Polyphenols Extracted from Enteromorpha clathrata Induce Apoptosis in Hepa1-6 Cell by Activating the Mitochondrial Apoptosis Signaling Pathways

Green alga Enteromorpha clathrata ( E. clathrata ) contains a variety of bioactive compounds, including polysaccharides, polyphenols and fat-soluble pigments etc., among which polyphenols exhibit a wide range of medicinal properties. E. clathrata polyphenols (ECPs) have shown various biological activities such as antioxidant, anti-inflammatory and antidiabetic effects; however, the potential of ECPs as an anti-cancer reagent remains unclear. The aim of this study was to investigate the anti-tumor activity and underlying mechanisms of ECPs on hepatocellular carcinoma. The cytotoxicity of Hepa1-6 cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. Flow cytometry and fluorescence microscope analysis of cell apoptosis after annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used for intracellular reactive oxygen species (ROS) detection. caspase-9 activity was determined using cspase-9 colorimetric assay. Mitochondrial transmembrane potential (Δψm) was measured using JC-1. Western blot and quantitative real-time PCR (qPCR) were used to assess the expressions of the apoptosis regulators Bax, Bcl-2, cytochrome c and caspase-3. It was found that ECPs showed a dose-dependent cytotoxicity against Hepa1-6 cells by inducing apoptosis. The apoptosis in ECPs-treated Hepa1-6 cells was accompanied by the loss of mitochondrial membrane potential, elevated ROS generation, increased release of mitochondrial cytochrome c, and up-regulation of caspase-9 and caspase-3. The expressions of Bax (pro-apoptotic molecule) and Bcl-2 (apoptosis suppressor) were up-regulated and down-regulated, respectively, at both mRNA and protein levels. These molecular alterations revealed that ECPs caused apoptosis of cells through the mitochondrial pathway, suggesting that ECPs are potential candidates to be developed for liver cancer treatment.