Caspase inhibition augmented oridonin-induced cell death in murine fibrosarcoma l929 by enhancing reactive oxygen species generation.
Oridonin, a diterpenoid isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, the growth-inhibitory activity of oridonin for L929 cells was exerted in a time-and dose-dependent manner. After treatment with oridonin for 24 h, L929 cells underwent both apoptosis and necrosis as measured by an lactate dehydrogenase (LDH) activity-based assay. A rapid generation of reactive oxygen species (ROS) was triggered by oridonin, and subsequently up-regulation of phospho-p53 (ser 15) expression and an increased expression ratio of Bax/Bcl-2 was observed. Furthermore, there was a significant fall in mitochondrial membrane potential (MMP) and increase in caspase-3 activity after exposure to oridonin for 24 h. Surprisingly, the pan-caspase inhibitor z-VAD-fmk and caspase3 inhibitor z-DEVD-fmk rendered L929 cells more sensitive to oridonin, rather than preventing oridonin-induced cell death. Oridonin and z-VAD-fmk co-treatment not only resulted in an even higher ROS production, but also made a more significant reduction in the MMP. Pretreatment of ROS scavenger N-acetylcysteine (NAC) led to a complete inhibition of oridonin-induced cell death, intracellular ROS generation, and MMP collapse. NAC treatment also reversed the potentiation of cell death by the pan-caspase inhibitor z-VAD-fmk. Taken together, these observations showed that oridonin-induced cell death in L929 cells involved intracellular ROS generation, activation of phospho-p53 (ser 15), and up-regulation of the Bax/Bcl-2 ratio; and the augmented cell death by z-VAD-fmk was dependent on an increased ROS production.
Wu
Jin-Nan
JN
China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang, China.
Huang
Jian
J
Yang
Jia
J
Tashiro
Shin-Ichi
S
Onodera
Satoshi
S
Ikejima
Takashi
T
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D045786:Diterpenes, Kaurane / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects; D019254:Genes, bcl-2 / Q000187:drug effects; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D051336:Mitochondrial Membranes / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000502:physiology
D000590:Amino Acid Chloromethyl Ketones; D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D045786:Diterpenes, Kaurane; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C011959:oridonin; D007770:L-Lactate Dehydrogenase