Reactive oxygen species (ROS) and cellular oxidant stress are considered inducers of carcinogenesis. However, the association of ROS with cancer is both complex and, at times, paradoxical. We assessed the effects of dihydromyricetin (DHM) on the induction of ROS accumulation and on the activation of the mitochondrial signaling pathway in human hepatoma HepG2 cells. The results indicated that DHM could reduce ROS accumulation in a concentration-dependent manner. Additionally, with increasing concentrations of DHM, the expression of proteins that participate in the cell apoptosis program increased in a concentration-dependent manner. Furthermore, we found that a low dose of H₂O₂ (10 nM) could reverse DHM-induced cell apoptosis. We observed the following critical issues: first, the cellular redox balance is vital in DHM-induced apoptosis of human hepatocellular carcinoma (HCC) cells, and second, ROS could function as a redox-active signaling messenger to determine DHM-induced cell apoptosis. In this study, we demonstrated that low levels of ROS are also critical for the function of HCC cells.

Dihydromyricetin alleviates carbon tetrachloride-induced acute liver injury via JNK-dependent mechanism in mice

Chrysin is a major active ingredient of flavonoids, known to exhibit protective effects against various types of cancer. However, the anticancer role of chrysin against hepatocellular carcinoma (HCC) and the underlying molecular mechanisms remain unclear. In order to evaluate the effects of chrysin on cell viability and apoptosis in human HCC, HepG2 and QGY7701 cells were used in the present study. Cell viability was monitored using an MTT assay. In addition, an Annexin V‑fluorescein isothiocyanate/propidium iodide kit was used for the labeling of the apoptotic cells, which were then measured using flow cytometry. Western blotting was used to examine the protein expression of p53, B‑cell lymphoma‑2 (Bcl‑2), Bcl‑2‑associated X (Bax), Bcl‑2‑associated death promoter (Bad), Bcl‑2 homologous antagonist/killer (Bak), caspases‑3 and ‑9, and cleaved‑caspases‑3 and ‑9. The results of the present study revealed that chrysin suppressed the cell viability of HepG2 and QGY7701 cells in a concentration‑dependent manner. In addition, chrysin induced significant apoptosis in HepG2 and QGY7701 cells. Furthermore, it was demonstrated that chrysin treatment increased the expression of proapoptotic proteins, including p53, Bax, Bad and Bak, while it decreased the protein level of antiapoptotic protein Bcl‑2. It was also demonstrated that chrysin induced apoptosis in the HCC cells by regulating the p53/Bcl‑2/caspase‑9 signaling pathway. In conclusion, the results of the present study suggested that chrysin may be a potential candidate agent for the induction of cell apoptosis in human HCC.

AIM: To investigate the effects of dihydromyricetin (DHM) on the migration and invasion of human hepatic cancer cells. METHODS: The hepatoma cell lines SK-Hep-1 and MHCC97L were used in this study. The cells were cultured in RPIM-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO₂ incubator. DHM was dissolved in dimethyl sulfoxide and diluted to various concentrations in medium before applying to cells. MTT assays were performed to measure the viability of the cells after DHM treatment. Wound healing and Boyden transwell assays were used to assess cancer cell motility. The invasive capacity of cancer cells was measured using Matrigel-coated transwell chambers. Matrix metalloproteinase (MMP)-2/9 activity was examined by fluorescence analysis. Western blot was carried out to analyze the expression of MMP-2, MMP-9, p-38, JNK, ERK1/2 and PKC-δ proteins. All data were analyzed by Student’s t tests in GraphPad prism 5.0 software and are presented as mean ± SD. RESULTS: DHM was found to strongly inhibit the migration of the hepatoma cell lines SK-Hep-1 (without DHM, 24 h: 120 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 65 ± 10 μmol/L, P < 0.001) and MHCC97L (without DHM, 24 h: 126 ± 7 μmol/L vs 100 μmol/L DHM, 24 h: 74 ± 6 μmol/L, P < 0.001). The invasive capacity of the cells was reduced by DHM treatment (SK-Hep-1 cells without DHM, 24 h: 67 ± 4 μmol/L vs 100 μmol/L DHM, 24 h: 9 ± 3 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 117 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 45 ± 2 μmol/L, P < 0.001). MMP2/9 activity was also inhibited by DHM exposure (SK-Hep-1 cells without DHM, 24 h: 600 ± 26 μmol/L vs 100 μmol/L DHM, 24 h: 100 ± 6 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 504 ± 32 μmol/L vs 100 μmol/L DHM 24 h: 156 ± 10 μmol/L, P < 0.001). Western blot analysis showed that DHM decreased the expression level of MMP-9 but had little effect on MMP-2. Further investigation indicated that DHM markedly reduced the phosphorylation levels of p38, ERK1/2 and JNK in a concentration-dependent manner but had no impact on the total protein levels. In addition, PKC-δ protein, a key protein in the regulation of MMP family protein expression, was up-regulated with DHM treatment. CONCLUSION: These findings demonstrate that DHM inhibits the migration and invasion of hepatoma cells and may serve as a potential candidate agent for the prevention of HCC metastasis.

Dihydromyricetin (DHM) is a major active ingredient of flavonoids compounds. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigated whether p53 is involved in DHM-triggered viability inhibition and apoptosis induction in cancer cells. MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was employed to evaluate the viability of HepG2 cells after DHM treatment. Meanwhile, p53 small interfering RNA (siRNA) was adopted to silence p53 expression. Protein level of p53 and Bax/Bcl-2 were evaluated by western blot analysis. Cell counting assay showed that DHM inhibited HepG2 cell growth effectively in a time- and dose-dependent manner. P53 expression was significantly increased after DHM treatment, whereas Bcl-2 was reduced potently. Furthermore, after co-treatment with Pifithrin-α (PFT-α, p53 inhibitor), Bcl-2 expression was reversed. The expression of Bax was no significant change, which was also observed after p53 silence. These findings defined and supported a novel function that DHM could induce human hepatocellular carcinoma HepG2 cells apoptosis by up-regulating Bax/Bcl-2 expression via p53 signal pathway.