The Affiliated Qingdao Hiser Hospital of Qingdao University, Qingdao, Shandong, China.
The Affiliated Qingdao Hiser Hospital of Qingdao University, Qingdao, Shandong, China.
Life Sci. 2020 Apr 15;247:117425. doi: 10.1016/j.lfs.2020.117425. Epub 2020 Feb 11.
Glioma is the most common type of malignant tumor of the nervous system, and aggressiveness and recurrence are major obstacles for treatment. This study is designed to explore the effects of amentoflavone (AF) on glioma, and to investigate the underlying mechanism of the anti-cancer activities of AF.
Cell morphology was recorded under microscopy. Cell viability and cell death ratio were determined by CCK-8 assay and lactate dehydrogenase (LDH) release assay, respectively. Cell cycle progression was assessed by flow cytometry. The levels of iron, MDA (malondialdehyde), lipid ROS, and GSH (reduced glutathione) were assessed by ELISA kit. The cycle-related proteins, ferroptosis-related protein, autophagy-related protein, and the phosphorylation of AMPK, mTOR and p70S6K were analyzed by western blotting. The autophagic flux was observed by transfecting cells with mRFP-GFP-LC3 plasmids. The xenograft murine models were established to analyze the effects of amentoflavone in vivo. The immunohistochemistry assay was performed to analyze the expression of LC3B, Beclin1, ATG5, ATG7, and ferritin heavy chain (FTH).
Our results showed that AF treatment led to reduction in cell viability and cell death. In addition, AF was found to block cell cycle progression in a dose-dependent manner in vitro. Following treatment with AF, the intracellular levels of iron, MDA, and lipid OS were increased, and the levels of GSH and the mitochondrial membrane potential were reduced. In addition, our results showed that AF promoted the autophagic by regulating autophagy-relevant proteins. Our results also showed that the autophagy-induction by AF was associated with regulation of AMPK/mTOR signaling. Mechanistically, the inhibition effects of AF on glioma cell were reversed by DFO, ferreostatin-1 as well as upregulation of FTH. Meanwhile, the FTH levels were increased by compound C and knockdown of ATG7. Moreover, both autophagy inhibitor Baf A1 and knockdown of ATG7 were able to compromising AF-induce ferroptosis and cell death. In vivo, the tumor growth was suppressed by AF in a dose-dependent manner. The level of MDA in the tumor tissue was increased while the level of GSH in tumor tissue was decreased by AF in a dose-dependent manner. Furthermore, the expression of LC3B, Beclin1, ATG5, ATG7 were increased, and the expression of FTH were decreased by AF in a dose-dependent manner in vivo. Conclusion These results demonstrate that AF triggered ferroptosis in autophagy-dependent manner. Our results suggest that AF has the potential to be considered as a novel treatment agent in glioma.
神经胶质瘤是最常见的神经恶性肿瘤，侵袭性和复发是治疗的主要障碍。本研究旨在探讨杨梅素（AF）对神经胶质瘤的作用，并探讨 AF 抗癌活性的潜在机制。
显微镜下记录细胞形态。通过 CCK-8 测定法和乳酸脱氢酶（LDH）释放测定法分别测定细胞活力和细胞死亡率。通过流式细胞术评估细胞周期进展。通过 ELISA 试剂盒评估铁、MDA（丙二醛）、脂质 ROS 和 GSH（还原型谷胱甘肽）的水平。通过 Western blot 分析周期相关蛋白、铁死亡相关蛋白、自噬相关蛋白以及 AMPK、mTOR 和 p70S6K 的磷酸化水平。通过转染 mRFP-GFP-LC3 质粒观察自噬流。建立异种移植小鼠模型以分析杨梅素在体内的作用。通过免疫组织化学分析测定 LC3B、Beclin1、ATG5、ATG7 和铁蛋白重链（FTH）的表达。
我们的结果表明，AF 处理导致细胞活力降低和细胞死亡。此外，AF 被发现以剂量依赖性方式在体外阻断细胞周期进展。用 AF 处理后，细胞内铁、MDA 和脂质 OS 水平升高，GSH 和线粒体膜电位水平降低。此外，我们的结果表明，AF 通过调节自噬相关蛋白促进自噬。我们的结果还表明，AF 诱导的自噬与 AMPK/mTOR 信号的调节有关。在机制上，DFO、ferreostatin-1 以及上调 FTH 可逆转 AF 对神经胶质瘤细胞的抑制作用。同时，用化合物 C 和敲低 ATG7 可增加 FTH 水平。此外，自噬抑制剂 Baf A1 和敲低 ATG7 均可削弱 AF 诱导的铁死亡和细胞死亡。体内，AF 以剂量依赖性方式抑制肿瘤生长。AF 以剂量依赖性方式增加肿瘤组织中 MDA 的水平，降低肿瘤组织中 GSH 的水平。此外，AF 以剂量依赖性方式增加体内 LC3B、Beclin1、ATG5、ATG7 的表达，降低 FTH 的表达。结论：这些结果表明，AF 以自噬依赖的方式触发铁死亡。我们的研究结果表明，AF 有潜力成为神经胶质瘤的一种新型治疗剂。
