Effects Of Triptolide On Migration,invasion And EMT-like Phenotype Of Glioma Cells

Objective:To observe the effects of triptolide on proliferation,migration,invasion and EMT-like phenotype of glioma cells,and to explore possible mechanisms of phenotypic changes in EMT.Method:Triptolide was applied to U87 and U251 glioma cell lines,and CCK-8method was used to detect the IC50 of TP on the two cells and the inhibition of cell proliferation at IC50 concentration.The cell scratch test was used to detect the migration ability of the cells,and the invasion ability of the cells was detected by transwell invasion assay.Western Blot analyzed epidermal mesenchymal?EMT?markers E-Cadherin,N-Cadherin,Vimentin,ZEB1,Snail,Slug expression levels,also detecting autophagy markers LC3B,Beclin-1,Atg-7.The pEGFP-C2-LC3B plasmid was transfected into glioma cells,and the effect of TP on autophagy was detected by immunofluorescence.Subcutaneous tumor test in nude mice was used to detect the inhibition of TP cell proliferation in vivo,and immunohistochemistry was used to detect the expression level of EMT markers in tumor tissues.Result:Triptolide significantly inhibited the proliferation of U87 and U251 cells in a dose and time-dependent manner.The IC₅₀ of U87 cells was 20 ng/ml,while the IC₅₀ of U251 cells was 50 ng/ml.Triptolide had a significant inhibitory effect on the migration and invasion of glioma cell lines U87 and U251.Western Blot experiments showed that TP reversed the process of EMT in glioma cells——the expression of epithelial marker E-Cadherin was up-regulated,while the expression of mesenchymal markers N-Cadherin,Vimentin,ZEB1,Snail and Slug were down-regulated.Triptolide caused increased autophagy in glioma cells,increased LC3B II/I ratio,and up-regulated Beclin-1 and Atg-7 expression.Cellular immunofluorescence showed that the number of autophagosomes increased significantly after TP was applied to cells.In nude mice,subcutaneous tumor experiments demonstrated the inhibitory effect of TP on proliferation of glioma cells in vivo.Immunohistochemistry confirmed that the expression of EMT marker E-Cadherin was up-regulated in tumor tissues,and the expression of mesenchymal markers N-Cadherin and Vimentin was down-regulated.Conclusion:Triptolide can inhibit glioma cell proliferation,migration,invasion,and reverse epithelialization?EMT?progression,and the possible mechanism of EMT reversal is that triptolide induces autophagy in glioma cells:Autophagy mediates E-cadherin and Vimentin expression through pathways such as ZEB1 and Snail,thereby inhibiting EMT progression.