Yang Yi, Zhang Li-hui, Yang Bing-xian, Tian Jin-kui, Zhang Lin
Department of Pharmacology, Hangzhou Key Laboratory of Medical Neurobiology, School of Medicine, Hangzhou Normal University, Hangzhou, China; College of Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou, China.
J Cell Mol Med. 2015 May;19(5):1055-64. doi: 10.1111/jcmm.12498. Epub 2015 Feb 20.
We aim to investigate the effect of aurantiamide acetate isolated from the aerial parts of Clematis terniflora DC against gliomas. Human malignant glioma U87 and U251 cells were incubated with different concentrations (0-100 μM) of aurantiamide acetate. Aurantiamide acetate greatly decreased the cell viability in a dose- and time-dependent manner. It induced moderate mitochondrial fragmentation and the loss of mitochondrial membrane potential. No significant difference was found in the alternation of other intracellular organelles, although F-actin structure was slightly disturbed. Apparent ultrastructure alternation with increased autophagosome and autolysosome accumulation was observed in aurantiamide acetate-treated cells. The expression of LC3-II was greatly up-regulated in cells exposed to aurantiamide acetate (P < 0.05 compared with control). The cytoplasmic accumulation of autophagosomes and autolysosomes induced by aurantiamide acetate treatment was confirmed by fluorescent reporter protein labelling. Administration of chloroquine (CQ), which inhibits the fusion step of autophagosomes, further increased the accumulation of autophagosomes in the cytoplasm of U87 cells. Autophagy inhibition by 3-methyladenine, Bafilomycin A1 or CQ had no influence on aurantiamide acetate-induced cytotoxicity, whereas autophagy stimulator rapamycin significantly suppressed aurantiamide acetate-induced cell death. The anti-tumour effects of aurantiamide acetate were further evaluated in tumour-bearing nude mice. Intratumoural injection of aurantiamide acetate obviously suppressed tumour growth, and increased number of autophagic vacuoles was observed in tumour tissues of animals receiving aurantiamide acetate. Our findings suggest that aurantiamide acetate may suppress the growth of malignant gliomas by blocking autophagic flux.
我们旨在研究从三叶铁线莲地上部分分离得到的醋酸橙黄酰胺对胶质瘤的作用。将人恶性胶质瘤U87和U251细胞与不同浓度（0 - 100 μM）的醋酸橙黄酰胺一起孵育。醋酸橙黄酰胺以剂量和时间依赖性方式显著降低细胞活力。它诱导了适度的线粒体碎片化和线粒体膜电位的丧失。尽管F - 肌动蛋白结构略有紊乱，但在其他细胞内细胞器的变化方面未发现显著差异。在醋酸橙黄酰胺处理的细胞中观察到明显的超微结构改变，自噬体和自溶酶体积累增加。在暴露于醋酸橙黄酰胺的细胞中，LC3 - II的表达显著上调（与对照组相比，P < 0.05）。通过荧光报告蛋白标记证实了醋酸橙黄酰胺处理诱导的自噬体和自溶酶体在细胞质中的积累。给予抑制自噬体融合步骤的氯喹（CQ），进一步增加了U87细胞细胞质中自噬体的积累。用3 - 甲基腺嘌呤、巴弗洛霉素A1或CQ抑制自噬对醋酸橙黄酰胺诱导的细胞毒性没有影响，而自噬刺激剂雷帕霉素显著抑制醋酸橙黄酰胺诱导的细胞死亡。在荷瘤裸鼠中进一步评估了醋酸橙黄酰胺的抗肿瘤作用。瘤内注射醋酸橙黄酰胺明显抑制肿瘤生长，并且在接受醋酸橙黄酰胺的动物肿瘤组织中观察到自噬泡数量增加。我们的研究结果表明，醋酸橙黄酰胺可能通过阻断自噬流来抑制恶性胶质瘤的生长。
