Autophagy Is Involved In Olanzapine-mediated Cytotoxic Effects In Human Glioma Cells

Aim: The aim of this study was to investigate the effects of olanzapine on growthinhibition as well as autophagy induction in glioma cells in vitro and in vivo.Methods: Human glioma cell lines LN229and T98were tested in this study. Todetermine cell viability of cultured glioma cells in response to olanzapine, MTT assaywas used. Western blotting was used to analyze the expression levels of LC3, p62andBcl-2proteins. Immunofluorescence using confocal microscopy was performed toidentify the formation of autophagosomes during autophagy. The apoptotic cells wereobserved using Hoechst33258staining followed by fluorescence microscopy, and thepercentage of dead cells was quantified using Annexin V-FITC/PI staining and flowcytometry. The in vivo studies were performed by subcutaneous injection of LN229glioma cells into BALB/c nude mice (male,6weeks old). The expression levels of LC3,p62and Bcl-2proteins in glioma xenografts were analyzed by western blotting. Forimmunohistochemical study, antibodies specific for Ki-67and caspase-3were used todetect the proliferation and apoptosis of xenografts, respectively.Results: LN229and T98cells were treated with olanzapine at a dose range of0,0.1,0.2,0.4,0.6,0.8or1.0mΜ for24,48or72h, respectively. As a result, the cellgrowth of LN229and T98were both suppressed in a concentration-dependent andtime-dependent manner. Moreover, the punctates of microtubule-associated protein LC3,indicative of autophagy, and the expression level of membrane-bound LC3-II weresignificantly increased in olanzapine-treated glioma cells starting at48h. Theexpression of protein p62, a substrate of autophagy, was decreased. Theolanzapine-induced autophagy was also accompanied by a decrease of Bcl-2, leading toapoptosis as evidenced by an increased staining of Hoechst33258and AnnexinV-FITC/PI of glioma cells. The growth inhibition of olanzaoine on glioma cells couldbe blocked by co-treatment with3MA (2mM). Furthermore, olanzapine effectivelyblocked the growth of subcutaneous xenograft of LN229glioma cells in vivo as tumorvolume curves described. The increased level of protein LC3-II followed by a decreased level of Bcl-2, suggesting that autophagy may contribute to apoptosis. In addition,reduced proliferation of glioma cells was shown by a decrease of Ki-67staining andincreased caspase-3staining indicative of apoptosis in mouse xenografts.Conclusion: Olanzapine inhibits the growth of glioma cells accompanied by theinduction of autophagy and apoptosis both in vitro and in vivo. The olanzapine-inducedautophagy plays a tumor-suppressing role in glioma.