Six soy isoflavones, daidzein (1), daidzin (2), genistein (3), genistin (4), glycitein (5) and glycitin (6), were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA; Fig. 1). The compounds, purified using HPLC, were of analytical grade. A chemically synthesized DNA template, poly(dA), was purchased from Sigma-Aldrich Inc. and a customized oligo(dT)₁₈ DNA primer was produced by Sigma-Aldrich Japan K.K. (Hokkaido, Japan). Radioactive nucleotide [³H]-labeled 2′-deoxythymidine-5′-triphosphate (dTTP; 43 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA, USA). Supercoiled pBR322 plasmid dsDNA was obtained from Takara Bio Inc. (Kyoto, Japan). All other reagents were analytical grade and were obtained from Nacalai Tesque Inc. (Kyoto, Japan).

Pol α was purified from calf thymus by immuno-affinity column chromatography, as described by Tamai et al (16). Recombinant rat pol β was purified from Escherichia coli JMpβ5, as described by Date et al (17). Human pol γ catalytic gene was cloned into pFastBac. The histidine-tagged enzyme was expressed using the BACTO-BAC HT Baculovirus Expression System, according to the supplier’s instructions (Life Technologies, Frederick, MD) and was purified using ProBond resin (Invitrogen Japan, Tokyo, Japan) (18). Human pols δ and ɛ were purified by nuclear fractionation of human peripheral blood cancer cells (Molt-4) using the second subunit of pol δ and ɛ-conjugated affinity column chromatography, respectively (19). A truncated form of human pol η (residues 1–511) tagged with His6 at its C-terminal was expressed in E. coli cells and was purified as described by Kusumoto et al (20). A recombinant mouse pol ι that was tagged with His6 at its C-terminal was expressed by E. coli and purified by Ni-NTA column chromatography (unpublished data). A truncated form of pol κ (residues 1–560) with 6X His-tags attached at the C-terminus was overproduced in E. coli and purified as described by Ohashi et al (21). Recombinant human His-pol λ was overexpressed in E. coli and purified according to a method described by Shimazaki et al (22). Recombinant human His-pol μ was overexpressed in E. coli BL21 and purified using Glutathione Sepharose™ 4B (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) column chromatography using the same method as for pol λ (22). Calf TdT, T7 RNA polymerase, T4 polynucleotide kinase, and Bovine pancreas deoxyribonuclease I were purchased from Takara Bio Inc. (Kyoto, Japan). Purified human placenta topos I and II were purchased from TopoGen Inc. (Columbus, OH, USA).

Reaction mixtures for calf pol α and rat pol β have been described previously (23,24); those for pol γ and for pols δ and ɛ were as described by Umeda et al (18) and Ogawa et al (25), respectively. Reaction mixtures for pols η, ι and κ were the same as for pol α and those for pols λ, μ and TdT were the same as for pol β. For the pol reactions, poly(dA)/oligo(dT)₁₈ (A/T, 2/1) and dTTP were used as the DNA template-primer substrate and nucleotide (dNTP; 2′-deoxynucleoside-5′-triphosphate) substrate, respectively. For TdT reactions, oligo(dT)₁₈ (3′-OH) and dTTP were used as the DNA primer substrate and nucleotide substrate, respectively.

Soy isoflavone compounds 1–6 were dissolved in distilled dimethyl sulfoxide (DMSO) to various concentrations and sonicated for 30 sec. Then, 4 μl aliquots were mixed with 16 μl of each enzyme (0.05 units) in 50 mM Tris-HCl at pH 7.5 that contained 1 mM dithiothreitol, 50% glycerol (by vol), and 0.1 mM ethylenediaminetetraacetic acid (EDTA). The mixtures were maintained at 0°C for 10 min. Next, 8 μl of each inhibitor-enzyme mixture was added to 16 μl of enzyme standard reaction mixture and incubated at 37°C for 60 min. The activity in samples without inhibitors was considered to be 100% and the activity was determined for each inhibitor concentration relative to the uninhibited activity. One unit of pol activity was defined as the amount of each enzyme that catalyzed the incorporation of 1 nmol dTTP into synthetic DNA template-primers in 60 min at 37°C and under normal reaction conditions (23,24).

The catalytic activity of topo I was determined by detecting supercoiled plasmid DNA (form I) in its nicked form (form II) (26). The topo I reaction was performed in a 20 μl reaction mixture that contained 10 mM Tris-HCl (pH 7.9), pBR322 DNA (250 ng), 1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin (BSA), 0.l mM spermidine, 5% glycerol, 2 μl of one of the 6 test compounds 1–6 dissolved in DMSO, and 2 units of topo I. The catalytic activity of topo II was analyzed in the same manner, except the reaction mixture contained 50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl₂, 0.5 mM ATP, 0.5 mM dithiothreitol, supercoiled pBR322 DNA (250 ng), and 2 units of topo II (26). The reaction mixtures were incubated at 37°C for 30 min, followed by digestion with 1% sodium dodecyl sulfate (SDS) and 1 mg/ml proteinase K. After digestion, 2 μl loading buffer, consisting of 5% sarkosyl, 0.0025% bromophenol blue, and 25% glycerol, was added. To study the binding of enzymes to DNA based on mobility shifts, the same procedure was followed, but SDS denaturation and proteinase K digestion were omitted. The mixtures were subjected to 1% agarose gel electrophoresis in Tris/borate/EDTA buffer. Agarose gel was stained with ethidium bromide (EtBr) and the DNA band shifts from form I to form II by topos I and II were detected using an enhanced chemiluminescence detection system (Perkin-Elmer Life Sciences Inc., Waltham, MA, USA). Zero-D scan (Version 1.0, M & S Instruments Trading Inc., Osaka, Japan) was used for densitometric quantitation.

Standard assays were used according to the manufacturer’s instructions to measure the activities of T7 RNA polymerase, mouse IMP dehydrogenase (type II), T4 polynucleotide kinase, and bovine deoxyribonuclease I, as described by Nakayama and Saneyoshi (27), Mizushina et al (28), Soltis and Uhlenbeck (29) and Lu and Sakaguchi (30), respectively.

Thermal profiles of the transition of dsDNA to single-stranded DNA (ssDNA) with or without genistein were obtained using a spectrophotometer (UV-2500, Shimadzu Corp., Kyoto, Japan) equipped with a thermoelectric cell holder, as described previously (31). Calf thymus DNA (6 μg/ml) was dissolved in 0.1 M sodium phosphate buffer (pH 7.0) that contained 1% DMSO. The solution temperature was equilibrated at 75°C for 10 min, and then increased by 1°C at 2-min intervals for each measurement point. Any change in the absorbance of the compound at each temperature point was automatically subtracted from that of the combined absorbance of the DNA and the compound by the spectrophotometer.

A human colon carcinoma cell line, HCT116, was obtained from the American Type Culture Collection (Manassas, VA, USA). HCT116 cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humid atmosphere of 5% CO₂/95% air. For the cell viability assay, cells were plated at 1×10⁴ into each well of a 96-well microplate with various concentrations of genistein. Cell viability was determined by the WST-1 assay (32).

Cellular DNA content for cell cycle analysis was determined as follows: aliquots of 3×10⁵ HCT116 cells were added to a 35-mm dish and incubated with a medium that contained genistein [94.0 μM based on the 50% lethal dose (LD₅₀) value] for 24 h. Cells were then washed with ice-cold PBS 3 times by centrifugation, fixed with 70% (v/v) ethanol, and stored at −20°C. DNA was stained with PI (3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide) staining solution for at least 10 min at room temperature in the dark. Intensity of fluorescence was measured using a FACSCanto flow cytometer in combination with FACSDiVa software (Becton-Dickinson Co., Franklin Lakes, NJ, USA).

The molecular structures of compounds were constructed using Discovery Studio (DS) 3.5 modeling software (Accelrys Inc., San Diego, CA, USA). Energy minimization was achieved using a salvation model and was calculated by the GBSW (Generalized Born with simple switching) parameter using the Minimization and Dynamics protocols contained within DS. The calculation used a CHARMm (Chemistry at HARvard Macromolecular Mechanics) force-field.

The inhibitory activity of each soy isoflavone toward mammalian pols was investigated using calf pol α, rat pol β, human pol γ, and human pol κ. Pols α, β, γ and κ were used as representatives of the B, X, A, and Y families of pols, respectively (6,7). Assessment of the relative activity of each pol at a set concentration (100 μM) of the 6 soy isoflavones showed that none of the compounds had any effect on pol inhibition, as no compound resulted in <90% relative activity of the 4 pols (Fig. 2). These results suggest that soy isoflavones do not influence the activities of mammalian pol species. When activated DNA (bovine deoxyribonuclease I-treated DNA) was used as the DNA template-primer substrate instead of synthesized DNA [poly(dA)/oligo(dT)₁₈ (A/T=2/1)] and dNTP was used as the nucleotide substrate instead of dTTP, the inhibitory effects of these compounds did not change (data not shown).

The inhibitory effects of each soy isoflavone were examined against human topos I and II, which have ssDNA and dsDNA nicking activity, respectively (8). None of the soy isoflavones at 100 μM influenced topo I nicking activity (Fig. 3). Even at concentration of greater than 100 μM, these compounds had no effect on topo I activity (data not shown). In contrast, 100 μM of genistein (6) completely inhibited the nicking activity of topo II, while the other compounds inhibited topo II to a lesser extent or not at all (Fig. 3). These results suggest that genistein is a potent human topo II inhibitor, but there are no topo I inhibitors among the 6 soy isoflavones tested. Genistein was therefore selected for further study.

Genistein did not affect the activity of any of the eleven mammalian pol species tested in vitro (Table I). Genistein inhibited the activity of human topo II with a 50% inhibitory concentration (IC₅₀) value of 37.5 μM (Table I).

Genistein had no influence on the activity of other DNA metabolic enzymes such as T7 RNA polymerase, mouse IMP dehydrogenase (type II), T4 polynucleotide kinase, and bovine deoxyribonuclease I (Table I). These results indicate that genistein should be specifically classified as an inhibitor of human topo II.

Specific assays were performed to determine whether genistein-induced inhibition resulted from the ability of the compound to bind to DNA or to the enzyme. The interaction of genistein with dsDNA was investigated by studying its thermal transition. To accomplish this, the melting temperature (Tm) of dsDNA in the presence of an excess of genistein (200 μM) was measured using a spectrophotometer equipped with a thermoelectric cell holder. As shown in Fig. 4, when a typical intercalating compound, EtBr (15 μM), was used as a positive control, a clear thermal transition (i.e., Tm) was observed from 75 to 90°C. However, no such thermal transition was observed when genistein was heated with dsDNA.

The question of whether the inhibitory effect of genistein resulted from non-specific adhesion to human topo II or from its selective binding to specific sites was investigated by determining whether an excessive amount of nucleic acid [poly(rC)] or protein (BSA) prevented the inhibitory effect of genistein. Poly(rC) and BSA had little or no influence on the inhibition of topo II by genistein (data not shown), suggesting that this compound selectively bound to the topo II enzyme molecule. These observations indicate that genistein does not act as a DNA intercalating agent or as a template-primer substrate. Instead, this compound binds directly to topo II and inhibits its activity.

Collectively, these results suggest that genistein may be a potent and specific inhibitor of human topo II. We therefore investigated in more detail whether topo II inhibition by genistein results in decreased human cancer cell proliferation.

Topos have recently emerged as important cellular targets for chemical intervention in the development of anticancer agents. Genistein could therefore be useful in chemotherapy, and thus, we investigated the cytotoxic effect of this compound against the HCT116 human colon carcinoma cultured cell line. As shown in Fig. 5A, 24 h of treatment with genistein treatment suppressed HCT116 cell growth in a dose-dependent manner, with a 50% lethal dose (LD₅₀) of 94.0 μM. This LD₅₀ is 2.5-fold higher than the IC50 for topo II. This suggests that genistein may be able to penetrate the cell membrane and reach the nucleus, where it may inhibit the activity of topo II, which leads to suppression of cell growth.

Next, we analyzed whether genistein affected the cell cycle distribution of compound-treated HCT116 cells. The cell cycle fraction was recorded after 24 h of treatment with a concentration of genistein equal to its LD₅₀. The ratio of cells in each of 3 phases (i.e., G1, S, and G2/M) in the cell cycle is shown in Fig. 5B. Treatment with genistein significantly increased the population of cells in the G2/M phase (2.57-fold increase of cells in G2/M phases), did not significantly change the proportion of cells in the G1 phase, and greatly decreased the percentage of cells in the S phase. Etoposide, which is a classic topo II inhibitor, arrested the cell cycle in the G2/M phase (1.80-fold increase of cells in G2/M phase, data not shown). These results suggest that genistein may be an effective inhibitor of topo II and halts the cell cycle at the G2/M phase.