Study On The Regulation Of Cinobufagin On Proliferation,Apoptosis,and Autophagy Of Oral Squamous Cell Carcinoma Cells Based On Network Pharmacology

Objectives:Oral squamous cell carcinoma(OSCC)has a high incidence rate and poor prognosis.At present,no ideal drug treatment has been found.Cinobufotalin is extracted from the skin of Chinese giant salamander or black eyed toad,and can promote hydration,reduce swelling,clear heat and detoxify,and soften and disperse nodules.In terms of anti-tumor effects,it has various mechanisms such as inhibiting tumor cell proliferation,invasion,and metastasis,promoting cell apoptosis,inducing autophagy,and regulating immune response.It has the potential to treat OSCC.This study determined the effect of cinobufagin on the proliferation and apoptosis of OSCC cells through network pharmacology analysis and in vitro cell experiment validation,and explored the effect of cinobufagin on OSCC cells by regulating the expression of autophagy related gene m RNA.Materials and Methods:Part 1: Network Pharmacological Analysis of the Effect of Huachansu on OSCC1.Obtain potential therapeutic targets for OSCC through Gene Cards and OMIM databases;Identify the chemical structural formulas of cinobufalin and its chemical components in the Pub Chem database,and predict target genes through the Swiss Target Prediction website;Obtain the intersection target of Huachansu OSCC on Venn website;2.Use STRING database to obtain PPI network and use Cytoscape software to draw network diagram;3.Conduct GO enrichment analysis and KEGG pathway analysis on intersecting targets using the Metascape database;4.Analyze the binding force between Huachansu and core targets through molecular docking technology.Part 2: In vitro experimental study on the effect of cinobufalin on OSCC cells1.Detect the proliferation of CAL27 and HOK cells through the CCK-8experiment;2.Flow cytometry was used to detect the apoptosis of CAL27 cells;3.RT-PCR method is used to detect the expression levels of core targets and autophagy related gene m RNA in CAL27 cells,and to explore the possible pathway of Huachansu in treating OSCC.Results:Part 1: Network Pharmacological Analysis1.The target was obtained by screening OSCC in the Gene Cards database with a Relevance score of ≥ 50.The OSCC target in the OMIM database was supplemented to obtain 76 related targets for the action of cinobufalin on OSCC;2.PPI analysis revealed that the core targets of Huachansu in the treatment of OSCC are STAT3,SRC,AKT1,PIK3R1,and MAPK3;3.GO enrichment analysis includes 56 biological processes,including 20 molecular functions,20 biological processes,and 16 cellular components,mainly involving protein phosphorylation,receptor complexes,protein kinase activity,etc;The KEGG pathway enriched and screened 20 signaling pathways,with the Pathways in cancer signaling pathway having the highest enrichment level,followed by the PI3K-Akt signaling pathway;4.Molecular docking results showed that the binding ability between cinobufagin and core target was good.Part 2: In vitro Cell Experiment Research1.The CCK-8 experiment results showed that when the concentration of cinobufalin was less than 4mg/ml,there was no significant inhibitory effect on HOK cell proliferation(P>0.05),but it could inhibit OSCC cell proliferation in a time and dose dependent manner(P<0.0001).When the concentration of cinobufalin was ≥4mg/ml,it had an inhibitory effect on HOK cell proliferation(P<0.05),but it had a more significant inhibitory effect on CAL27 cell proliferation(P<0.0001);2.The results of flow cytometry experiments showed that compared with the untreated group,cinobufagin induced OSCC cell apoptosis in a concentration dependent manner;The RT-PCR experiment results showed that at a concentration of 4mg/ml,cinobufagin significantly upregulated the m RNA expression of autophagy related genes Beclin1(P<0.001)and LC3(P<0.0001),and downregulated the expression of core targets STAT3(P<0.001),SRC(P<0.001),AKT1(P<0.0001),PIK3R1(P<0.0001),and MAPK3(P<0.0001).Conclusions:1.Through network pharmacology analysis,a total of 76 targets were identified for the action of cinobufalin on OSCC,with core targets being STAT3,SRC,AKT1,PIK3R1,and MAPK3.Molecular docking showed that cinobufalin binds well to the core target.KEGG analysis shows that the PI3K-AKT pathway is the core pathway for the action of cinobufalin on OSCC.2.The results of cell experiments indicate that cinobufagin can inhibit the proliferation of OSCC cells and promote cell apoptosis,increase the expression level of autophagy related gene m RNA in OSCC cells,induce cell autophagy,and inhibit the expression level of core target m RNA.This discovery provides theoretical support and scientific basis for the anticancer treatment of OSCC with Huachansu.