Suppression Of Autophagy Augments The Radiosensitizing Effects Of STAT3 Inhibition On Human Glioma Cells

Purpose: In this study, human malignant glioma cells U251 and A172 were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA(sh RNA) plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The potential mechanisms of STAT3 inhibition-induced radiosensitizing effects in glioma cells were investigated.Methods and Materials: STAT3 expression was inhibited by WP1066 or sh RNA-STAT3 and the efficiency of inhibition was confirmed by western blotting. Clonogenic survival assays were administrated to evaluate the STAT3 inhibition-induced radiosensitization on U251 cells. Terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) assay and Annexin V-PE assay were performed to detect apoptosis of U251 cells. Expressions of cleaved Caspase 3,cleaved PARP1, Belin 1,ATG5, LC3II/I were evaluated by immunoblotting to identify the potential mechanisms of radiosensitization. To e^lore the role of autophagy autophagy inhibitor 3- Methyladenine(3-MA) or si ATG5 was used to suppress autophagy induced by both STAT3 inhibition and ionizing radiation(IR) ? GFP-LC3 transient transfection was used to observe cellular autophagy. Apoptosis related proteins(Caspase 3,cleaved PARP1) and autophagy related proteins(Belin 1,ATG5 LC3II/I) were evaluated by western blot assay.Results: Both pharmacological and genetic inhibition of STAT3 significantly decreased the surviving fraction of cells compared with that of untreated cells. Moreover, inhibition of STAT3 could enhance IR-induced apoptosis and expression of apoptosis related proteins(Caspase 3,cleaved PARP1). Meanwhile, STAT3 inhibition could induce activity of cytoprotective autophagy and expression of Belin 1,ATG5 LC3II/I. Furthermore, suppression of STAT3 inhibition-induced cytoprotective autophagy by 3-MA and si ATG5 could induce more apoptotic cells and contribute to the radiosensitivity of cells treated with STAT3 inhibition and X-rays.Conclusion: Inhibition of STAT3 and autophagy radio sensitized U251 glioma cells, and the underlying mechanism may be involved the induction of apoptosis and cytoprotective autophagy. Inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells.