Investigation of the possible mechanism of two kinds of sterols extracted from Leucocalocybe mongolica in inducing HepG2 cell apoptosis and exerting anti-tumor effects in H22 tumor-bearing mice

Abstract

Sterols are one of the main components of medicinal fungi with an anti-tumor effect. In this study, ergosta-4, 6, 8(14), 22-tetraen-3-one (ET) and (22E, 24R)-ergosta-7, 22-dien-3β, 5α, 6β-triol (ED) were obtained from Leucocalocybe mongolica and were used for the first time to study their ability to induce apoptosis in HepG2 cells and their anti-tumor effects and related mechanism in H22 tumor-bearing mice. MethodThe chemical structures were defined by IR and NMR. In vitro, the CCK8 assay was used as a cytotoxicity assay. Flow cytometry was used for the HepG-2 cell apoptosis analysis, which was examined via annexin V-FITC/PI double staining, and the related expression levels of the apoptosis-associated proteins were determined by western blot analysis. In vivo, ICR male mice were randomly assigned to eight groups: the model group, CTX (25 mg/kg/d) group, and ET and ED groups, which were treated with three different concentrations of each compound (0.025, 0.05, and 0.1 mmol/kg/d). Relevant biochemical indicators were detected by ELISA assay, H & E staining, TUNEL assay, immunohistochemical staining and western blot. ResultsIn vitro, ET and ED showed significant cytotoxic effects against HepG2, MCF–7, and HeLa cells, especially HepG-2 cells, and both ED and ET demonstrated a good effect in inhibiting the proliferation of HepG-2 cells. In vivo, ET and ED significantly decreased the tumor volume and VEGF levels but increased the serum cytokine levels of IFN-γ, IL-2, IL-6 and TNF-α. H & E staining, TUNEL assay, immunohistochemical analysis, and western blotting indicated that the both ET and ED exhibited anti-tumor activity in vivo by promoting apoptosis and inhibiting angiogenesis. Conclusion: These results indicated that both ET and ED have a strong inhibitory effect on the proliferation of HepG-2 cells in vitro and an anti-H22 tumor effect in vivo.