β-sitosterol induces reactive oxygen species-mediated apoptosis in human hepatocellular carcinoma cell line

Objective: It is of interest to investigate the anti-proliferative effect of β-sitosterol (BS) on human hepatocellular carcinoma (HepG۲) cell line.Materials and Methods: β-sitosterol treatments (۰.۶ and ۱.۲ mM/ml) were done in HepG۲ and after ۲۴ hr, cell viability was evaluated by MTT assay. Reactive oxygen species (ROS) accumulating potential of BS was assessed by dichloro-dihydro-fluorescein diacetate staining. Morphology related to apoptosis was investigated by acridine orange and ethidium bromide dual staining. Cytochrome c and caspase ۳ expressions were evaluated by immunofluorescence and western blot analyses.Results: β-sitosterol induced cytotoxicity (p<۰.۰۰۱) and intracellular ROS in HepG۲ cells in a dose-dependent manner.BS treatments accumulated induced intracellular ROS accumulation which led to membrane damage and mitochondrial toxicity. At the molecular level, BS treatments induced cytochrome c release from mitochondria and enhanced the protein expressions (p<۰.۰۵ vs ۰.۶ mM/ml and p<۰.۰۰۱ vs ۱.۲ mM/ml) of both caspase ۳ and cleaved caspase ۳.Conclusion: β-sitosterol induced ROS accumulation which plays a critical role in apoptosis via the intrinsic pathway in HepG۲ cells. The present investigation paves the way for further in vivo studies.

Keywords:

Liver cancer , β-sitosterol , Reactive Oxygen Species , Apoptosis , Caspase