Inhibition Of Malignant Growth Of Gliomas By Marizomib

Objective: 1.To investigate the inhibition of malignant growth of glioma cells by marizomib and screen related signaling pathways using proteasome inhibitor marizomib to intervene U251,U87,U118,T98 G glioma cells.2.Using proteasome inhibitor marizomib to intervene in human glioma implant nude mice model,to explore marizomib inhibited malignant growth of glioma implant tumors in nude mice and analyze the changes of its signaling pathway.Methods: 1.A variety of human glioma cells(U251,U87,U118,T98G)were cultured,and different concentrations of marizomib(0 nmol/ L,100 nmol / L,400 nmol / L,800 nmol / L,1000 nmol / L)were used to intervene with human glioma cells for 48 h,then the cell morphological changes were observed and photographed microscopically and recorded;The apoptosis of glioma cells was detected using annexin V-APC / 7-AAD assay.After treatment with 400 nmol / L marizomib with or without intervention in human glioma T98 G cells for 48 hours,tandem mass tag(TMT)-based proteomics was applied to detect protein expression changes in glioma cells treated with and without marizomib intervention,and bioinformatics tools were used to analyze signaling pathways acting on glioma cells.2.Establishment of U251 glioma subcutaneous implant tumor nude mice model,divided into experimental and control groups,each group of six male 6-week-old nude mice,the experimental group was treated with the proteasome inhibitor marizomib for 3 weeks,the control group was treated with solvent for 3 weeks.The body weight,tumor size,skin cleanliness,diet,and activity of the two groups of nude mice were observed.After tumor implantation on the 10 th day,tumor diameter and transverse diameter were measured at regular intervals and tumor volume was calculated.Twenty one days later,tumor tissues were harvested to define histopathological properties of tumors by HE staining,immunohistochemistry,and metabolomics was used to determine metabolite and metabolic pathway changes in subcutaneously implanted glioma tissues from the marizomib intervention and control groups.Results: 1.After treatment of U251,U87,T98 G,and U118 glioma cells with marizomib(0 nmol / L,100 nmol / L,400 nmol / L,800 nmol /L,and 1000 nmol / L)for 48 h,compared with the 0 nmol / L blank control group,the cells of the four groups gradually ruptured,and the culture solution showed suspended necrotic cells and cell debris,as well as slow growth of glioma cells.The apoptosis rate of U251,U87,T98 G,U118 glioma cell lines increased gradually as the concentration gradient of marizomib increased.The proteomic results showed 220 up-regulated proteins and 265 down regulated proteins in the glioma T98 G cells treated with marizomib intervention compared with the non intervention human glioma T98 G cells,and the differential protein enrichment showed that the main focus was on DNA replication,cell cycle,lipid metabolism and so on.2.12 all the tumors grew out in the axilla of nude mice(100%success rate of implanting tumors),and it was visible that the implanting tumors protruded from the surface of the mouse skin,which was round,oval or irregular,touched harder and less mobile.After marizomib treatment,the nude mice moved well,fed normally,the skin color was good,the subcutaneous tumor growth was significantly slowed,the tumor volume was small,the gross view of the tissue was round or oval or irregularly lobulated,no obvious envelope could be observed,the color of the tumor was deeper than the surrounding normal tissue,it was erosive fish flesh like,and the control group showed tissue hemorrhage and necrosis.Microscopic observation of subcutaneous implanted tumor tissue,he staining: magnification 200 × Posteriorly,the glioma cells were round or spindle shaped and densely arranged with large nucleoli with inverted nuclear cytoplasmic ratio and atypical nuclei on light microscopy.In the control group,haemorrhagic necrosis and blood vessel proliferation were obvious.Immunohistochemistry: magnification 400 ×Lower,GFAP、Ki-67、EGFR、ATRX、CHI3L1、SOX2、CD133 、PDL1 staining in the glioma tissue was significantly lower in the experimental group than in the control group.3.Metabolomics of subcutaneous implant tumors the results showed that a total of nine endogenous metabolites(glycerol,taurine,myristic acid,pentadecanoic acid,9-hexadecenoic acid,palmitic acid,petroleum sulfonic acid,4-hydroxybenzyl alcohol,cholesterol)were changed in the experimental group compared with the control group;And screening resulted in three differential metabolic pathways(primary bile acid biosynthesis,fatty acid biosynthesis,and taurine and low taurine metabolism).Conclusions: 1.Marizomib can induce glioma cells to undergo apoptosis and affect DNA replication,cell cycle,lipid metabolism in glioma cells.2.Marizomib inhibited glioma malignant growth and affected lipid metabolism of glioma tissues in tumor growing nude mice,the mechanism of which needs to be further explored.