Prediction and verifcation of the key ingredients and molecular targets of Guizhi Fuling capsule against tumour metastasis and resistance

Purpose:The well-known traditional Chinese formula Guizhi Fuling capsule(GFC)has been reported to reverse ovarian cancer drug resistance.Extrachromosomal DNA(ecDNA)plays an important role in tumour metastasis and resistance.The purpose of this study was to investigate the potential mechanisms by which GFC blocks tumour metastasis and reverses drug resistance by targeting ecDNA.Methods:CNKI and PubMed were used to obtain pharmacokinetic research data on GFC in rats,and the bioactive ingredients detected in rat serum or plasma were collected.Network databases were used to screen the abnormally expressed genes in ecDNA,tumour metastasis genes,resistance genes,and the active ingredient targets of GFC.The KOBAS3.0 database was used to enrich the KEGG pathways and GO functions;the STRING platform was used to construct the core protein interaction network;and the molecular docking online tool SwissDock was used to analyse the binding activity of the core targets and the active ingredients.RT-qPCR,Western blotting and laser confocal microscopy were used to verify the efect of the sera containing GFC on ecDNA,mRNA and protein expression of key targets.Results:Twenty-three bioactive ingredients of GFC were retrieved from PubMed and CNKI.Nine shared targets were simultaneously involved in abnormal genes in ecDNA,tumour metastasis and resistance and the active ingredient targets of GFC.GO functional analysis indicated that the cotargets involved cell proliferation,apoptotic regulation,nuclear functions,etc.The potential pathways involved in the reversal of tumour metastasis and drug resistance of GFC were the PI3K-Akt signalling,cancer,and platinum drug resistance pathways.Three shared proteins targeting ecDNA(AKT1,EGFR and MYC)stand out from the top 20 PPI targets,and all of the bioactive ingredients of GFC have strong binding afnity to the three proteins.The active ingredients can reduce the expression of MYC,EGFR and AKT1 mRNA and protein and the amount of ecDNA in drug-resistant OC cells.Conclusions:GFC targeting ecDNA to reverse tumour metastasis and drug resistance has the characteristics of multiple ingredients,multiple targets,and multiple pathways,which provides a new perspective for the development of new drugs targeting ecDNA to beneft tumour treatment.