The Research On The Apoptotic Effect Of Sarsasapogenin On HepG2 Cells

Sarsasapogenin is a steroidal sapogenin with antitumor property. Apoptosis was confirmed by cell cycle analysis and LDH assay. To explain the mechanism of its apoptotic effect, mitochondrial activity was assessed via a 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow Cytometry (FCM) was used to estimate the changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, and cellular-reduced glutathione (GSH) level. Laser scanning confocal microscope (LSCM) recorded instantaneous ROS burst after the application of sarsasapogenin. The relationship between intracellular Ca²⁺ concentration and ROS burst was examined by LSCM. Western blotting was used to determine the expression level and intracellular distribution of cytochrome c (cyt c) and p-Akt. Results of this study demonstrate that during apoptosis, calcium dependent ROS burst acted as an early event followed by depolarization of MMP, prolonged ROS generation, and significantly declined GSH level. Cyt c was up-regulated and released from mitochondria to cytosol and p-Akt was declined during the process. These new findings show that mitochondrial ROS burst was an early upstream apoptotic signal which may trigger the mitochondrial apoptotic pathway and played a vital role in sarsasapogenin-induced HepG2 cell apoptosis.