The Inhibitory Action and Mechanisms of Marsdenia Tenacissima Oral Liquid on the Proliferation of Gastric Cancer Cells Based on Network Pharmacology and in vitro Experiments and the Sensitization Effect of Combining Erlotinib and Apatinib
Objective To investigate the potential mechanism of Marsdenia Tenacissima(MT)oral liquid in inhibiting gastric cancer(GC)by network pharmacology,molecular docking and in vitro cell experiments.Methods The chemical components and target genes of MT were collected by literature searching,TCMSP and Swiss Target Prediction databases.Disease-related targets were obtained by GeneCard database.The protein interaction(PPI)network of intersection targets was constructed by STRING platform and Cytoscape software.GO functional enrichment and KEGG signaling pathways were obtained by Metascape network platform,molecular docking validation of some active components and potential targets was performed by AutoDock Vina and other software.Methyl thiazolyl tetrazolium(MTT)method was used to detect the inhibiting effect of MT oral liquid on the proliferation of GC cells and the synergistic effects of MT oral solution combined with Erlotinib and Apatinib on GC cells were also observed.Flow cytometry was used to detect the apoptosis induced by MT oral liquid and its effect on the cell cycle of GC cells.RT-qPCR was used to detect the effects of MT oral liquid on the cell cycle and apoptosis-related gene expression in GC cells.Western blot was used to verify the targets screened by network pharmacology method and molecular docking.Results A total of 17 main active components,1 880 key targets of GC and 167 main KEGG pathways were obtained.The MTT tests showed that MT oral liquid with the concentration of 40～200 mg/mL had an inhibitory effect on the proliferation of GC cells in a concentration-dependent and time-dependent manner(P<0.01).When combined with Erlotinib and Apatinib,it had a synergistic effect(P<0.01).The results of flow cytometry showed that MT oral liquid with the concentration of 40～160 mg/mL could change the cell cycle distribution of GC block it in G0/G1 phase and induce its apoptosis(P<0.01).RT-qPCR results showed that MT oral liquid could up-regulate the expression of Bcl-2 associated X protein(BAX)and down-regulate the expressions of Bcl-2 apoptosis regulator(Bcl-2),cyclin dependent kinase(CDK)4 and CDK6(P<0.05).The results of WB showed that MT oral liquid could downregulate the phosphorylation expression levels of p-EGFR,p-PI3K,p-AKT,and p-mTOR,thereby reducing the ratio of p-EGFR/EGFR,p-PI3K/PI3K,p-AKT/AKT1,and p-mTOR/mTOR,up-regulate the expressions of Bax,P53 and P21,and down-regulate the expressions of Bcl-2,CDK4 and CDK6 significantly(P<0.05).Conclusion MT oral liquid could inhibit the proliferation of GC cells effectively,arrest cell cycle,induce apoptosis of GC cells,and improve the sensitivity of small molecule target drugs including Erlotinib and Apatinib in the treatment of GC.The mechanism is related to the up-regulation of Bax,P53 and P21 proteins,and down-regulation of Bcl-2,CDK4 and CDK6 proteins and inhibition of EGFR signaling pathway via regulating PI3K/AKT/mTOR signaling pathway.The experimental results had verified the results of network pharmacology and molecular docking.
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