Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II.
Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topo II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.
Kawiak
Anna
A
Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.
Piosik
Jacek
J
Stasilojc
Grzegorz
G
Gwizdek-Wisniewska
Anna
A
Marczak
Lukasz
L
Stobiecki
Maciej
M
Bigda
Jacek
J
Lojkowska
Ewa
E
D017209:Apoptosis / Q000187:drug effects*; D020552:Comet Assay; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D029787:Drosera / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D005434:Flow Cytometry; D008401:Gas Chromatography-Mass Spectrometry; D018922:HL-60 Cells; D006801:Humans; D009285:Naphthoquinones / Q000302:isolation & purification / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D017382:Reactive Oxygen Species; D059005:Topoisomerase II Inhibitors; C014758:plumbagin