Kawiak Anna, Piosik Jacek, Stasilojc Grzegorz, Gwizdek-Wisniewska Anna, Marczak Lukasz, Stobiecki Maciej, Bigda Jacek, Lojkowska Ewa
Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.
Toxicol Appl Pharmacol. 2007 Sep 15;223(3):267-76. doi: 10.1016/j.taap.2007.05.018. Epub 2007 Jun 7.
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.
活性氧（ROS）已被公认为关键分子，它可以选择性地修饰蛋白质，从而调节包括细胞凋亡在内的细胞信号传导。白花丹醌是一种具有抗肿瘤活性的萘醌，已知其能产生活性氧，并且已发现它通过稳定拓扑异构酶II（Topo II）-DNA可裂解复合物来抑制Topo II的活性。本研究的目的是阐明活性氧和Topo II抑制在白花丹醌介导的细胞凋亡诱导中的作用。通过彗星试验测定，白花丹醌诱导HL-60细胞中的DNA裂解，而在Topo II活性降低的细胞系HL-60/MX2中，DNA损伤水平显著降低。与细胞内活性氧的产生相比，HL-60细胞中DNA链断裂形成的起始延迟。在HL-60/MX2细胞中，活性氧以相似的速率产生，而检测到DNA损伤水平显著降低。用N-乙酰半胱氨酸（NAC）预处理细胞可减轻白花丹醌诱导的DNA损伤，表明活性氧的产生参与了可裂解复合物的形成。这些结果表明，白花丹醌诱导的活性氧不会直接损伤DNA，但需要Topo II的参与。此外，使用光谱学进行的实验表明白花丹醌与DNA之间没有直接相互作用。HL-60/MX2细胞中细胞凋亡的诱导显著延迟，表明Topo II抑制参与了白花丹醌介导的细胞凋亡。因此，这些发现强烈表明活性氧介导的Topo II抑制是白花丹醌诱导细胞凋亡特性的重要机制。
