Mitochondrial pathway mediated by reactive oxygen species involvement in α-hederin-induced apoptosis in hepatocellular carcinoma cells.

Aim: To investigate the antitumor activity of α-hederin in hepatocellular carcinoma (HCC) cells and its underlying mechanisms and .

Methods: SMMC-7721, HepG-2 and Huh-7 HCC cells were cultured and treated with α-hederin (0, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L, 25 μmol/L, 30 μmol/L, 35 μmol/L, 40 μmol/L, 45 μmol/L, 50 μmol/L, 55 μmol/L, or 60 μmol/L) for 12 h, 24 h, or 36 h, and cell viability was then detected by the Cell Counting Kit-8. SMMC-7721 cells were treated with 0, 5 μmol/L, 10 μmol/L, or 20 μmol/L α-hederin for 24 h with or without DL-buthionine-,-sulfoximine (2 mmol/L) or -acetylcysteine (5 mmol/L) pretreatment for 2 h, and additional assays were subsequently performed. Apoptosis was observed after Hoechst staining. Glutathione (GSH) and adenosine triphosphate (ATP) levels were measured using GSH and ATP Assay Kits. Intracellular reactive oxygen species (ROS) levels were determined by measuring the oxidative conversion of 2',7'-dichlorofluorescin diacetate. Disruption of the mitochondrial membrane potential was evaluated using JC-1 staining. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C were detected by western blotting. The antitumor efficacy of α-hederin was evaluated in a xenograft tumor model.

Results: The α-hederin treatment induced apoptosis of HCC cells. The apoptosis rates in the control, low-dose α-hederin (5 μmol/L), mid-dose α-hederin (10 μmol/L) and high-dose α-hederin (20 μmol/L) groups were 0.90% ± 0.26%, 12% ± 2.0%, 21% ± 2.1% and 37% ± 3.8%, respectively ( < 0.05). The α-hederin treatment reduced intracellular GSH and ATP levels, induced ROS, disrupted the mitochondrial membrane potential, increased the protein levels of Bax, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C, and decreased Bcl-2 expression. The α-hederin treatment also inhibited xenograft tumor growth .

Conclusion: The α-hederin saponin induces apoptosis of HCC cells the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.