Ethanol Extract of Hizikia fusiforme Induces Apoptosis in B16F10 Mouse Melanoma Cells through ROS-Dependent Inhibition of the PI3K/Akt Signaling Pathway

Background: 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. Methods: 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. Results: 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. Conclusion: 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.