DNA damage, c-myc suppression and apoptosis induced by the novel topoisomerase II inhibitor, salvicine, in human breast cancer MCF-7 cells

Abstract

Salvicine, a diterpenoid quinone compound, possesses potent in vitro and in vivo antitumor activity. Salvicine is a novel non-intercalative topoisomerase II poison. In this study salvicine induced evident DNA damage, which was further characterized as double-strand breaks mainly in MCF-7 human breast cancer cells. The degree of damage was highly correlated with growth inhibition of MCF-7. Using a PCR-stop assay we demonstrated that this damage was selective. Preferential damage occurred in the p2 promoter region, but not the 3'-end of the protooncogene c-myc. The expression of oncogenes, such as c-myc and c-jun, was additionally investigated. Salvicine induced a dose-dependent decrease in c-myc gene transcription, concomitant with an increase in c-jun expression. Furthermore, reverse-transcription PCR and Western blotting data revealed that salvicine failed to stimulate the mRNA and protein levels of p53 and its downstream targets p21 and bax. The phosphorylation degree of serine 15 of p53, which is thought to be an active form of p53 in response to cellular DNA damage, remained in a steady state. In view of these results, we propose that the downregulation of c-myc resulting from selective damage plays a role in apoptosis signaling. Moreover, salvicine-induced apoptosis in MCF-7 subsequent to DNA damage seems to be mediated through a p53-independent pathway.

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

“…The cytotoxicity of salvicine against MCF-7 cells was evaluated by sulforhodamine B (SRB) dye assay, as described previously (Lu et al, 2005). In brief, 100 l of cells (5 ϫ 10 4 /ml) per well were seeded in 96-well plates.…”

“…DNA DSBs were evaluated using the neutral single-cell gel electrophoresis assay (the neutral comet assay), as described previously (Olive et al, 1990) with minor modifications (Lu et al, 2005). In brief, MCF-7 cells (5 ϫ 10 5 /ml) were seeded into six-well plates, incubated overnight, and then treated with various concentrations of salvicine for the indicated times.…”

“…1). Salvicine possesses potent in vitro and in vivo activities against malignant tumor cells, particularly in some human solid tumor models (Qing et al, 1999), and induces apoptosis in various human tumor cell lines (Qing et al, 2001;Liu et al, 2002;Lu et al, 2005). It is noteworthy that salvicine shows prominent anti-multidrug resistance activities associated with down-regulation of mdr1 gene expression via the activation of c-jun .…”

“…This suggests that salvicine might promote DNA strand breaks by stabilizing the cleavable complex (Meng et al, 2001b). Further work in human breast carcinoma MCF-7 cells demonstrated that salvicine was responsible for inducing DNA double-strand breaks (DSBs), which have been proposed to be responsible for cell death (Lu et al, 2005).…”

The Telomeric Protein TRF2 Is Critical for the Protection of A549 Cells from Both Telomere Erosion and DNA Double-Strand Breaks Driven by Salvicine

“…DNA DSBs were evaluated using the neutral single-cell gel electrophoresis (neutral comet assays) as described previously (Olive et al, 1990) with minor modifications (Lu et al, 2005a). Quantification was performed by analyzing at least 50 randomly selected comets per slide with the Komet 5.5 software (Kinetic Imaging Ltd., Nottingham, UK).…”