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 (HepG2) cell line. Materials and Methods: β-sitosterol treatments (0.6 and 1.2 mM/ml) were done in HepG2 and after 24 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 3 expressions were evaluated by immunofluorescence and western blot analyses. Results: β-sitosterol induced cytotoxicity (p<0.001) and intracellular ROS in HepG2 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<0.05 vs 0.6 mM/ml and p<0.001 vs 1.2 mM/ml) of both caspase 3 and cleaved caspase 3. Conclusion: β-sitosterol induced ROS accumulation which plays a critical role in apoptosis via the intrinsic pathway in HepG2 cells. The present investigation paves the way for further in vivo studies.