Mitochondrial ROS burst as an early sign in sarsasapogenin-induced apoptosis in HepG2 cells.
Sarsasapogenin is a steroidal sapogenin with antitumor properties. 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 application of sarsasapogenin. Western blotting was used to determine the expression level and intracellular distribution of cytochrome c (cyt c). It is demonstrated that during apoptosis, ROS burst acted as an early event followed by depolarization of MMP, prolonged ROS generation, and significantly declined GSH level. Cyt c was upregulated and released from mitochondria to cytosol during the process. These findings show that a mitochondrial ROS burst is an early upstream apoptotic signal which may trigger the mitochondrial apoptotic pathway and play a vital role in sarsasapogenin-induced HepG2 cell apoptosis.
Ni
Yuan
Y
Institute of Biochemistry, Zhejiang University, Hangzhou, PR China.
Gong
Xing-guo
XG
Lu
Min
M
Chen
Han-min
HM
Wang
Yong
Y
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013150:Spirostans / Q000494:pharmacology*; D013997:Time Factors
D000972:Antineoplastic Agents, Phytogenic; D017382:Reactive Oxygen Species; D013150:Spirostans; D045304:Cytochromes c; C046297:sarsasapogenin; D005978:Glutathione