Analysis Of The Mechanism Of Action Of Cordycepin Combined With Doxorubicin Against Triple-Negative Breast Cancer By Network Pharmacology

Cordyceps militaris(L.)Fr.is a precious medicinal and edible mushroom in China.As one of the main active ingredient Cordyceps militaris,cordycepin has natural advantage of low toxicity,high safety and strong activity and gains extensive attention for obvious anti-tumor effects.Triple-negative breast cancer(TNBC),as the strongest "killer" of female breast cancer,is still mainly treated with chemotherapy.It is one of the important directions of current treatment to find natural products or drugs for combination to enhance the effect of chemotherapy.Consequently,cordycepin was used in combination with the first-line anti-tumor drug(doxorubicin)in the study,which enriches pharmacodynamic basic theory of cordycepin of Cordyceps militaris medicinal substance and provides new ideas for clinical therapeutics of TNBC.In present study,the combined effect was first detected.Secondly,the effects of cell viability,cell proliferation,cell metastasis and cell apoptosis and toxicity of drug combination on TNBC was explored in vivo and in vitro.Then,the molecular mechanism of drug combination was analyzed by network pharmacology.Finally,the mechanism was preliminarily verified through molecular docking and biological experiments.The main research results are as follows:(1)Combination index(CI)showed that CI value of cordycepin combined with doxorubicin against TNBC were all less than 1,indicating synergistic effects at experimental concentration.Especially CI value is only 0.665 at cordycepin(80 μM)combined with doxorubicin(1 μM),and cell inhibiting rate reach 60.31%±1.06,which more than median lethal rate.(2)The results of cell experiment in vitro showed that drug combination make cells become round,gradually shed and broken.Cloning information assay suggested that drug combination inhibited cell proliferation,and cell proliferation rate was only 7.03%±1.19.Motility assay showed that cell migration was inhibited,and cell migrating rate was only 18.82%±2.43.Hochest 33258 and DNA Ladder assay showed that cell apoptosis was induced,and cell apoptotic rate up to 78.52%±11.18,and broken DNA bands of cell apoptosis occurred.(3)In vivo experiment of xenograft tumor in nude mice showed that tumor weight of control group was 3.57 times that of combined group.Tumor inhibiting rate of combined group was 79.53%±12.63,which was1.21 and 1.34 times that of cordycepin group and doxorubicin group respectively.We further observed weight evenly of animal,ruddy color,uniform texture and better elasticity of liver in combined group.Liver index and hepatic function index ALT,AST was normal compared with the control group,with no significant difference(P>0.05).These results showed that drug combination significantly inhibited the growth of TNBC in vivo,with no obvious toxicity.(4)Trough network pharmacology explored the mechanism of drug combination,we gained the potential targets of cordycepin and doxorubicin 147 and 2469 respectively.Drug combination against TNBC involved with 76 targets,which were mainly focused on cell apoptosis,cell metastasis and cell cycle by GO analysis.Analysis of KEGG and Enrichr releaved that 76 targets were main enrichment in 6 signaling pathway of relating to tumor growth including TNF,IL-17,AGE-RAGE and apoptotic pathway.TNF signaling pathway was most significantly and crosstalk with other enrichment pathways,indicating the molecular mechanism of drug combination associated with the regulatory for 11 targets enrichment in TNF pathway including CFLAR、NFKB1、MAP3K7、MAPK8、CASP3、ICAM1、MMP3、MMP9、JUNB、CXCL1、CCL2.(5)Molecular docking showed that the binding energy of cordycepin and doxorubicin with 11 targets all less than 0,indicating 2 compound both could spontaneously bind with targets.Cordycepin and doxorubicin could bond with amino acid residues of important proteins,such as CFLAR,MMP3 and MAP3K7,through hydrogen bond,PI bond and H-PI bond to stably bind in active pocket of protein.Drug combination also accordingly increased CASP3 and MAPK8;and decreased CFLAR,NFKB1,MAP3K7,ICAM1,MMP3,MMP9,JUNB,CXCL1 and CCL2 on gene and protein levels,which consistent with analysis of network pharmacology.These results indicated that drug combination regulated 11 targets of TNF pathway to inhibit the growth of TNBC.Cordycepin combined with doxorubicin synergistically inhibits the growth of TNBC in vivo and in vitro,with no obvious toxicity,which the key mechanism associated with the regulatory for drugs on multiple targets of TNF pathway.These provides scientific basis to enrich pharmacodynamic basic theory of cordycepin of Cordyceps militaris medicinal substance...