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.
Highlights
Glioblastoma (GBM) is the most common and devastating tumor in the central nervous system
Gli1 activity correlates with methylguanine DNA methyltransferase (MGMT) expression in primary GBM tissues As nuclear staining of Gli1 is a reliable marker of HH pathway activity in brain glioma [16], the percentage of Gli1 nuclear staining was used to divide the 48 surgically resected GBM samples into Gli1-positive or Gli1-negative groups
The MGMT promoter methylation status showed a stronger correlation with MGMT level (p < 0.0001)
Summary
Glioblastoma (GBM) is the most common and devastating tumor in the central nervous system. O6-methylguanine DNA methyltransferase (MGMT), is able to reverse the anti-tumor effect of TMZ, and mainly contributes to the chemoresistance in a fraction of patients. Our previous study showed that Gli was a potential target to alleviate multidrug resistance of human glioma by regulating the transcription of a series of chemoresistance-associated genes [12], but the relationship between HH/Gli signaling and MGMT expression in GBM is yet to be clarified. Inhibition of the HH/Gli 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.
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