Preliminary Exploration Of The Effect And Mechanism Of Cynaropicrin Inhibiting The Growth Of Melanoma Cells

Objective Melanoma is a type of cancer that is easy to metastasize at an early stage and has a high mortality rate.At present,the disease is mainly treated with drugs.Early in situ melanoma can be cured by surgery.Although there are currently advanced melanoma treatment drugs available Immunization and targeted drug therapy,but their use has been restricted due to the low response rate and drug side effects.Cynaropicrin is a sesquiterpene lactone derived from a variety of plants.It has good biological effects and has potential anti-cancer effects.In this project,by observing the inhibitory effect of cynaropicrin on human melanoma cells and studying the mechanism,it explored the possibility that cynaropicrin can be used as a melanoma therapeutic drug.Methods Treat human melanoma A375 cells and A2058 cells with 0,5,10,20,40,60,and 80 μM cynara,respectively,observe cell growth and proliferation,draw cell viability diagrams,and calculate IC50(the number of surviving cells is inhibited by the drug The concentration of the drug at half the original number of cells).Western Blot was used to detect the expression of apoptosis-related proteins BAX,BCL-2 and PARP,etc.,to observe whether cynaropicrin induces cell apoptosis,and to detect the expression levels of cell cycle-related proteins Cyclin B1 and CDK1 to determine The effect of cynaropicrin on the cell cycle of melanoma.After planting A375 cells in nude mice subcutaneously to form tumors,they were divided into 4 groups and injected intraperitoneally with 0(control group),10 mg/Kg,20 mg/Kg,40 mg/Kg cynaropicrin(concentrations are IC50)After treatment,the nude mice were slaughtered and tumor specimens were taken to observe the growth of the tumor.Transcriptome sequencing was performed on the human melanoma A375 cells treated with cynaropicrin for 24 hours and the control group treated with DMSO(solvent),the differential gene expression of the two groups was analyzed,and GO,KEGG annotation,and KEGG signal pathway enrichment were performed for preliminary Explore the key genes and possible mechanisms of cynaropicin inhibiting the growth of melanoma cells.Results The growth of human melanoma A375 cells and A2058 cells treated with different concentrations of cynaropicrin was inhibited,with IC50 of 27.4μM and 24.2μM,respectively.The results of Western Blot detection of BAX protein,BCL-2 protein and PARP protein showed that the higher the concentration of intervened cynaropicrin,the expression of BAX protein and PARP protein was significantly up-regulated,and the expression of BCL-2 protein was significantly downregulated.Western Blot was used to detect cyclin.The results of B1(Cyclin B1)and cyclindependent kinase 1(CDK1)showed that the higher the concentration of intervened cynaropicrin,the expression of Cyclin B1 was significantly up-regulated,and the expression of CDK1 was significantly down-regulated.In animal experiments,the higher the dose of cynaropicin injected into the intraperitoneal cavity,the more obvious the inhibition of the growth of tumors in nude mice with subcutaneous tumor formation,and no significant effect on the body weight of nude mice.Transcriptome sequencing of A375 cells after the intervention of cynaropicrin and A375 cells that were not treated with cynaropicrin showed that there were a total of 348 differentially expressed genes,of which 272 differentially expressed genes were up-regulated and differentially down-regulated.76.According to protein interaction network analysis,IL-6,CXCL-8 and SERPINE are the differentially up-regulated proteins.The transcribed proteins are the most connected proteins.CENPA,B3GAT1,AURKB are the significantly down-regulated differential genes.The best protein has an important position in the protein network of melanoma,and may have a very important relationship with the growth and proliferation of melanoma.According to the scatter diagram of KEGG signal pathway enrichment,the enriched signal pathways include TNF signaling pathway,VEGF signaling pathway,TGF-β signaling pathway,FOXO signaling pathway,NF-kappa b signaling pathway and Erb B signaling pathway.Conclusions 1.In vitro cynaropicrin may inhibit the growth and proliferation of melanoma A375 and A2058 cells by promoting apoptosis and blocking cell cycle progression,and its IC50 is 27.4μm and 24.2μm,respectively.2.Cynaropicrin can significantly inhibit the growth of melanoma tumors in the subcutaneous tumor model of nude mice induced by human melanoma A375 cells in vivo.3.The mechanism of cynaropicin inhibiting the growth of melanoma cells may be related to the TNF signaling pathway,VEGF signaling pathway,TGF-β signaling pathway,FOXO signaling pathway,NF-kappa b signaling pathway,and Erb B signaling pathway.