Reactive Oxygen Species Elicit Apoptosis by Concurrently Disrupting Topoisomerase II and DNA-Dependent Protein Kinase

Abstract

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 salvicineinduced 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.

R16, a novel amonafide analogue, induces apoptosis and G2-M arrest via poisoning topoisomerase II

“…Neutral Single-Cell Gel Electrophoresis Assay HL-60 cells (5 Â 10 5 /mL) were treated with either R16 or amonafide for 1 h. DNA double-strand breaks (DSB) were evaluated by neutral single-cell gel electrophoresis assay, which was done according to the method developed by Singh (21) with slight modifications (22). A fluorescence microscope (Olympus BX51) was used to capture the images.…”

“…Trapped in Agarose DNA Immunostaining Assay Trapped in agarose DNA immunostaining (TARDIS) assay was done as previously reported (22,24). Briefly, untreated or treated cells were harvested and mixed with low-melting gel spreading on slides, followed by placing the slides in lysis buffer containing protease inhibitors.…”

“…R16 Induces Stable DNA -topo II Complexes. Next, the TARDIS assay was used to detect the formation of topo II -DNA cleavage complexes at the cellular level (22,24). Heat-induced reversibility is a unique property of topo II cleavage complexes (29).…”

Important differences between topoisomerase-I and -II targeting agents

“…Similar attenuation of double-strand breaks upon pretreatment with NAC and exposure to other established topoisomerase-II targeting drugs, like doxorubicin and newer agents like salvacine have been reported previously, but the mechanism of ROS generation in this metabolic context is not fully understood. 17,18 Mitoxantrone is not unique in its capacity as a ROS-generation facilitator, and this quality is shared with other groups of drugs, for example farnesyl-transferase inhibitors. 19 A limiting factor in the use of topoisomerase-II targeting drugs is the significant toxicity that develops with the cumulative use of these drugs.…”

“…Thus, there is an urgent need for the development of future treatment regimens that may reduce the total dose of these drugs by combining them with inhibitors of DNA repair, currently under investigation. [18][19][20] The study by Huang and colleagues provides important new insight into the molecular mechanism of action of these drugs, thus facilitating the development of new therapy protocols.…”