Triptolide reverses epithelial-mesenchymal transition in glioma cells via inducing autophagy.

BACKGROUND

To observe the effects of triptolide (TP) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of glioma cells, and to explore the possible mechanisms of phenotypic changes in EMT.

METHODS

The U87 and U251 glioma cell lines were treated TP. The Cell Counting Kit-8 (CCK-8) method was used to detect the half-maximal inhibitory concentration (IC) of TP in these two cell lines and the inhibition of cell proliferation at the IC concentration. The wound-healing experiment and Transwell invasion assay were used to detect the cells' migration and invasion abilities, respectively. Using western blot protocol, the expression levels of the EMT markers were analyzed, and the levels of the autophagy markers were also detected. The pEGFP-C2-LC3B plasmid was transfected into glioma cells, and the effect of TP on autophagy was detected by immunofluorescence. A subcutaneous tumor model in nude mice was established to observe the effect of TP on cell proliferation , and immunohistochemistry (IHC) was used to detect the expression levels of EMT markers in mouse tumor tissues.

RESULTS

TP significantly inhibited the proliferation of U87 and U251 cells in a dose- and time-dependent manner. TP had a significant inhibitory effect on the migration and invasion of U87 and U251 cells. Western blot showed that TP reversed the process of EMT in glioma cells, which was evidenced by the upregulated expression of the epithelial marker E-cadherin, and the downregulated expression of the mesenchymal markers N-cadherin, Vimentin, ZEB1, Snail, and Slug. TP increased autophagy in glioma cells, increased the LC3B II/I ratio, and upregulated Beclin-1 and Atg-7 expression. Immunofluorescence showed that the number of autophagosomes increased significantly after TP was applied to cells. In the nude mouse subcutaneous tumor model, experiments revealed an inhibitory effect of TP on glioma cell proliferation . IHC confirmed that the expression of E-cadherin was upregulated in mouse tumor tissues, while the expression levels of N-Cadherin and Vimentin were downregulated.

CONCLUSIONS

TP can inhibit glioma cell proliferation, migration, and invasion, and reverse EMT progression. The possible mechanism of EMT reversal in glioma cells is that TP induces autophagy.