基金项目：国家自然科学基金项目（No. 81072656, 81102466, 81373430）
通信联系人：梁中琴(1954-), 女, 教授,肿瘤药理,神经药理.
摘要：目的：本文旨在研究奥氮平（olanzapine）对胶质瘤细胞的抑制作用，及其可能的作用机制。方法：实验选用胶质瘤T98 和LN229 细胞。MTT 法检测不同浓度的奥氮平作用不同时间后对两个胶质瘤细胞增殖率的影响。Western blot 检测自噬相关蛋白LC3、p62 和凋亡相关蛋白Bcl-2 表达水平，免疫荧光检测自噬荧光颗粒的形成。Hoechst 33258 染色以及Annexin V-FITC/PI 实验检测不同浓度奥氮平对细胞凋亡影响。MTT 和集落形成实验分析自噬抑制剂3MA 对奥氮平抑瘤作用的影响。结果：MTT 结果显示，奥氮平对LN229 和T98均有抑制作用。LC3、p62 的蛋白表达情况以及免疫荧光检测LC3 的颗粒形成与分布情况都表明奥氮平诱导了自噬。Western blot 分析Bcl-2 蛋白表达、Hoechst 33258 染色实验以及V-FITC/PI 染色实验表明，奥氮平促进LN229 和T98 细胞凋亡。MTT 和集落形成实验表明，自噬抑制剂3MA 减弱了奥氮平对这两株胶质瘤细胞的增殖抑制作用。结论：奥氮平能够抑制胶质瘤细胞生长，其抗胶质瘤作用的机制可能与自噬诱导相关。
Autophagy is involved in olanzapine-mediated cytotoxic effects in human glioma cells
WANG Yixuan, XU Shuqing, CHEN Xianghui, LIU Ruisi, LIANG Zhongqin
(Department of pharmocology, College of Pharmaceutical Sciences, Soochow University,SuZhou,215000)
Abstract: Aim: The aim of this study was to investigate the effects of olanzapine on growth inhibition as well as autophagy induction in glioma cells. Methods: Human glioma cell lines LN229 and T98 were tested in this study. MTT assay was used to determine cell viability of cultured glioma cells in response to olanzapine,. Western blotting was performed to analyze the expression levels of LC3, p62 and Bcl-2 proteins. Immunofluorescence using confocal microscopy was used to identify the formation of autophagosomes during autophagy. The apoptotic cells were observed using Hoechst 33258 staining followed by fluorescence microscopy, and the percentage of dead cells was quantified using annexin V-FITC/PI staining and flow cytometry. Results:LN229 and T98 cells were treated with olanzapine at a dose range of 0, 0.1, 0.2, 0.4, 0.6, 0.8 or1.0 mΜ for 24, 48 or 72h, respectively. As a result, the cell growth of LN229 and T98 were both suppressed in a concentration-dependent and time-dependent manner. Moreover, the punctates of microtubule-associated protein LC3, indicative of autophagy, and the expression level of membrane-bound LC3-II were significantly increased in olanzapine-treated glioma cells starting at48 h. The expression of protein p62, a substrate of autophagy, was decreased. The olanzapine-induced autophagy was also accompanied by a decrease of Bcl-2, leading to apoptosis as evidenced by an increased staining of Hoechst 33258 and Annexin V-FITC/PI of glioma cells.The growth inhibition of olanzaoine on glioma cells could be blocked by co-treatment with 3MA(2 mM). Conclusion: Olanzapine inhibits the growth of glioma cells accompanied by the induction of autophagy. The olanzapine-induced autophagy plays a tumor-suppressing role in glioma cells.
Key words: pharmacology; olanzapine; autophagy; apoptosis; glioma
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