Hedgehog/Gli1 signaling pathway regulates MGMT expression and chemoresistance to temozolomide in human glioblastoma

Abstract

BackgroundChemoresistance of glioblastoma (GBM) is a feature of this devastating disease. This study is to determine the relationship between Hedgehog (HH)/Gli1 signaling pathway and chemoresistance to temozolomide (TMZ) in human GBM.MethodsWe analyzed Gli1 nuclear staining and O6-methylguanine DNA methyltransferase (MGMT) expression in 48 cases of primary GBM tissues by immunohistochemistry. Quantitative PCR, western blot, methylation-specific PCR, cell proliferation and apoptosis assay were used to investigate changes of MGMT expression and chemosensitivity to TMZ after manipulating HH/Gli1 signaling activity in A172 and U251 GBM cell lines. Chromatin immunoprecipitation assay was utilized to identify potential Gli1 potential binding sites in MGMT gene promoter region. We established GBM xenografts using U251 cells to assess whether inhibiting HH/Gli1 signaling activity restored chemosensitivity to TMZ.ResultsO6-Methylguanine DNA methyltransferase-positive GBM tissues had a significantly higher rate of Gli1 nuclear staining than MGMT-negative ones (67.7% vs. 32.3%, p = 0.0159). Activation of HH/Gli1 signaling by pcDNA3.1-Gli1 cell transfection in A172 cells led to increased MGMT expression and enhanced resistance to TMZ treatment. Inhibition of the HH/Gli1 signaling by cyclopamine in U251 cells resulted in decreased MGMT expression and increased sensitivity to TMZ treatment. Both ways altered MGMT levels without changing the MGMT promoter methylation. The potential binding site of Gli1 in the MGMT gene promoter region was located at – 411 to − 403 bp upstream the transcriptional start site. The in vivo study revealed a synergistic effect on tumor growth inhibition with the combined administration of cyclopamine and TMZ.ConclusionsThis study shows that HH/Gli1 signaling pathway regulates MGMT expression and chemoresistance to TMZ in human GBM independent from MGMT promoter methylation status, which offers a potential target to restore chemosensitivity to TMZ in a fraction of GBM with high MGMT expression.

Cyclopamine sensitizes Glioblastoma cells to Temozolomide treatment through Sonic Hedgehog pathway

“…Thus, in 'exchange condition', the GBM cells were first submitted to cyclopamine followed by TMZ treatment (Cyclo/TMZ) or vice-versa (TMZ/Cyclo). In all combined treatment conditions, we observed a significant decrease in cell viability, as described in previous studies [25,42]. The pharmacological combination of TMZ and cyclopamine (TMZ+Cyclo) showed a cooperative effect, corroborating the idea that the pharmacological combination of TMZ with other drugs, such as SHH inhibitor GANT61, are more successful than monotherapy by decreasing the cell viability of GBM cells [1,[60][61][62].…”

“…As expected, a significant viability reduction in all GBM cell lines treated with 200μM TMZ for 144 hours was observed (6 days-time relatively similar to chemotherapy treatment) [56]. Previous reports have demonstrated the effectiveness of the combination of higher concentrations of TMZ with other compounds, such as cyclopamine in GBM cells [25,57]. However, the need to use lower concentrations of TMZ associated with other compounds to better reduce the side effects as compared to TMZ monotherapy has been exploited [27,58,59].…”

“…It has already been demonstrated that high levels of Gli1 are associated with upregulation of O-6-methylguanine-DNA methyltransferase (MGMT) and consequently induces the glioma chemoresistance to TMZ [25]. Therefore the inhibition of the SHH pathway could be a promising therapeutic approach by restoring the chemosensitivity of GBM cells to TMZ.…”

“…The GBM03 cell line has higher clonogenic potential. The clonogenicity assay was performed in GBM cells submitted to different combinations of cyclopamine25 and TMZ. The total number of colonies formed was observed using phase-contrast microscopy (A to P).…”

1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine Treatment After Brain Irradiation Preserves Cognitive Function in Mice

“…Recent publications indicated that deregulated developmental pathways play a key role in GBM progression and tumorigenesis by conferring drug resistance to the tumor cells and that inhibition of Hedgehog pathway induced apoptosis in GBM cells 16-18 . We had previously demonstrated that Compound #5 activates the Hedgehog pathway through binding to the transmembrane domain of Smoothened 7 .…”