tumor-burdened the survival time of mice,cut the Ki-67 proliferation related markers associated with angiogenesis marker CD34 expression,and lowered thethe expression of p-EGFR,while t-EGFR expression did not see significant changes,taking Huaier extract well tolerated and nude mice.Conclusions: Huaier may inhibit the activation of EGFR,VEGFR2 and downstream pathways,and inhibit NSCLC proliferation and angiogenesis.Based on the characteristics of low toxicity and multiple targets of Huaier and the fact that Huaier inhibits the proliferation and angiogenesis of NSCLC in vivo and in vitro experiments,Huaier may become a traditional Chinese medicine for the adjuvant treatment of lung cancer.These findings provide new insights into the molecular function of Huaier extract and demonstrate its potential as a therapeutic agent for NSCLC.