TRAIL and Celastrol Combinational Treatment Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells via Targeting Wnt/ß-catenin Signaling Pathway.

OBJECTIVE: To investigate the mechanistic basis for the anti-proliferation and anti-invasion effect of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) and celastrol combination treatment (TCCT) in glioblastoma cells. METHODS: Cell counting kit-8 was used to detect the effects of different concentrations of celastrol (0-16 µmol/L) and TRAIL (0-500 ng/mL) on the cell viability of glioblastoma cells. U87 cells were randomly divided into 4 groups, namely control, TRAIL (TRAIL 100 ng/mL), Cel (celastrol 0.5 µmol/L) and TCCT (TRAIL 100 ng/mL+ celastrol 0.5 µmol/L). Cell proliferation, migration, and invasion were detected by colony formation, wound healing, and Transwell assays, respectively. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to assess the levels of epithelial-mesenchymal transition (EMT) markers (zona occludens, N-cadherin, vimentin, zinc finger E-box-binding homeobox, Slug, and ß-catenin). Wnt pathway was activated by lithium chloride (LiCl, 20 mol/L) and the mechanism for action of TCCT was explored. RESULTS: Celastrol and TRAIL synergistically inhibited the proliferation, migration, invasion, and EMT of U87 cells (P<0.01). TCCT up-regulated the expression of GSK-3ß and down-regulated the expression of ß-catenin and its associated proteins (P<0.05 or P<0.01), including c-Myc, Cyclin-D1, and matrix metalloproteinase (MMP)-2. In addition, LiCl, an activator of the Wnt signaling pathway, restored the inhibitory effects of TCCT on the expression of ß-catenin and its downstream genes, as well as the migration and invasion of glioblastoma cells (P<0.05 or P<0.01). CONCLUSIONS: Celastrol and TRAIL can synergistically suppress glioblastoma cell migration, invasion, and EMT, potentially through inhibition of Wnt/ß-catenin pathway. This underlies a novel mechanism of action for TCCT as an effective therapy for glioblastoma.

Celastrol enhances TRAIL-induced apoptosis in human glioblastoma via the death receptor pathway.

PURPOSE: Glioblastoma is the most common, malignant and devastating type of primary brain tumor. Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is characterized by its lethality to precancerous and cancerous cells. However, many kinds of tumor cells, including most glioma cells, tend to evade TRAIL-induced apoptosis. Celastrol is a pleiotropic compound from a traditional Chinese medicine that has proven to be useful as a sensitizer for TRAIL treatment. However, the underlying mechanism and role of celastrol in the sensitization of glioma cells remain to be elucidated. METHODS: The viability of glioma cell lines was examined by the CCK-8 assay. The expression of DR5 was detected by reverse transcriptase quantitative real-time PCR. The protein expression of DR5, cleaved caspase-8, cleaved caspase-3 and PARP were measured by western blot. The apoptosis rates and the sub-G1 population were detected by flow cytometry. The cellular morphological changes were assessed by TUNEL apoptosis and Hoechst 33258 staining assays. The knockdown of DR5 expression was conducted by siRNA. RESULTS: In this study, we observed that celastrol treatment inhibited cell viability in a dose-dependent manner, while glioma and normal human astroglial cell lines were resistant to TRAIL treatment. We also observed that the antiproliferative effects of TRAIL in combination with a noncytotoxic concentration of celastrol were significantly greater than those of celastrol or TRAIL alone. In addition, cell death induced by the combination treatment was apoptotic and occurred through the death receptor pathway via activation of caspase-8, caspase-3, and PARP. Furthermore, celastrol upregulated death receptor 5 (DR5) at the mRNA and protein levels, and siRNA-mediated DR5 knockdown reduced the killing effect of the combination drug treatment on glioma cells and reduced the activation of caspase-3, caspase-8 and PARP. CONCLUSIONS: Taken together, the results of our study demonstrate that celastrol sensitizes glioma cells to TRAIL via the death receptor pathway and that DR5 plays an important role in the effects of this cotreatment. The results indicate that this cotreatment is a promising tumor-killing therapeutic strategy with high efficacy and low toxicity.