Effect And Mechanism Of Sodium Cantharidinate And Vitamin B6 Combined With Eutidrone In Inhibiting Triple-negative Breast Cance

Purpose:To study the effects of sodium cantharide and vitamin B6 combined with Utidelone on the proliferation,cell cycle and apoptosis of human breast cancer MDA-MB-231 cell line,and to analyze the potential mechanism of cantharides in the treatment of breast cancer by means of network pharmacology and molecular docking research.Method:1.The CCK-8 method was used to detect the effects of sodium cantharidate,vitamin B6 and Utidelone on the proliferation of human breast cancer MDA-MB-231 cell line,and the IC50 values were calculated respectively.Select the appropriate concentration of sodium cantharide and vitamin B6 to combine with Utidelone,and test the inhibition rate of the combined group.The effects of sodium cantharidate and vitamin B6,eutidelol alone and sodium cantharidate and vitamin B6 combined with eutidelol on cell cycle and apoptosis of human breast cancer MDA-MB-231 cell line were detected by flow cytometry.2.The effective chemical components and targets of cantharides were screened by searching TCMSP,the related target genes of breast cancer were searched in Genecards database,the"compound-target"network diagram and protein network interaction analysis（PPI）of cantharides were constructed by using Cytoscape3.7.1software,GO and KEGG enrichment analysis was carried out by David database,molecular docking was carried out by using Auto Dock Vina,and finally Py MOL and Lig Plot+were used.3.The Western Blot method was used to verify bcl-2 and BAX in seven genes screened by network pharmacology.In addition,the signal pathway proteins p21,p27,cyclin D1 and bcl-xl related to cell cycle and apoptosis were detected to explore the changes of molecular mechanism.Results:1.Sodium cantharidate and vitamin B6 injection:IC₅₀24h:10.05μmol/L,IC₅₀48h:7.485μmol/L.Utidelone IC₅₀24h:37.61μmol/L,IC₅₀48h:20.26μmol/L.2.When sodium cantharidate and vitamin B6 injection with a concentration of10μmol/L were combined with 0.1μmol/L,1μmol/L,10μmol/L,100μmol/L,respectively,the IC5024h of Utidelone was as follows:1.022μmol/L,IC5048h:1.01μmol/L.3.The results of cell cycle test showed that compared with the control group,the combined group accounted for 46.8%in G2/M phase,which was significantly higher than that of the single drug group,21.2%higher than that of the cantharidin single drug group,and 27%higher than that of Utidelone single drug group.Both sodium cantharidin and Utidelone could block the cells in G2/M phase,and sodium cantharidin could enhance Utidelone..4.The results of flow cytometry showed that the apoptosis rates of cells induced by10μ/L sodium cantharidate and vitamin B6 injection and 0.1μmol/L Utidelone injection were 8.91%and 13.8%,respectively.Compared with the control group,sodium cantharidate single drug group,and Utidelone single drug group,the apoptosis rate induced by sodium cantharidate and vitamin B6 combined with Utidelone was78.4%.5.According to network pharmacology and molecular docking analysis,there are 5active components of Mylabris,31 related targets,1123 breast cancer targets,and 79core targets by topological analysis.GO analysis includes 81 biological processes,12cell components,23 molecular functions,and 59 KEGG enrichment results,involving Pathways in cancer,Micro RNAs in cancer and other signal pathways.The molecular docking results showed that the binding energy of cantharidin with TP53,Bcl-2 and PTGS2 was low,which indicated that they had good binding activity,suggesting that Bax,Bcl-2 and PTGS2 were the important targets of cantharides.6.Western Blot showed that the expression of Bax was weak in the control group and single drug group,but significantly increased in the combined group,while the expression of Bcl-2 and Bcl-xl was significantly down-regulated in the combined group.P21 and P27,as cell cycle inhibitors,were significantly up-regulated in the combined group.The expression of cyclin D1 was down-regulated in the combined group.Conclusion:Sodium cantharidate can inhibit the proliferation of breast cancer MDA-MB-231cells,and sodium cantharidate combined with enhanced chemotherapy drug,Utidelone,can inhibit the growth and proliferation of breast cancer MDA-MB-231cells,and the cells are mainly blocked in G2/M phase.Sodium cantharidate can also enhance the induction of early apoptosis by Utidelone.Network pharmacology and molecular docking predict that cantharides can regulate TP53 and Bcl-2 through cancer signals and other pathways,and has anti-breast cancer effect.In vitro experiments have proved that the molecular mechanism of cantharides inhibiting the growth and development of human breast cancer MDA-MB-231 cells may be related to its effective regulation of Bcl-2 and Bax proteins.This study explored the effect and mechanism of sodium cantharidate and vitamin B6 combined with Utidelone in inhibiting triple negative breast cancer,which provided a theoretical basis for further research on its optimal treatment strategy for breast cancer.