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华蟾素抗胶质瘤的作用及机制研究
The Research on the Anti-glioma Effect and Mechanism of Cinobufagin
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摘要: 目的 研究华蟾素对U87胶质瘤细胞增殖、凋亡的影响，并初步探讨其中的分子机制。方法 不同浓度的华蟾素（0.5~20 μmol/L）处理U87细胞6 h、12 h、24 h、48 h后，用CCK-8试剂盒进行做细胞活性检测，观察华蟾素对U87胶质瘤细胞活性的影响。不同浓度的华蟾素（1~20 μmol/L）分别处理12 h、24 h后，用Hochest33342试剂检测U87胶质瘤细胞的凋亡情况。免疫荧光染色观察华蟾素处理24 h后与细胞增殖相关蛋白p-AKT(T308)的表达情况。Western blot检测不同浓度的华蟾素（1~20 μmol/L）处理24 h后与细胞增殖相关蛋白｛磷酸化的磷脂酰肌醇激酶（p-PI3K）、苏氨酸308位点磷酸化的蛋白激酶B〔p-AKT(T308)〕、丝氨酸473位点磷酸化的蛋白激酶B〔p-AKT(S473)〕、磷酸化核糖体S6蛋白激酶（PS6）、磷酸化的真核细胞翻译起始因子4E结合蛋白1(p-4EBP1)｝和凋亡相关蛋白（BAX、cleaved-caspase 3、cleaved-caspase 9）的表达情况。结果 华蟾素可抑制U87 胶质瘤细胞的活性、诱导U87胶质瘤细胞的凋亡，呈浓度和时间依赖性反应。华蟾素作用后可使凋亡相关蛋白cleaved-caspase 3、BAX 的表达水平增高， PI3K/AKT信号通路相关蛋白p-AKT(T308)的表达水平降低。结论 华蟾素可抑制U87 胶质瘤细胞的增殖、诱导U87胶质瘤细胞的凋亡，这可能经由影响PI3K-AKT-4EBP1与BAX-caspase的信号通路实现。
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关键词:
- 华蟾素 胶质瘤 生长抑制 凋亡
Abstract: Objective To study the effect of cinobufagin (CB) on the proliferation inhibition and induction of apoptosis in glioblastoma cell lines U87 and its molecular mechanism. Methods A gradient concentration (0-20 μmol/L) of CB was used to treat the U87 glioma cells for 6 h, 12 h, 24 h and 48 h, respectively. Cell viabilities were determined by CCK-8 assay to discover the effects of different concentrations of CB on the proliferation of glioma cells. Different concentrations (1-20 μmol/L) of CB were used to treat the U87 glioma cells for 12 h and 24 h, hochest33342 staining assay was used to assess the apoptosis levels. Immunofluorescence staining was used to determine the expression of growth related proteins phospho-protein kinase B(T308)〔 p-AKT(T308)〕 in U87 glioma cells after being treated with CB for 24 h. Western blot was used to determine the apoptotic related proteins (BAX, cleaved-caspase 3, cleaved-caspase 9) and growth related proteins 〔phospho-inositide 3-kinase (p-PI3K), p-AKT(T308), p-AKT(S473), phospho-ribosomal protein S6 kinase (PS6), phospho-4E-binding protein 1 (p-4EBP1)〕. Results A significant effect of CB on the proliferation inhibition and induction of apoptosis in U87 glioma cells in a time- and dose-dependent manner was observed. Treatment with CB induced the expression levels of apoptosis-related protein, cleaved-caspase 3 and BAX, and the PI3K-AKT-4EBP1 signaling pathway related proteins p-AKT(T308) and p-4EBP1 were decreased. Conclusion CB can inhibit U87 glioma cells growth and induce apoptosis, which may involve the PI3K-AKT-4EBP1 and BAX-caspase signaling pathways. -
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期刊类型引用(5)
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5. 于秀荣，陈钧泽，李慧杰，齐元富. 华蟾素抗肿瘤研究. 中医学报. 2019(06): 1174-1178 . 百度学术
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