Molecular mechanisms underlying Warburgia salutaris effects on oxidative stress and apoptotic parameters in Human Hepatoma Cells

Abstract

This study aims to determine the molecular effects of Warburgia salutaris extract in HepG2 cells and elucidate the possible mechanisms. The MTT assay was employed to determine cell viability and the half maximal inhibitory concentration (IC50) of Warburgia salutaris-treated in HepG2 cells (0-5mg/ml). Extracellular lactate dehydrogenase and ATP were also quantified as a measure of cell viability. The production of reactive oxygen species (ROS) was assessed by quantifying lipid peroxidation and oxidative DNA damage, and reactive nitrogen species (RNS) in treated HepG2 cells. The cells response to free radicals was assessed by measuring GSH. Stress response antioxidant and apoptotic markers were detected using western blotting and /or qPCR. Cell death parameters assayed included annexin V, caspase activity and necrosis. Single-cell gel electrophoresis (SCGE) was used to visualise DNA damage in the HepG2 cells and confirmed with DNA fragmentation assay. The Hoechst assay allowed the visualisation of the nucleus to assess cell growth and apoptosis. Decreased cell viability was associated with a decreased level of ATP. The presence of oxidative stress was suggested by increased HSP70 and Nrf2 protein expression and confirmed by increase ROS, RNS, GPx and catalase; and a corresponding decrease of SOD2 and glutathione. Caspase 8 showed no significant difference between treatment concentrations, caspase 9 was decreased and caspase 3/7 increased. A reduction in p53 correlated with chromatin changes, increase in comet lengths and DNA fragmentation. NF{kappa}B protein was significantly decreased at the IC50, along with decreased cMyc protein expression. Our findings shows that Warburgia salutaris promotes apoptosis by inducing oxidative stress in HepG2 cells and may be a potential anti-cancer agent that would serve as an alternative to conventional therapeutic agents.