Reactive oxygen species elicit apoptosis by concurrently disrupting topoisomerase II and DNA-dependent protein kinase.
Reactive oxygen species (ROS) are produced by all aerobic cells and have been implicated in the regulation of diverse cellular functions, including intracellular signaling, transcription activation, proliferation, and apoptosis. Salvicine, a novel diterpenoid quinone compound, demonstrates a broad spectrum of antitumor activities. Although salvicine is known to trap the DNA-topoisomerase II (Topo II) complex and induce DNA double-strand breaks (DSBs), its precise antitumor mechanisms remain to be clarified. In this study, we investigated whether salvicine altered the levels of ROS in breast cancer MCF-7 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that salvicine stimulated intracellular ROS production and subsequently elicited notable DSBs. The addition of N-acetyl cysteine (NAC), an antioxidant, effectively attenuated the salvicine-induced ROS enhancement and subsequent DNA DSBs. Heat treatment reversed the accumulation of DNA DSBs, and the addition of NAC attenuated the Topo II-DNA cleavable complexes formation and the growth inhibition of salvicine-treated JN394top2-4 yeast cells, collectively indicating that Topo II is a target of the salvicine-induced ROS. On the other hand, when examining the impact of salvicine on DNA repair pathways, we unexpectedly observed that salvicine selectively down-regulated the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) protein levels and repressed DNA-PK kinase activity; both of these effects were attenuated by NAC pretreatment of MCF-7 cells. Finally and most importantly, NAC attenuated salvicine-induced apoptosis and cytotoxicity in MCF-7 cells. These results indicate that apart from its direct actions, salvicine generates ROS that modulate DNA damage and repair, contributing to the comprehensive biological consequences of salvicine treatment, such as DNA DSBs, apoptosis, and cytotoxicity in tumor cells. The finding of salvicine-induced ROS provides new evidence for the molecular mechanisms of this compound. Moreover, the effects of salvicine-induced ROS on Topo II and DNA-PK give new insights into the diverse biological activities of ROS.
Lu
Hua-Rui
HR
Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Peoples Republic of China.
Zhu
Hong
H
Huang
Min
M
Chen
Yi
Y
Cai
Yu-Jun
YJ
Miao
Ze-Hong
ZH
Zhang
Jin-Sheng
JS
Ding
Jian
J
D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D045744:Cell Line, Tumor; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009285:Naphthoquinones / Q000494:pharmacology; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D059005:Topoisomerase II Inhibitors
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D009687:Nuclear Proteins; D017382:Reactive Oxygen Species; D059005:Topoisomerase II Inhibitors; C122096:salvicine; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D000111:Acetylcysteine