Mechanism Of Guben Jiedu Prescription In Inhibiting The Metastasis Of Lung Adenocarcinoma Based On TMCO1/PI3K/Akt Axis

Research BackgroundLung cancer is one of the most common malignancies and has become the leading cause of death from malignant tumors worldwide.Lung cancer metastasis is the main reason for treatment failure in clinical patients.Traditional Chinese medicine can exert anticancer activity by regulating genetics,epigenetics,tumor microenvironment and cancer stem cells.In our previous experiments,we found that TMCO1 expression was elevated in primary and metastatic lung cancer foci,and knockdown of TMCO1 inhibited the viability and migration of lung cancer A549 cells.Further transcriptome sequencing revealed that TMCO1 was associated with various biological processes and pathways such as development,tumor metastasis invasion,stem cells,tumor angiogenesis,EMT,TGFβ,PI3K/Akt,and HIF1α.Guben Jiedu Prescription is an experienced formula summarized by PIAO Bing-kui Professor of Guang’anmen Hospital of CACMS after long-term clinical practice,which can effectively inhibit lung cancer metastasis and prolong the survival time of patients.Our preliminary pre-experiment found that the prescription can inhibit the viability and migration force of lung cancer A549 cells and may be related to TMCO1,but it deserves further study.Part Ⅰ Mechanism of Guben Jiedu Prescription against Lung cancer based on network pharmacology,molecular dockingObjectives:Predicting the targets and pathways of Guben Jiedu Prescription in inhibiting lung cancerMethods:network pharmacology and molecular docking:The active components of Guben Jiedu Prescription and their targets were screened by the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform(TCMSP),High Throughput Experiment and Reference Database(HERB)of Traditional Chinese Medicine,PubChem,SwissTargetPrediction and UniProt databases.Cytoscape 3.8.0 were employed to construct Guben Jiedu Prescription-active components-target network.The targets related to lung cancer were obtained from GEO,Genecards,OMIM and Malacards databases.The intersection targets were obtained by using Venny database.The protein-protein interaction(PPI)network was established using STRING database,and the core targets of PPI network were screened using CytoNCA plug-in of Cytoscape 3.8.0.GO analysis and KEGG pathway enrichment analysis were performed using DAVID 6.8.Autodock vina was used for molecular docking.Results:using network pharmacology predicted 237 active ingredients of Guben Jiedu Prescription,880 targets of drug intersection with lung cancer,core targets of PPI network were AKT1,EGFR,TNF,IL6,STAT3,PIK3CA,mTOR,etc.GO analysis showed that biological processes were mainly enriched in protein tyrosine/serine phosphorylation and proliferation,apoptosis,inflammatory response.The KEGG pathway enrichment analysis revealed that the treatment of lung cancer with Guben Jiedu Prescription was associated with the regulation of cancer-related signaling pathways,especially PI3K/Akt signaling pathway.Molecular docking results showed that the main active ingredients of Guben Jiedu Prescription had good binding strength to AKT1,PIK3CA and GSK-3β.Part Ⅱ the effect and mechanism of the Guben Jiedu Prescription for the inhibition of metastatic tumors in immunodeficient miceObjectives:to verify the effect and mechanism of the Guben Jiedu Prescription for the inhibition of lung adenocarcinoma metastatic tumors in immunodeficient mice Methods:The animal model of lung cancer metastasis was established by tail vein injection of NVSG rat A549 cells,and the experiment was divided into blank group,model group,high,medium and low dose groups of Guben Jiedu Prescription,and positive drug group,which were given saline and Guben Jiedu Prescription high,medium and low dose Chinese medicine gavage,and positive drug intraperitoneal injection.After 23 days of administration,lung tissues were taken for paraffin sectioning,HE staining and immunohistochemistry to detect β-catenin,vimentin,TMCO1,PI3K and Akt protein expression.Results:After modeling,the mice in each group were in good mental condition and had normal food and water.Compared with the model group,the body mass of mice in Guben Jiedu Prescription group decreased slightly,while that of mice in the positive drug group decreased significantly.HE staining results showed that compared with the model group,the tumor cells in the high,medium and low dose groups of Guben Jiedu Prescription were reduced in number,arranged into small nesting clusters or single scattered distribution,with some cells degenerating;the high dose group had the best effect,accompanied by tumor cell remnants.Immunohistochemical results showed compared with the model group,there was mildly elevated in β-catenin protein expression in the middle-dose group of Guben Jiedu Prescription(P<0.05),and significantly elevated in the high-dose and positive drug group(P<0.01);compared with the model group,there was mildly reduced in vimentin protein expression in the middle-dose group of Guben Jiedu Prescription(P<0.05),and significantly reduced in the high-dose and positive drug group(P<0.01);compared with the model group,there was mildly reduced in TMCO1 protein expression in the middle-dose group of Guben Jiedu Prescription(P<0.05),and significantly reduced in the high-dose and positive drug group(P<0.01);compared with the model group,there was reduced in PI3K protein expression in the high-dose group and positive drug group(P<0.05);compared with the model group,there was mildly reduced in Akt protein expression in the middle-dose and high-dose group(P<0.05),and significantly reduced in positive drug group(P<0.01).Part Ⅲ:the the effect and mechanism of the Guben Jiedu Prescription-containing serum for the inhibition of metastasis of lung adenocarcinoma A549 cellsObjectives:to verify the the effect and mechanism of the Guben Jiedu Prescription-containing serum for the inhibition of metastasis of lung adenocarcinoma A549 cellsMethods:prepare the drug-containing serum of Guben Jiedu Prescription.The experiments were divided into normal serum group(10%blank serum)、(2.5%、5%、10%)of Guben Jiedu Prescription serum、SC79 group、high dose of Guben Jiedu Prescription group and high dose+SC79 group、A549-TMCO1-29 cell group and A549-TMCO1-33 cell group;MTT assay was performed to detect the survival rate of A549 cells;scratch assay was performed to detect the migration force of A549 cells;Western blot to detect the expression of proteins;Real-time PCR to detect the expression of mRNA.Results:MTT and scratch assays showed that Guben Jiedu Prescription-containing serum inhibited the viability and migration ability of A549 cells(P<0.05).Real-time PCR results showed that N-cadherin and Vimentin mRNA expression was reduced in Guben Jiedu Prescription-containing serum group compared with the normal serum group(P<0.01).Western blot results showed that E-cadherin protein expression was elevated in Guben Jiedu Prescription-containing serum group compared with the normal serum group,N-cadherin、Vimentin、p-Akt/Akt、p-GSK-3β/GSK-3β protein expressiona were reduced((P<0.05,P<0.01).This indicates that Guben Jiedu Prescription-containing serum inhibits EMT and PI3K/Akt pathways.Rescue results showed that E-cadherin protein expression was reduced in high dose+SC79 group compared with high dose of Guben Jiedu Prescription group,N-cadherin、Vimentin、p-Akt/Akt、p-GSK-3β/GSK-3β protein expressiona were elevated(P<0.01).This indicates that the addition of SC79 attenuated the inhibitory effect of Guben Jiedu Prescription-containing serum on EMT.Moreover,western blot results showed that E-cadherin protein expression was elevated in A549-TMCO1-29 cell group and A549-TMCO1-33 cell group compared with the normal serum group,N-cadherin、Vimentin、p-Akt/Akt、p-GSK-3β/GSK-3β protein expressiona were reduced((P<0.05,P<0.01).This indicates that knockdown of TMCO1 inhibits EMT and PI3K/Akt pathways.Conclusions1.Through network pharmacology and molecular docking,there are multi-components and multi-targets in Guben Jiedu Prescription,and PI3K/Akt signaling pathway may be one of the main pathway for its action.2.Based on the results of in vivo and in vitro cellular assays,Guben Jiedu Prescription inhibited lung adenocarcinoma metastasis and epithelial mesenchymal transition(EMT)via the TMCO1/PI3K/Akt axis.