Differentiation of bel-7402 human hepatocarcinoma cells induced by aqueous extracts of fresh gecko (AG) and its anti-tumor activity in vivo

Ethnopharmacological relevance: Gecko, a kind of reptile, has been widely used as a traditional Chinese medicine to treat various diseases including cancer in China for thousands of years. The aim of this study was to investigate the anti-tumor effect of AG (aqueous extracts of fresh gecko) on human hepatocellular carcinoma cell Bel-7402 in vitro and mouse H22 hepatocellular in vivo. Further to underlie the molecular mechanism of AG inducing the differentiation of Bel-7402 cells. Materials and methods: AG was obtained by water extracting method and qualitatively analyzed through High Performance Liquid Chromatography. The total protein concentration of AG was measured by BCA (bicinchoninic acid disodium) assay. The anti-tumor activities in vivo were analyzed through H22 (mouse hepatocellular carcinoma cell line H22) tumor xenografts mice. The cytotoxic activity of AG on Bel-7402 cells was evaluated by MIT assays. AFP (alpha fetoprotein) was detected by radioimmunoassay. ALB (albumin), ALP (alkaline phosphatase) and gamma-GT (gamma-glutamyl transpeptidase) were detected by biochemical methods with commercial kits. While morphological changes were observed through an inverted microscope. Moreover, the expression level of the proteins involved in MAPK (mitogen-activated protein kinase) signal pathway which was closely related to cellular differentiation was assessed by Western blot. Results: AG showed obviously anti-tumor activity in vivo and anti-proliferative activity on Bel-7402 cells in vitro both dose-dependently. The number of clones of Bel-7402 cells treated with AG reduced and the cells were displaying differentiation state such as relatively bigger size and dispersed growth. The biochemical function markers of the cells were significantly changed after being treated with AG. The data showed that AFP secretion of the cells decreased 42.5%, ALB secretion increased 58.9%, the activity of ALP and gamma-GT markedly decreased 67.0% and 48.5% separately when the concentration of AG was 10 pm], and those effects were all in a dose-dependent manner. The major original and phosphorylated signal proteins (ERK1/2 (extracellular sigal-regualted kinase 1/2), P38 (p38 MAPK) and JNK1/2 (c-Jun N-terminal kinase 1/2)) involved in MAPK signal pathway were measured and the results showed that AG activated the ERK1/2 of Bel-7402 cells. Conclusions: AG has anti-tumor activity in vivo and inhibits Bel-7402 cell proliferation in vitro through inducing cell differentiation, and the mechanism involves the activation of ERK1/2. (C) 2014 Elsevier Ireland Ltd. All rights reserved.