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. 2013 Jan 8;52(1):221-7.
doi: 10.1021/bi3014455. Epub 2012 Dec 26.

Oxidative metabolites of curcumin poison human type II topoisomerases

Adam C Ketron  1 , Odaine N Gordon, Claus Schneider, Neil Osheroff
Affiliations

Oxidative metabolites of curcumin poison human type II topoisomerases

Adam C Ketron et al. Biochemistry. .

Abstract

The polyphenol curcumin is the principal flavor and color component of the spice turmeric. Beyond its culinary uses, curcumin is believed to positively impact human health and displays antioxidant, anti-inflammatory, antibacterial, and chemopreventive properties. It also is in clinical trials as an anticancer agent. In aqueous solution at physiological pH, curcumin undergoes spontaneous autoxidation that is enhanced by oxidizing agents. The reaction proceeds through a series of quinone methide and other reactive intermediates to form a final dioxygenated bicyclopentadione product. Several naturally occurring polyphenols that can form quinones have been shown to act as topoisomerase II poisons (i.e., they increase levels of topoisomerase II-mediated DNA cleavage). Because several of these compounds have chemopreventive properties, we determined the effects of curcumin, its oxidative metabolites, and structurally related degradation products (vanillin, ferulic acid, and feruloylmethane) on the DNA cleavage activities of human topoisomerase IIα and IIβ. Intermediates in the curcumin oxidation pathway increased the level of DNA scission mediated by both enzymes ~4-5-fold. In contrast, curcumin and the bicyclopentadione, as well as vanillin, ferulic acid, and feruloylmethane, had no effect on DNA cleavage. As found for other quinone-based compounds, curcumin oxidation intermediates acted as redox-dependent (as opposed to interfacial) topoisomerase II poisons. Finally, under conditions that promote oxidation, the dietary spice turmeric enhanced topoisomerase II-mediated DNA cleavage. Thus, even within the more complex spice formulation, oxidized curcumin intermediates appear to function as topoisomerase II poisons.

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Figures

Figure 1
Figure 1
Oxidative transformation of curcumin. Adapted from Griesser et al.
Figure 2
Figure 2
Enhancement of topoisomerase II-mediated DNA cleavage by curcumin in the presence of oxidant. The effects of curcumin on the cleavage of negatively supercoiled plasmid DNA by human topoisomerase IIα (left) and topoisomerase IIβ (right) were determined in the absence (open circles, – Oxidant) or presence (closed circles, + Oxidant) of 50 μM K3Fe(CN)6. Error bars represent standard deviations for three independent experiments.
Figure 3
Figure 3
Effects of K3Fe(CN)6 on curcumin oxidation and the DNA cleavage activity of human topoisomerase IIα. Left: The effects of K3Fe(CN)6 on the cleavage of negatively supercoiled plasmid DNA by human topoisomerase IIα were determined in the absence (open circles) or presence (closed circles) of 25 μM curcumin. Error bars represent standard deviations for three independent experiments. Right: Ultraviolet/visible spectroscopic analysis of the loss of curcumin (maximum wavelength at 430 nm) and appearance of oxidized products (peak at 263 nm) in Tris-HCl buffer (pH 7.9) (top) and in topoisomerase II assay buffer (pH 7.9) (bottom). Scans were obtained at a frequency of one per min. K3Fe(CN)6 was added to the reaction in topoisomerase II assay buffer at 10 min.
Figure 4
Figure 4
DNA cleavage induced by oxidized curcumin is reversible and protein-linked. Assay mixtures contained enzyme in the absence of curcumin or oxidant (TIIα), 50 μM curcumin and 50 μM K3Fe(CN)6 in the absence of enzyme [−TIIα + Curcumin + Oxidant], or complete reactions treated with SDS prior to adding EDTA (SDS). To determine whether DNA cleavage induced by oxidized curcumin was reversible, reactions were incubated with EDTA prior to trapping cleavage complexes with SDS (EDTA). To determine whether DNA cleavage induced by oxidized curcumin was protein-linked, proteinase K treatment was omitted (−ProK). Error bars represent standard deviations for three independent experiments. A representative agarose gel is shown at the top. DNA lanes correspond to the bars shown in the graph. The positions of negatively supercoiled (form I, FI), nicked (form II, FII), and linear (form III, FIII) plasmid DNA are indicated.
Figure 5
Figure 5
Effects of 4′,4″-dimethylcurcumin on topoisomerase II-mediated DNA cleavage. The effects of 4′,4″-dimethylcurcumin (structure at top, 4′- and 4″-methyl groups highlighted in red) in the absence (open circles, – Oxidant) or presence (closed circles, + Oxidant) of 50 μM K3Fe(CN)6 on the cleavage of negatively supercoiled plasmid DNA by human topoisomerase IIα were determined. Data for oxidized curcumin (dashed line from Figure 2) are included for comparison. Error bars represent standard deviations for three independent experiments.
Figure 6
Figure 6
Effects of bicyclopentadione on topoisomerase II-mediated DNA cleavage. The effects of bicyclopentadione (structure shown in Figure 1) in the absence (open circles, – Oxidant) or presence (closed circles, + Oxidant) of 50 μM K3Fe(CN)6 on the cleavage of negatively supercoiled plasmid DNA by human topoisomerase IIα were determined. Inset: curcumin was incubated in the absence (−) or presence (+) of K3Fe(CN)6 for 10 min before addition to topoisomerase IIα-DNA cleavage assay mixtures that contained 50 μM K3Fe(CN)6. Error bars represent standard deviations for three independent experiments.
Figure 7
Figure 7
Oxidized curcumin intermediates act as redox-dependent topoisomerase II poisons. Left: The effects of DTT on the enhancement of topoisomerase IIα-mediated DNA cleavage by oxidized curcumin intermediates were determined. Reaction mixtures contained DNA and human topoisomerase IIα in the absence or presence of 50 μM curcumin, 50 μM K3Fe(CN)6, or 250 μM DTT. In some reactions, DTT was added after the establishment of enzyme-DNA cleavage complexes (Post-DTT). Middle: The effects of oxidized curcumin intermediates on topoisomerase IIα activity when compounds were incubated with the enzyme prior to the addition of DNA. Human topoisomerase IIα was incubated with a combination of 50 μM curcumin and 50 μM K3Fe(CN)6 for 0-10 min prior to the addition of DNA to initiate 6 min cleavage reactions. Right: The effects of oxidized curcumin intermediates on the enhancement of DNA cleavage by human wild-type topoisomerase IIα (WT) and mutant quinone-resistant topoisomerase IIαC392A/C405A (C392A/C405A) were determined. Error bars for all three panels represent standard deviations for three independent experiments.
Figure 8
Figure 8
Effects of curcumin degradation products on topoisomerase II-mediated DNA cleavage. The effects of vanillin (left), ferulic acid (middle), or feruloylmethane (right) on the cleavage of negatively supercoiled plasmid DNA by human topoisomerase IIα were determined in the absence (open circles, – Oxidant) or presence (closed circles, + Oxidant) of 50 μM K3Fe(CN)6. Data for curcumin (dashed line from Figure 2) are included in the left panel for comparison. Error bars represent standard deviations for three independent experiments.
Figure 9
Figure 9
Effects of turmeric on topoisomerase II-mediated DNA cleavage. The effects of turmeric on the cleavage of negatively supercoiled plasmid DNA by human topoisomerase IIα were determined in the absence (open circles, – Oxidant) or presence (closed circles, + Oxidant) of 50 μM K3Fe(CN)6. The turmeric stock solution was determined to contain ~2.7% curcumin. On the basis of this concentration, DNA cleavage results for 5 and 25 μM oxidized curcumin intermediates (asterisks) are overlaid at the associated turmeric concentrations for comparison. Error bars represent standard deviations for three independent experiments.
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