Department of Biochemistry, Dong-eui University College of Korean Medicine and Anti-Aging Research Center, Dong-eui University, Busan, 47227, Republic of Korea; Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan, 46241, Republic of Korea.
Department of Biochemistry, Dong-eui University College of Korean Medicine and Anti-Aging Research Center, Dong-eui University, Busan, 47227, Republic of Korea.
Arch Biochem Biophys. 2021 Jan 15;697:108688. doi: 10.1016/j.abb.2020.108688. Epub 2020 Nov 21.
Coptisine is isoquinoline alkaloid derived from Coptidis Rhizoma and is known to have potential anti-cancer activity toward various carcinomas. Targeting autophagy is one of the main approaches for cancer therapy, but whether the anti-cancer efficacy of coptisine involves autophagy is still unclear. Therefore, this study investigated the effect of coptisine on autophagy in hepatocellular carcinoma (HCC) Hep3B cells, and identified the underlying mechanism. Our results showed that coptisine increased cytotoxicity and autophagic vacuoles in a concentration-dependent manner. Furthermore, the expressions of light chain 3 (LC3)-I/II, Beclin-1 and autophagy genes were markedly increased by coptisine, while the expression of p62 decreased. In addition, we found that pretreatment with bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, markedly reduced coptisine-mediated autophagic cell death, but 3-methyladenine, an inhibitor for autophagosome formation did not. Moreover, our results showed that although coptisine up-regulated AMP-activated protein kinase (AMPK) that partially induced LC3-I/II, coptisine-mediated AMPK signaling did not directly regulate autophagic cell death. Additionally, we found that coptisine suppressed the phosphorylation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR), and this effect was notably enhanced by PI3K inhibitor LY294002. Meanwhile, coptisine significantly increased both the production of mitochondrial reactive oxygen species (ROS) and the recruitment of mitophagy-regulated proteins to mitochondria. Furthermore, N-acetylcysteine, a potential ROS scavenger, substantially suppressed the expression of mitophagy-regulated proteins and LC3 puncta by coptisine. Overall, our results demonstrate that coptisine-mediated autophagic cell death was regulated by PI3K/Akt/mTOR signaling and mitochondrial ROS production associated with mitochondrial dysfunction. Taken together, these findings suggest that coptisine exerts its anti-cancer effects through induction of autophagy in HCC Hep3B cells.
小檗碱是从小檗属植物中提取的一种异喹啉生物碱，已知其对多种癌具有潜在的抗癌活性。靶向自噬是癌症治疗的主要方法之一，但小檗碱的抗癌功效是否涉及自噬尚不清楚。因此，本研究探讨了小檗碱对肝癌 Hep3B 细胞自噬的影响，并确定了其潜在机制。结果表明，小檗碱呈浓度依赖性地增加细胞毒性和自噬小体。此外，小檗碱显著增加了 LC3-I/II、Beclin-1 和自噬基因的表达，同时降低了 p62 的表达。此外，我们发现用自噬体-溶酶体融合抑制剂巴弗洛霉素 A1 预处理可显著降低小檗碱介导的自噬细胞死亡，但自噬体形成抑制剂 3-甲基腺嘌呤则没有。此外，结果表明，虽然小檗碱上调了部分诱导 LC3-I/II 的 AMP 激活蛋白激酶（AMPK），但小檗碱介导的 AMPK 信号通路并未直接调节自噬细胞死亡。此外，发现小檗碱抑制了磷酸肌醇 3-激酶/蛋白激酶 B/雷帕霉素靶蛋白（PI3K/Akt/mTOR）的磷酸化，而这种作用可被 PI3K 抑制剂 LY294002 显著增强。同时，小檗碱显著增加线粒体活性氧（ROS）的产生和线粒体自噬调节蛋白向线粒体的募集。此外，ROS 清除剂 N-乙酰半胱氨酸可显著抑制小檗碱诱导的自噬调节蛋白和 LC3 斑点的表达。总之，这些结果表明，小檗碱介导的自噬细胞死亡受 PI3K/Akt/mTOR 信号和与线粒体功能障碍相关的线粒体 ROS 产生的调节。综上所述，这些发现表明小檗碱通过诱导 HCC Hep3B 细胞自噬发挥其抗癌作用。
