Inhibition of STAT3 Enhances the Radiosensitizing Effect of Temozolomide in Glioblastoma Cells in vitro and in vivo

Abstract

Introduction: Despite aggressive treatment with radiation therapy plus concurrent and adjuvant temozolomide (TMZ), the prognosis for glioblastoma remain poor. We investigated the potential of targeting signal transducer and activator of transcription-3 (STAT3) to improve the therapeutic outcome of combined radiotherapy and TMZ and its associated mechanisms in glioblastoma. Methods: We evaluated the preclinical potential of a STAT3 inhibitor, Cpd188 combined with temozolomide and radiation by using two established glioblastoma cell lines (U251, U87) and two patients-derived glioblastoma cell lines (GBL12, GBL28). Clonogenic assay was performed to determine the surviving fraction of cells after treatment, apoptosis was evaluated by caspase-3/7 assay and annexin V-FITC/PI double staining, modified Boyden chamber assay, wound healing assay, vasculogenic assay were performed to establish the association of epithlial-mesenchymal transition (EMT)-related mechanism. In vivo studies using 6- to 8-week old BALB/c nude mice bearing intracranial U251 xenografts, bioluminescence imaging was used to evaluate tumor growth. Markers associated with tumor microenvironmet were assessed by immunohistochemical analysis. Results: Cpd188 potentiated the radiosensitizing effect of TMZ in U251 glioblastoma cell line which has high levels of p-STAT3 expression and in vitro. Increased radiosensitizing effects of TMZ were associated with the induction of apoptosis and the reversion of epithelial-mesenchymal transition (EMT). Cpd188 delayed in vivo tumor growth in combination with fractionated radiation and TMZ with improved survival rates compared to control (P=0.003) and radiation only group (P=0.007). Immunohistochemical staining of tumor sections showed that Cpd188 decreased the expression of CD31 (a marker of endothelial proliferation), vascular endothelial growth factor, and hypoxia-inducible factor-1α, suggesting that Cpd188 also has anti-angiogenic effects. We also confirmed the radiosensitizing effect of Cpd188 of GBL28 cell line which was originated from a patient who had a glioblastoma and also was confirmed high level of STAT3 expression and unmethylated MGMT. Cpd188 selectively sensitized glioblastoma cells to the cytotoxic effects of radiotherapy plus TMZ while not effecting the cell death of normal astrocytes. Conclusions: These data indicate that Cpd188 has the potential to improve the therapeutic outcome of combined radiotherapy and TMZ in human glioblastoma, especially in patients whose tumor has a high level of STAT3 expression regardless of MGMT methylation status.