A Quantitative Proteomics Study On The Eeffects And Mechanism Of Artemisinin And Its Derivatives Treated Glioblastoma Cells

Glioma is the most common primary tumor of the central nervous system(CNS),which is divided into four grades(Ⅰ-Ⅳ)by the World Health Organization(WHO).Among them,the highest degree of malignant WHO grade IV glioblastoma(Glioblastoma,GBM)showed invasive growth,difficult to completely remove by the surgery,and then supplemented with standard chemoradiotherapy drugs,patients with poor prognosis and easy recurrence.Therefore,it is a medical problem that finding a safe,effective and non-toxic side-effect therapeutic drug is urgently needed to be solved.In recent years,there have been reports in the literature on the anti-tumor pharmacological effects of Artemisinin(ART)and its derivatives,combined with the characteristics of simple administration,fast absorption and permeability through the blood-brain barrier,which has brought hope to the treatment of GBM.It also allows such "old medicines" to find new uses.Although artemisinin compounds have a broad-spectrum anti-tumor effect,there are few studies on the treatment of GBM,and the mechanism of action is not clear.This study mainly explores how artemisinin derivatives can inhibit the role of human malignant glioma GBM cells,and how it will be applied to the clinical treatment of GBM.Therefore,this study is based on quantitative proteomics techniques and bioinformatics analysis,and integrates a clinical sample of the Cancer Genome Atlas(TCGA)database to study on the effects and mechanism of artemisinin and its derivatives treated GBM,and verified at the cellular and molecular levels.First,four drugs(ARS,ARM,ARS,and DHA)were tested against three human malignant glioma GBM cells(U87MG,U251MG,and T98G)using the Cell Counting Kit-8(CCK-8).Secondly,using quantitative proteomics based on mass spectrometry to identify differentially expressed proteins in U87MG cells treated with DHA,and further systematic analysis of differential proteins for bioinformatics,revealing artemisinin-derived the potential target and mechanism of action of GBM.Thirdly,based on the bioinformatics analysis results of the clinical samples of the TCGA database,the mechanism by which this derivative treats GBM extends to clinical applications.Finally,PI single staining combined with flow cytometry,DAPI staining,Transwell migration chamber and Western Blot experiments to verify the mechanism of DHA treatment of GBM cells in cell cycle,apoptosis and tumor migration and invasion.And reveal their changes at the molecular level.The results showed that the first DHA has the highest inhibitory activity against GBM cells among the four drugs of artemisinin,and U87MG cells are most sensitive to such drugs.Secondly,based on proteomics technology,there were 4842 quantitative protein quantified in U87MG cells treated with DHA,and 176 expression up-regulation and 179 expression down-regulation in the protein with 1.2-fold difference.Third,bioinformatics analysis of differential proteins showed that up-regulated proteins are mainly distributed on mitochondria,which are involved in oxidoreductase activity and participate in cell metabolism and redox processes and down-regulated proteins are mainly distributed in the nucleus,which is related to DNA polymerase activity.Participate in DNA replication and cell cycle processes.Among them,ubiquitin-conjugating enzyme E2C(UBE2C)is mainly involved in the regulation of cell cycle progression and protein degradation,which may be a potential target for DHA treatment of GBM.According to literature research,Forkhead box protein M1(FoxM1)is the upstream target of UBE2C.Fourth,based on clinical sample analysis in the TCGA database,compared with normal brain tissue(n=5),there were 6822 genes up-regulated and 5513 genes down-regulated in GBM(n=169)tissues.The GO enrichment of the first 5%of the up-regulated genes showed that GBM promoted the malignant transformation of tumor cells mainly by upregulating and expressing cell cycle-associated proteins.Secondly,FoxM1 and UBE2C are highly expressed in GBM tissues and are positively correlated.These results together reveal that the mechanism of action of DHA in the treatment of GBM is to down-regulate the expression of UBE2C in the downstream target by down-regulating FoxM1,thereby affecting the cell cycle progression of GBM.Fifth,combined with the above results,this study demonstrated at the cellular and molecular levels that DHA can significantly increase the G2/M phase ratio of U87MG cells,up-regulate the cyclin B1 and downregulate cyclin D1;and activate the apoptotic protein caspase 3 to cause nuclear fragmentation of U87MG cells and increase the percentage of subpopulations of Sub-G0 cells;significantly reduce the number of U87MG cells passing through the migration chamber.In conclusion,the results of this study indicate that DHA mainly down-regulates FoxM1 to regulate the down-regulation of downstream target UBE2C,thereby blocking the cell cycle of GBM in G2/M phase,and also induces apoptosis of GBM cells and inhibits migration and invasion of GBM cells.ability.It can be seen that artemisinin and its derivatives have the potential to treat GBM,which lays a foundation for the subsequent drug optimization and application research based on anti-GBM of this derivatives.