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, 46 (20), 6097-108

Bioflavonoids as Poisons of Human Topoisomerase II Alpha and II Beta

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Bioflavonoids as Poisons of Human Topoisomerase II Alpha and II Beta

Omari J Bandele et al. Biochemistry.

Abstract

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.

Figures

Figure 1
Figure 1
Structures of selected bioflavonoids. Flavones, flavonols, and isoflavones are shown.
Figure 2
Figure 2
Genistein-induced DNA cleavage is mediated by human topoisomerase IIα (hTIIα) and IIβ (hTIIβ). Ethidium bromide-stained agarose gels are shown. The reversibility of genistein-induced DNA cleavage complexes was determined by adding EDTA to reaction mixtures before these complexes were trapped by SDS (+EDTA; lanes 4 and 9). To determine whether DNA cleavage induced by genistein was protein-linked, proteinase K treatment was omitted (−ProK; lanes 5 and 10). Control reactions contained DNA alone (DNA; lanes 1 and 6), DNA and enzyme in the absence of genistein (none; lanes 2 and 7), or reaction mixtures treated with SDS prior to EDTA (Genistein; lanes 3 and 8). The mobility of negatively supercoiled DNA (form I, FI), nicked circular plasmid (form II, FII), and linear molecules (form III, FIII) are indicated. Data are representative of at five least independent experiments.
Figure 3
Figure 3
Effects of bioflavonoids on double-stranded DNA breaks generated by human topoisomerase IIα and IIβ. Data for topoisomerase IIα– (hTIIα; open bars) and IIβ–(hTIIβ; closed bars) mediated DNA cleavage in the presence of 50 μM flavones, flavonols, and isoflavones are shown in the bar graph. The inset shows a titration for DNA cleavage meditated by topoisomerase IIα (open circles) and IIβ (closed circles) in the presence of 0–200 μM genistein. Error bars represent standard deviations for three independent experiments.
Figure 4
Figure 4
Effects of bioflavonoids on DNA cleavage site utilization by human topoisomerase IIα (hTIIα) and IIβ (hTIIβ). Autoradiograms of polyacrylamide gels are shown. DNA cleavage reactions contained no compound (none), 25 μM etoposide, or 50 μM bioflavonoid. A DNA control is shown in the far left lane of each autoradiogram (DNA). Data are representative of two independent experiments.
Figure 5
Figure 5
Contributions of the 5-OH and 4′-OH moieties to the activity of genistein. Effects of daidzein (lacking 5-OH; closed circles), biochanin A (containing a 4′-methoxy group in place of the 4′-OH; open circles), and chrysin (a flavone that lacks the 4′-OH; closed squares) on the ability of genistein to enhance DNA cleavage mediated by human topoisomerase IIα (hTIIα) and IIβ (hTIIβ) are shown. DNA cleavage reactions were carried out in the presence of 50 μM genistein and 0–500 μM competing bioflavonoid. Competition was quantified by the loss of genistein-induced linear DNA molecules. DNA cleavage was set to 100% in the absence of competitor. Error bars represent standard deviations for three independent experiments.
Figure 6
Figure 6
Contributions of the 5-OH and 4′-OH moieties to the binding of genistein to topoisomerase IIα. Effects of daidzein (lacking 5-OH; closed circles), biochanin A (containing a 4′-methoxy group in place of the 4′-OH; open circles), chrysin (a flavone that lacks the 4′-OH; closed squares) and unlabeled genistein (open squares) on the ability of [14C]genistein to bind to human topoisomerase IIα are shown. Nitrocellulose filter binding assays were carried out in the presence of 25 μM [14C]genistein and 0–250 μM competing bioflavonoid. Competition was quantified by the loss of enzyme bound [14C]genistein. Enzyme binding was set to 100% in the absence of competitor. Error bars represent standard deviations for three independent experiments.
Figure 7
Figure 7
Effects of bioflavonoids on DNA ligation mediated by human topoisomerase IIα (hTIIα, top panels) and IIβ (hTIIβ, bottom panels). Left panels: DNA ligation was examined in the absence of compounds (closed circles), or in the presence of 50 μM genistein (open squares) or etoposide (closed squares). Samples were incubated at 37 °C to establish DNA cleavage/ligation equilibria and were shifted to 0 °C to initiate the ligation reaction. The amount of DNA cleavage observed at equilibrium for was set to 100% at time zero. Ligation was quantified by the loss of linear cleaved molecules. Right panels: Representative DNA ligation data for reactions containing no compound, 50 μM bioflavonoid, or 50 μM etoposide at 20 s after shifting samples to 0 °C are shown. Error bars represent standard deviations for three independent experiments.
Figure 8
Figure 8
Dithiothreitol does not impair the ability of genistein to enhance DNA cleavage mediated by human topoisomerase IIα (hTIIα) or IIβ (hTIIβ). Ethidium bromide-stained agarose gels are shown. The positions of supercoiled (FI), nicked circular (FII), and linear (FIII) DNA molecules are labeled as in Figure 2. The gels show reactions in which 50 μM genistein was incubated without dithiothreitol (−DTT; lane 4) or with 500 μM dithiothreitol prior to its addition to cleavage reactions (+DTT; lane 5). Parallel control reactions mediated by topoisomerase IIα or IIβ in the absence of genistein are shown in lanes 2 and 3. A DNA standard is shown in lane 1. Results are representative of three independent experiments.
Figure 9
Figure 9
Genistein does not inactivate human topoisomerase IIα (hTIIα) or IIβ (hTIIβ) in the absence of DNA. Ethidium bromide-stained agarose gels of DNA cleavage reactions are shown. Enzymes were incubated simultaneously with plasmid DNA and 50 μM genistein (Same; lane 3) or with 50 μM genistein for 5 min prior to the addition of DNA (Pre; lane 4). A DNA standard (DNA; lane 1) and reactions mediated by topoisomerase IIα or IIβ in the absence of genistein (hTII; lane 2) are shown. The positions of supercoiled (FI), nicked circular (FII), and linear (FIII) DNA are labeled as in Figure 2. Results are representative of three independent experiments.
Figure 10
Figure 10
htop2αG474A displays decreased sensitivity to genistein. Levels of DNA cleavage for the wild-type human enzyme (hTIIα, closed circles) and hTop2αG474A (G474A, open circles) were quantified and expressed as the relative increase in linear molecules in the presence of 0–200 μM genistein. DNA cleavage for yeast topoisomerase II(yTII, closed squares) is shown for comparison. Error bars represent standard deviations for three independent experiments.
Figure 11
Figure 11
Bioflavonoids enhance DNA cleavage mediated by topoisomerase IIα and IIβ in cultured human CEM cells. The ICE bioassay was used to monitor levels of cleavage complexes in cells treated with selected bioflavonoids. DNA (3 μg) from cell cultures treated for 1 h in the absence of compound (none) or in the presence of 50 μM bioflavonoid or 50 μM etoposide was blotted onto a nitrocellulose membrane. Immunoblots were probed with a polyclonal antibody directed against either human topoisomerase IIα or IIβ, respectively. A representative immunoblot is shown. The bar graph shows quantified data for topoisomerase IIα (hTIIα; open bars) or IIβ (hTIIβ; closed bars). Levels of covalently bound topoisomerase II (expressed in ng) are based on standards of purified human type II topoisomerases. Error bars represent the standard deviation for three independent experiments.
Figure 12
Figure 12
Contribution of the 5-OH and 4′-OH moieties to the ability of genistein to enhance DNA cleavage mediated by human topoisomerase IIα or IIβ in cultured human CEM cells. The ICE bioassay was used to monitor the level of cleavage complexes in CEM cells treated with 25 or 50 μM genistein (Gen) in the absence or presence of 250 μM biochanin A (BA) or 500 μM daidzein (Daid), respectively. DNA (3 μg) from cells treated for 1 h were blotted onto a nitrocellulose membrane. Immunoblots were probed with a polyclonal antibody directed against either human topoisomerase IIα or IIβ, respectively. A representative immunoblot is shown. The bar graph shows quantified data for topoisomerase IIα (hTIIα; open bars) or IIβ (hTIIβ; closed bars). Levels of covalently bound topoisomerase II (expressed in ng) are based on standards of purified human type II topoisomerases. Error bars represent the standard deviation of three independent experiments.

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