Research On The Mechanism Of OCF Against NSCLC Based On DTL-PDCD4/JNK Signaling Pathway

Background:Lung cancer still accounts for a large proportion of cancer deaths worldwide,with the majority of patients suffering from non-small cell lung cancer(NSCLC).Over the years,numerous clinical and experimental studies have led to decline in the death rate of NSCLC to a certain extent.However,it is still necessary to develop effective drugs to treat lung cancer to improve the survival rate and quality of life of patients with lung cancer.Traditional Chinese medicine(TCM)is playing an increasingly important role in the treatment of cancer.Treating both symptoms and root causes,nourishing ’qi’ eliminating phlegm and removing blood stasis,eliminating pathogenic and detoxifying and other treatment methods are widely used in clinical practice.Based on the principle of activating blood circulation and removing blood stasis combined with nourishing ’qi’,our research group created the optimizing component formula(OCF)extracted from Salvia miltiorrhiza and Panax ginseng.Preliminary studies have proved that it has effect on cancers,but its pharmacodynamic substance basis and internal mechanism need further verification and research.From the new perspective of biomarker and ubiquitination,this study is expected to verify the anti-non-small cell lung caner effect of OCF and its internal molecular mechanism by using network pharmacology technology and biological experiments.Objective:To investigate whether optimizing component formula(OCF)can play an antinon-small cell lung caner effect by regulating DTL-PDCD4/JNK pathwayMethods:1.MTT assay was used to detect the effects of optimizing component formula(OCF)on the activity of normal lung cells and non-small cell lung caner cells.2.The effects of OCF on the proliferation of normal lung cells and non-small cell lung caner cells were detected by colony formation assay.3.The effects of OCF on the migration of non-small cell lung caner cells were detected by wound-healing assay.Transwell assay was used to detect the effects of OCF on the migration and invasion of non-small cell lung caner cells.4.Molecular docking technique was used to predict the binding ability of OCF and DTL protein.5.Bio-Layer Interferometry was used to verify the interaction between OCF and DTL protein.6.Network pharmacology screening of active ingredients,target and pathway prediction of OCF:TCMSP,HERB,SymMAP database combined with literature screening of active ingredients of OCF;NPASS and DRUGBANK database to predict active ingredient targets;KEGG and GO enrichment analysis to predict the pathway of active ingredient targets.IPA(Ingenuity Pathway Analysis)analyzes the association between important targets and their corresponding components and DTL.8.RT-qPCR assay was used to detect the effects of OCF on the expression levels of genes related to DTL-PDCD4/JNK signaling pathway in A549 non-small cell lung caner cells.9.Western-blot assay was used to detect the effects of OCF on the protein expression levels of DTL-PDCD4/JNK signaling pathway related proteins in A549 non-small cell lung caner cells.Results:1.MTT assay showed that OCF could inhibit the activity of A549,H1299,95D,H1975 and H460 non-small cell lung caner cells,but had no significant effect on BEAS-2B normal lung cells.2.Colony formation assay showed that the OCF could inhibit the proliferation of A549,H1299,95D,H1975 and H460 non-small cell lung caner cells,but had no significant effect on BEAS-2B normal lung cells.3.Wound-healing assay showed that OCF could inhibit the migration of A549,H1299,95D,H1975 and H460 non-small cell lung caner cells.Transwell experiment showed that OCF could inhibit the migration and invasion of non-small cell lung caner cells A549,H1299,95D,H1975 and H460.4.Molecular docking predicted that the small molecular components of OCF could interact with DTL protein,and salvianolic acid A,salvianolic acid Y and ginsenoside Rf had strong binding ability with DTL protein.5.Bio-Layer Interferometry verified that DTL protein had strong interaction with OCF and its components,including total salvianolic acid,total ginsenside and ginseng polysaccharide.6.Results of targets and pathways predicted by network pharmacology:(1)A total of 40 active ingredients of OCF were screened by TCMSP,HERB and SymMAP database combined with literature;(2)A total of 192 active ingredient prediction targets were collected from NPASS and DRUGBANK databases;(3)KEGG enrichment analysis showed that the targets of OCF active ingredients were mainly enriched in cancer pathway,MAPK signaling pathway,relaxin signaling pathway and prolactin signaling pathway;(4)716 targets related to NSCLC were obtained from DisGeNet,DrugBank and Genecards databases;(5)There were 38 intersection targets between OCF targets and NSCLC targets,and protein-protein interaction analysis showed that EGFR,AKT1,MAPK1,ESR1 and SRC had a high degree of connectivity.(6)IPA analysis showed that there was a direct or indirect relationship between PDCD4 and DTL and the target genes of OCF active components on MAPK pathway.8.RT-qPCR showed that OCF down-regulated the mRNA expression levels of DTL,MAP4K1,JNK,c-Jun and c-Myc genes,and up-regulated the mRNA expression levels of PDCD4 and p53 genes in A549 non-small cell lung caner cells.9.Western-blot showed that OCF significantly down-regulated the protein expression levels of DTL,MAP4K1,phosphorylated JNK and phosphorylated c-Jun,and up-regulated the protein expression levels of PDCD4 and p53 in A549 non-small cell lung caner cells.Conclusion:OCF can affect the activity of NSCLC cells and inhibit their proliferation,migration and invasion,which may be related to the regulation of DTL-PDCD4/JNK signaling pathway.