Department of Molecular Biology, College of Natural Sciences, Dong-eui University, Busan 47340, Republic of Korea.
Anti-Aging Research Center, Dong-eui University, Busan 47340, Republic of Korea.
Asian Pac J Cancer Prev. 2020 May 1;21(5):1275-1282. doi: 10.31557/APJCP.2020.21.5.1275.
Previous studies have reported that Hizikia fusiforme, an edible brown seaweed, has diverse health-promoting effects; however, evidence for its anti-cancer potential is still lacking. In this study, we examined the effect of ethanol extract of H. fusiforme (EHF) on the proliferation of B16F10 mouse melanoma cells.
Analyses of cell viability and apoptosis were performed to study the actions of EHF on B16F10 cells. Cellular reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were measured using a flow cytometer. Western blot analysis was carried out to measure apoptosis and phosphoinositide 3-kinase (PI3K)/Akt signaling related proteins.
EHF treatment significantly decreased B16F10 cell viability, which was associated with induction of apoptosis. EHF activated caspase-8 and caspase-9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. In addition, EHF destroyed the integrity of mitochondria and increased Bax/Bcl-2 ratio, which contributed to cytosolic release of cytochrome c. EHF further enhanced intracellular levels of ROS and the addition of N-acetyl cysteine (NAC), a ROS inhibitor, significantly diminished EHF-induced mitochondrial dysfunction and growth inhibition. Moreover, EHF inactivated the PI3K/Akt signaling pathway and LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of EHF. However, increased apoptosis and reduced cell viability by simultaneous treatment of EHF and LY294002 were significantly attenuated in the presence of NAC.
These results indicate that EHF induces apoptosis through activation of extrinsic and intrinsic apoptotic pathways and ROS-dependent inactivation of PI3K/Akt signaling in B16F10 cells.
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先前的研究报告称，可食用的褐藻马尾藻具有多种促进健康的作用；然而，其抗癌潜力的证据仍然不足。在这项研究中，我们研究了马尾藻乙醇提取物（EHF）对 B16F10 小鼠黑色素瘤细胞增殖的影响。
方法：通过细胞活力和细胞凋亡分析来研究 EHF 对 B16F10 细胞的作用。使用流式细胞仪测量细胞内活性氧（ROS）和线粒体膜电位（ΔΨm）。通过 Western blot 分析来测量凋亡和磷酸肌醇 3-激酶（PI3K）/Akt 信号相关蛋白。
结果：EHF 处理显著降低了 B16F10 细胞活力，这与诱导细胞凋亡有关。EHF 激活了半胱天冬酶-8 和半胱天冬酶-9，分别参与了外在和内在凋亡途径的起始，还增加了 caspase-3 活性，这是一种典型的效应半胱天冬酶，随后导致多聚（ADP-核糖）聚合酶裂解。此外，EHF 破坏了线粒体的完整性并增加了 Bax/Bcl-2 比值，这有助于细胞溶质中细胞色素 c 的释放。EHF 进一步增加了细胞内 ROS 的水平，添加 ROS 抑制剂 N-乙酰半胱氨酸（NAC）可显著减弱 EHF 诱导的线粒体功能障碍和生长抑制。此外，EHF 使 PI3K/Akt 信号通路失活，PI3K/Akt 抑制剂 LY294002 增加了 EHF 诱导的细胞凋亡作用。然而，在存在 NAC 的情况下，EHF 和 LY294002 同时处理时引起的凋亡增加和细胞活力降低明显减弱。
结论：这些结果表明，EHF 通过激活外在和内在凋亡途径以及 ROS 依赖性的 PI3K/Akt 信号失活诱导 B16F10 细胞凋亡。
