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 in vitro and in vivo. METHODS: SMMC-7721, HepG-2 and Huh-7 HCC cells were cultured in vitro 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-S,R-sulfoximine (2 mmol/L) or N-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 in vivo 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 (P < 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 in vivo. CONCLUSION: The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.

Comparative Metabolomic Profiling of Hepatocellular Carcinoma Cells Treated with Sorafenib Monotherapy vs. Sorafenib-Everolimus Combination Therapy.

BACKGROUND: Sorafenib-everolimus combination therapy may be more effective than sorafenib monotherapy for hepatocellular carcinoma (HCC). To better understand this effect, we comparatively profiled the metabolite composition of HepG2 cells treated with sorafenib, everolimus, and sorafenib-everolimus combination therapy. MATERIAL AND METHODS: A 2D HRMAS 1H-NMR metabolomic approach was applied to identify the key differential metabolites in 3 experimental groups: sorafenib (5 µM), everolimus (5 µM), and combination therapy (5 µM sorafenib +5 µM everolimus). MetaboAnalyst 3.0 was used to perform pathway analysis. RESULTS: All OPLS-DA models displayed good separation between experimental groups, high-quality goodness of fit (R2), and high-quality goodness of predication (Q2). Sorafenib and everolimus have differential effects with respect to amino acid, methane, pyruvate, pyrimidine, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism. The addition of everolimus to sorafenib resulted in differential effects with respect to pyruvate, amino acid, methane, glyoxylate and dicarboxylate, glycolysis or gluconeogenesis, glycerophospholipid, and purine metabolism. CONCLUSIONS: Sorafenib and everolimus have differential effects on HepG2 cells. Sorafenib preferentially affects glycerophospholipid and purine metabolism, while the addition of everolimus preferentially affects pyruvate, amino acid, and glucose metabolism. This phenomenon may explain (in part) the synergistic effects of sorafenib-everolimus combination therapy observed in vivo.