Delphinidin Modulates the DNA-Damaging Properties of

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Delphinidin Modulates the DNA-Damaging Properties of Topoisomerase II Poisons Melanie Esselen, Jessica Fritz, Melanie Hutter, and Doris Marko* Section of Food Toxicology, Institute of Applied Biosciences, UniVersita¨t Karlsruhe (TH), Adenauerring 20a, 76131 Karlsruhe, Germany ReceiVed August 5, 2008

The anthocyanidin delphinidin (DEL) has recently been shown to inhibit human topoisomerase I and II, without stabilizing the covalent DNA/topoisomerase intermediate [Habermeyer, M., Fritz, J., Barthelmes, H. U., Christensen, M. O., Larsen, M. K., Boege, F., and Marko, D. (2005) Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity. Chem. Res. Toxicol. 18, 1395-404]. In the present study, we demonstrated that DEL affects the catalytic activity of topoisomerase IIR in a redox-independent manner. Furthermore, this potent inhibitory effect is not limited to a cell-free system, but is also of relevance within intact cells. DEL at micromolar concentrations was found to significantly decrease the level of topoisomerase IIR/DNA intermediates stabilized by the topoisomerase II poison doxorubicin in the human colon carcinoma cell line (HT29). In addition, DEL diminished the DNA-damaging properties of topoisomerase II poisons in HT29 cells without affecting the level of sites sensitive to formamidopyrimidine-DNA-glycosylase. However, the preventive effect on DNA damage exhibited an apparent maximum at a concentration of 10 µM DEL, followed by a recurrence of DNA damage at higher DEL concentrations. Furthermore, the incubation of HT29 cells with 10 µM DEL resulted in a decrease of etoposide (ETO)-induced DNA strand breaks. However, the level of ETO-stabilized covalent topoisomerase/DNA intermediates did not affect DEL, indicating an additional mechanism of action. An impact of DEL on genes involved in the repair of DNA doublestrand breaks and the onset of apoptosis has to be considered. In conclusion, the natural food constituent DEL represents, depending on the concentration range, a protective factor against the DNA-damaging effects of topoisomerase II poisons in vitro. Further studies are needed to clarify whether in vivo a high DEL intake might compromise the therapeutic outcome of these anticancer agents. Introduction Anthocyanins represent a class of colored plant constituents, which occur in many fruits and vegetables of the daily diet. They are glycosides of the anthocyanidins, which differ with respect to the substitution pattern at the phenyl ring (B-ring, Scheme 1). The most abundant anthocyanidins in food are delphinidin, cyanidin, malvidin, peonidin, and pelargonidin (2). Anthocyanins have been suggested to possess antioxidative (3, 4), vasoprotective (5), anti-inflammatory (6, 7), and chemopreventive (8, 9) properties and antiobesity effects (10, 11). On the basis of these proposed positive health effects, anthocyaninrich preparations have gained increasing popularity in the fast expanding market of food supplements. However, information about their mechanism of action and solid epidemiological data are quite limited. An anthocyanin-rich extract and cyanidin-3glucoside, one of the most abundant anthocyanins in the diet, have been reported to decrease the number of adenomas in the * To whom correspondence should be addressed. Phone: +49-(0)7216082936. Fax: +49-(0)721-6087254. E-mail: [email protected]. 1 Abbreviations: DEL, delphinidin; DOX, doxorubicin; HT29, human colon carcinoma cell line; fpg, formamidopyrimidine-DNA-glycosylase; ETO, etoposide; CPT, camptothecin; DSMO, dimethyl sulfoxide; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; BSA, bovine serum albumin; MER, merbarone; CIDEA, cell-death-inducing DFFR-like effector A; NBN, nibrin, member of the NBS1(NBN)/MRE11/RAD50 DNA repair complex; BTG2, BTG2/TIS21 gene included in response to DNA damage and the p53 pathway; PCBP4, gene encoded for the poly(C)-binding protein 4; F17782, 2′′,3′′-bis[(pentafluorophenoxy)acetyl]-4′′,6′′-ethylideneβ-D-glucoside of 4′-phosphate-4′-(dimethylepipodophyllotoxin)-2N-methylglucamine salt; MRN, MRE11/NBN/RAD50 complex.

Scheme 1. Chemical Structure of the Anthocyanidin DEL

APCmin mouse model (12, 13). Furthermore, anthocyanincontaining preparations were found to diminish aberrant crypt foci formation in azoxymethan-treated rats (14). Among the aglycons, the anthocyanidin delphinidin (DEL;1 Scheme 1) was found to exhibit the highest growth inhibitory properties in vitro (15-19). We showed recently that anthocyanidins interfere with signaling cascades crucial for the regulation of tumor cell proliferation and the onset of apoptosis (18-20). In contrast, potentially adverse effects of the interference of flavonoids with human topoisomerases are discussed, which might be of relevance for the maintenance of DNA integrity. Topoisomerases are essential enzymes involved in central DNA processing steps such as replication, transcription, translation, and recombination. Type I topoisomerase is a monomeric enzyme that introduces a transient single strand break into the DNA, enabling, e.g., relaxation processes. Type II topoisomerase

10.1021/tx800293v CCC: $40.75  2009 American Chemical Society Published on Web 01/30/2009

Interference of Delphinidin with Topoisomerase Poisons

is a dimeric and ATP-dependent enzyme that breaks both DNA strands concomitantly, permitting the passage of a second DNA double helix (21-23). Topoisomerase-targeting compounds can affect the activity of topoisomerase I or II at several steps of the catalytic cycle. Catalytic inhibitors bind to the enzyme prior to DNA binding, thus inhibiting the formation of the transient covalent topoisomerase/DNA intermediate. However, many of the known topoisomerase inhibitors stabilize the enzyme/DNA intermediate by forming a ternary complex. These compounds are classified as topoisomerase poisons (24-26). Topoisomerase poisons represent potent inhibitors of cell growth, and many of these compounds are used as anticancer agents, e.g., the anthracycline doxorubicin (DOX) or the epipodophyllotoxin analogue etoposide (ETO) (27, 28). Natural food constituents have also been reported to interfere with human topoisomerases. Flavonoids such as the soy isoflavone genistein or the green tee catechin (-)-epigallocatechin-3-gallate have been described to act as topoisomerase poisons (29-35). In contrast, we showed recently that anthocyanidins bearing vicinal hydroxy groups at the B-ring (e.g., DEL) represent potent catalytic inhibitors of topoisomerase I and II which prevent the stabilization of the cleavable complex by the topoisomerase I poison camptothecin (CPT) in a cell-free system. Furthermore, DEL was found to diminish the level of CPT-induced DNA strand breaks in human colon carcinoma cells (1). In the present study, the question was addressed of whether the protective effect of DEL is limited to topoisomerase I poisons or whether the DNA-damaging properties of topoisomerase II poisons are modulated as well, as exemplified for DOX and ETO. Furthermore, we investigate whether the impact of DEL on DNA integrity is associated with the onset of apoptosis and DNA repair.

Experimental Procedures Materials. CPT, DOX, ETO, merbarone (MER), and menadione were purchased from Sigma-Aldrich (Taufkirchen, Germany). DEL was received from Extrasynthe`se (Genay, France). For all assays the compound solutions were solved in dimethyl sulfoxide (DMSO) before the experiment was started, without the use of stock solutions, to a final DMSO concentration in the different test systems of maximum 1%. Cell Culture. HT29 (human colon adenocarcinoma, ACC 299) cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). The cell culture was performed in humidified incubators (37 °C, 5% CO2). HT29 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/ v) penicillin/streptomycin. The cells were routinely tested for the absence of mycoplasm contamination. Cell culture media and supplements were obtained from Invitrogen Life Technologies (Karlsruhe, Germany). Decatenation Assay. The catalytic activity of topoisomerase IIR was detected using catenated kinetoplast DNA (kDNA) in a cell-free decatenation assay. kDNA is an aggregate of interlocked DNA minicircles (mostly 2.5 kb), which can be released by topoisomerase II. kDNA (200 ng) (TopoGen, Ohio) was incubated in a final volume of 30 µL containing 40 ng of topoisomerase IIR (50 mM Tris, pH 7.9, 120 mM KCl, 10 mM MgCl2, 1 mM ATP, 0.5 mM DTT, 0.5 mM EDTA, and 0.03 mg/mL BSA) at 37 °C for 1 h. The reaction was stopped by the addition of a 1/10 volume of 1 mg/mL proteinase K in 10% (w/v) SDS followed by incubation at 37 °C for 30 min. Gel electrophoresis was

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performed in the absence of ethidium bromide at 60 V for 3 h in a 1% (w/v) agarose gel with Tris acetate/EDTA (TAE) buffer (40 mM Tris, 1 mM EDTA, pH 8.5, and 20 mM acetic acid). Subsequently, the gel was stained in 10 µg/mL ethidium bromide solution for 20 min. The fluorescence of ethidium bromide was detected with the LAS-3000 system (Fujifilm, Raytest, Germany) with Image Analyzer software (AIDA 3.52) for quantification. Arbitrary light units were plotted as test over control (T/C, %). To analyze the potential redox-dependent inhibition of the activity of topoisomerase IIR, 500 µM dithiothreitol (DTT) was incubated with DEL prior to the decatenation assay according to ref 34. Cleavage Assay. The cleavage assay was performed according to a modified method of ref 36. Plasmid DNA (200 ng of pUC18) was incubated in a final volume of 20 µL (10 mM Tris, pH 7.9, 50 mM sodium chloride, 50 mM magnesium chloride, 0.1 mM EDTA, 2.5% (v/v) glycerol, and 4 mM ATP) with 1.36 µg of topoisomerase IIR and ETO (100 µM) alone or in combination with DEL for 6 min at 37 °C. The samples were subsequently treated with 4 µL of proteinase K (1 mg/mL) and 1 µL of EDTA (375 mM, pH 8.0) and incubated at 45 °C for 30 min. On the basis of the high intercalation of DOX, DOX was displaced from DNA by the addition of 0.5 µL of ethidium bromide (1 mg/mL) to the samples. After 10 min of incubation gel electrophoresis was performed at 10 V in 1% (w/v) agarose gel with 1 µg/mL ethidium bromide in Tris acetate/EDTA buffer (40 mM Tris, 1 mM EDTA, pH 8.5, and 20 mM acetic acid) for 16 h. The fluorescence of ethidium bromide was detected with the LAS-3000 system (Fujifilm, Raytest, Germany) with Image Analyzer software (AIDA 3.52) for quantification. Arbitrary light units were plotted as T/C (%). Single-Cell Gel Electrophoresis (Comet Assay). Single-cell gel electrophoresis was performed according to the method of Gedik et al. (37). HT29 cells (3 × 105 in 5 mL of serumcontaining medium) were spread into Petri dishes (d ) 5 cm) and allowed to grow for 48 h prior to treatment with drugs. In the experiments with single compounds HT29 cells were treated for 1 h with the solvent control (1%, v/v, DMSO), 100 µM CPT, 10 µM ETO, and 10 µM DOX or DEL in serum-free medium. For the coincubation experiments, HT29 cells were preincubated for 30 min with the solvent control (0.2%, v/v, DMSO) or the respective anthocyanidin (DEL), followed by 1 h of coincubation of the respective compounds with 100 µM CPT, 10 µM ETO, or 10 µM DOX. Thereafter, aliquots (70 µL ) 70 000 cells) were centrifuged (5 min, 200g). The resulting cell pellet was resuspended in 65 µL of low-melting-point agarose and distributed onto a frosted glass microscope slide, precoated with a layer of normal-melting-point agarose. The slides were coverslipped and kept at 4 °C for 10 min to allow solidification of the agarose. After removal of the coverglass, the slides were immersed for 1 h at 4 °C in lysis solution (89 mL of lysis stock solution, 2.5 mM sodium chloride, 100 mM EDTA, 10 mM Tris, 1% (w/v) N-laurylsarcosyl sodium salt, 1 mL of Triton-X-100, 10 mL of DMSO). For the additional detection of oxidative DNA damage, the slides were washed three times in enzyme buffer (40 mM HEPES, pH 8.0, 0.1 M potassium chloride, 0.5 mM EDTA, 0.2 mg/mL bovine serum albumin (BSA)), covered with 50 µL of either enzyme buffer or formamidopyrimidine-DNA-glycosylase (fpg enzyme), and incubated for 30 min at 37 °C. Subsequently, DNA was allowed to unwind (300 mM NaOH, 1 mM EDTA, pH 13.5, 20 min, 4 °C) followed by horizontal gel electrophoresis at 4 °C for 20 min (25 V, 300 mA). Thereafter, the slides were washed three times with 0.4 M Tris-HCl, pH 7.5, and stained with ethidium

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bromide (40 µL per coverslip, 20 µg/mL). Fluorescence microscopy was performed with a Zeiss Axioskop 20 (λex ) 546 ( 12 nm; λem g 590 nm). The slides were subjected to computer-aided image analysis (Comet Assay III System, Perceptive Instruments, Suffolk, Great Britain), scoring 50 images per slide randomly picked from each electrophoresis. For each concentration of drug two slides were independently processed and analyzed. The results were parametrized with respect to the tail intensity (intensity of the DNA in the comet tail calculated as the percentage of overall DNA intensity in the respective cell). Such quantitative data were always derived from at least three independent sets of experiments and from the evaluation of 100 individual cells per concentration (50 per slide) in each experiment. In parallel to the comet assay, the viability of the cells was determined by trypan blue exclusion. Isolating in Vivo Complexes of Enzyme to DNA (ICE Bioassay). The ICE bioassay was performed with slight modifications as described previously in ref 38. A total of 1.2 million HT29 cells were spread into Petri dishes (two Petri dishes for one concentration) and allowed to grow for 48 h. Thereafter the cells were incubated with the solvent control (1%, v/v, DMSO), DOX, merbarone, or DEL for 1 h under serumfree conditions. For the coincubation experiments, HT29 cells were treated for 30 min with the solvent control (0.2%, v/v, DMSO), MER, or DEL, followed by 1 h of coincubation of the respective compounds with 1 or 10 µM DOX. The medium was removed, and the cells were abraded at room temperature in 3 mL of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 1% (w/v) N-laurylsarcosyl sodium salt). The cell lysate was layered onto a cesium chloride gradient in polyallomer tubes (14 mL, SW40, Beckman Coulter GmbH, Krefeld, Germany). One gradient consisted of four layers (2 mL/layer) of cesium chloride with a decreasing density from the bottom to the top. The tubes were centrifuged at 100000g for 24 h at 20 °C. The gradients were fractionated (300 µL/fraction) from the bottom of the tubes. The DNA content in the single fractions was determined by measuring the absorbency at 260 nm using a NanoDrop spectrophotometer (PeqLab Biotechnologie GmbH, Erlangen, Germany), and all fractions were blotted onto a nitrocellulose membrane using a slot blot apparatus (Minifold II, Whatman/ Schleicher & Schuell, Dassel, Germany). Topoisomerase was detected using a rabbit polyclonal antibody against topoisomerase IIR (170 kDa) at a 1:500 dilution. An antirabbit IgG peroxidase conjugate (1:2000) was used as the secondary antibody. All antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The respective chemoluminescent signals (LumiGLO, Cell Signaling Technology) were analyzed using an LAS 3000 with AIDA Image Analyzer 3.52 software for quantification (Raytest, Straubenhardt, Germany). Arbitrary light units were plotted as T/C (%). RNA Preparation. A total of 700 000 HT29 cells were spread into Petri dishes (d ) 10 cm) and allowed to grow for 48 h. The cells were incubated with the solvent control (1%, v/v, DMSO) or DEL for 1.5 h under serum-free conditions. RNA was extracted from the cells using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) including an on-column DNase treatment step according to the manufacturer’s protocol. The concentration was measured with a NanoDrop spectrophotometer (PeqLab Biotechnologie GmbH, Erlangen, Germany). The purity was checked with an Agilent BioAnalyzer (Waldbronn, Germany) using an RNA 6000 Nano LabChip. Real-Time PCR Array (RT2 Profiler PCR Array System “Human DNA Damage Signaling Pathway”, SABiosciences, Frederick, MD). Reverse transcription was

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carried out using the RT2 PCR Array First Strand Kit (SABiosciences, Frederick, MD) following the manufacturer‘s instructions. A 1 µg portion of total RNA per sample was used. Realtime PCR was performed with a BioRad CFX96 (BioRad, Mu¨nchen, Germany) with the RT2 Real-Time SYBR Green PCR Master Mix (SABiosciences, Frederick, MD) following the manufacturer’s protocol. A melting-curve program was immediately run after the cycling program. In each reaction at temperatures greater than 70 °C only one peak appeared. Furthermore, a genomic DNA control, a reverse transcription control, and a positive PCR control were included in the realtime PCR. Relative transcript levels were calculated using the ∆∆Ct data analysis method.

Results Redox-Independent Inhibition of Topoisomerase IIr Activity. Several polyphenols have been reported to inhibit topoisomerase activity in a redox-dependent manner (34, 35). To investigate whether DEL affects topoisomerase IIR in a redox-dependent or redox-independent mechanism, the reducing agent DTT was included in the test protocol according to the method of ref 34. The catalytic activity of topoisomerase IIR was detected using the cell-free decatenation assay. DEL incubated with 500 µM DTT (lanes 12-16, Figure 1B) inhibited the activity of topoisomerase IIR at concentrations g7.5 µM, comparable to the results without DTT (lanes 3-7, Figure 1). The topoisomerase II poisons DOX and ETO served as positive controls (lanes 8 and 9 (without DTT) and lanes 17 and 18 (with DTT)). Protective Effect of DEL against Topoisomerase II Poisons. To investigate whether DEL interferes with the ETOor DEL-induced stabilization of the covalent topoisomerase/ DNA intermediate, a cell-free cleavage assay was performed according to ref 36. Plasmid (pUC18) DNA was incubated with ETO and DEL in the presence of topoisomerase IIR. ETO and DOX stabilized the cleavable complex, visible as linear plasmid DNA (Figure 2, lanes 3 and 10). By coincubation of ETO or DOX with DEL, the amount of linear DNA was diminished in a concentration-depended manner (Figure 2, lanes 4-6 and 11-13). At a concentration of 50 µM DEL the effect of ETO and DOX on the cleavable complex of topoisomerase IIR was significantly suppressed. Thus, in this cell-free test system the presence of DEL interfered effectively with the topoisomerasetargeting properties of these topoisomerase II poisons. The results raised the question of whether this interference is also of relevance within intact cells. If indeed DEL suppresses the stabilization of the cleavable complex in cells, a decrease of the DNA-damaging effects of ETO and DOX has to be postulated. Impact of DEL on the Strand-Breaking Properties of Topoisomerase II Poisons. To test this hypothesis, the effect of DEL on the DNA-damaging properties of ETO or DOX was determined by single-cell gel electrophoresis (comet assay). HT29 (human colon carcinoma) cells were pretreated for 30 min with DEL, followed by incubation with DEL in combination with ETO (10 µM, Figure 3A) or DOX (10 µM, Figure 3B) for 1 h. The incubation with ETO (Figure 3A) or DOX (Figure 3B) as single compounds for 1 h resulted in a significant increase of DNA damage. Furthermore, DEL itself enhanced the rate of DNA strand breaks at concentrations g50 µM (Figure 3A,B). In the presence of DEL, the DNA-damaging properties of ETO (Figure 3A) and DOX (Figure 3B) were significantly diminished with an apparent maximum at 10 µM DEL. At higher concentrations (50 µM DEL), the recurrence of DNA



Figure 1. Catalytic activity of recombinant human topoisomerase IIR determined as the decatenation of kDNA. Topoisomerase IIR was incubated for 60 min at 37 °C with DEL or the topoisomerase poison ETO or DOX in the absence or presence of DTT. The reaction was stopped with 1% (w/v) SDS, and after digestion with proteinase K, the samples were separated by 1% agarose gel electrophoresis. The ethidium bromide-labeled DNA was documented under UV light by digital photography. (A) Fluorescence signals of decatenated kDNA treated with topoisomerase IIR and DEL, ETO, or DOX were calculated as T/C (%) in comparison to the solvent control DMSO. The data show the means ( SD of at least four independent experiments (Student’s t test: *, p < 0.05; **, p < 0.01; ***, p < 0.001). (B) Representative gel of a decatenation assay with topoisomerase IIR. Lanes 1-9 show the samples without DTT and lanes 10-18 the samples with DTT. Lanes 1 and 10 represent catenated kDNA not exposed to topoisomerase IIR enzyme. Active topoisomerase II releases single free DNA circles from the catenated DNA network treated with the solvent control DMSO (lanes 2 and 11). Increasing concentrations of DEL show an inhibitory effect of the catalytic activity at g7.5 µM (lanes 3-7 and 12-16) as well as the positive controls ETO (lanes 8 and 17) and DOX (lanes 9 and 18).

strand breaks was observed, reaching a level mediated by ETO (Figure 3A) or DOX (Figure 3B) alone. Furthermore, the combination of 100 µM DEL with ETO (Figure 3A) enhanced the rate of DNA damage above the effects of ETO alone, equivalent to the calculated sum of the strand-breaking properties of the single compounds (DEL and ETO). The coincubation of DEL (100 µM) with DOX also resulted in a recurrence of the DNA damage, but did not exceed significantly the effect of DOX alone (Figure 3B). In addition to the topoisomerase-targeting effects, DOX is considered to induce oxidative DNA damage by hydroxy peroxide formation (39-41). Thus, the question was addressed of whether the modulation of the DNA-damaging properties of DOX is due to potential antioxidative effects of DEL. As a measure for oxidative DNA damage, postincubation treatment of the cells with the DNA repair enzyme fpg was included in the comet assay protocol. Fpg is involved in the first step of the base excision repair to remove specific modified bases from the DNA, creating apurinic or apyrimidinic sites, which are subsequently cleaved by the intrinsic AP-lyase activity of the

enzyme, generating a gap in the DNA strand (42). Under the alkaline conditions of the comet assay, these fpg-generated single-strand gaps are visible as additional DNA strand breaks. The redox cycling agent menadione was used as a positive control for oxidative stressors in the fpg-modified comet assay (Figure 3C). Incubation of HT29 cells with DOX for 1 h significantly enhanced the level of fpg-sensitive sites. In contrast to the results on the basic DNA damage (comet assay without fpg), DEL did not affect the formation of DOX-mediated fpgsensitive sites (Figure 3C, hatched bars). DEL Does Not Affect the Stability of the Cleavable Complex in HT29 Cells. We showed that DEL acts as a catalytic redox-independent inhibitor of human topoisomerases in cell-free test systems. In intact cells, no effect of DEL on the amount of free topoisomerase was observed, indicating that the stability of the cleavable complex is not affected (1). In the present study, the ICE bioassay (38) was used to directly determine the amount of topoisomerase covalently linked to the DNA. Cell lysates were loaded onto a cesium chloride gradient, separating proteins covalently linked to DNA from free proteins.

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Figure 2. Topoisomerase IIR DNA cleavage assay, coincubation experiments with DEL and ETO (100 µM) or DOX (10 µM). (A) Fluorescence signals of plasmid DNA treated with topoisomerase IIR and DEL in combination with ETO or DOX were calculated as rel. linear plasmid DNA in comparison to the solvent control DMSO. The data show the means ( SD of at least three independent experiments (Student’s t test: **, p < 0.01; ***, p < 0.001). (B) Representative gel of the cleavage assay with topoisomerase IIR. pUC18 plasmid DNA (200 ng) was incubated with 1.36 µg of recombinant topoisomerase IIR and ETO (100 µM, lanes 4-6) or DOX (10 µM, lanes 11-13) in combination with DEL for 6 min at 37 °C. Incubation of pUC18 and topoisomerase IIR with 100 µM ETO is shown in lane 3 and with DOX in lane 10. To characterize the linear form of DNA, pure linear plasmid was included in the testing (lanes 7 and 14).

The gradient fractions were transferred onto a nitrocellulose membrane followed by Western blot with an anti-topoisomerase IIR (170 kDa) antibody. The DNA content of the fractions was determined photometrically, obtaining a maximum (80 ng/µL) at fraction 9 (Figure 4A). Treatment of HT29 cells for 1 h with DOX (1 or 10 µM) or ETO (10 µM) clearly enhanced the level of topoisomerase IIR in the DNA peak fractions (fraction 4-10, Figure 4B), indicative of enhanced stabilization of the cleavable complex. In contrast, the catalytic inhibitor MER (43, 44) did not affect the extent of DNA/topoisomerase binding up to 400 µM (Figure 4B). DEL did not induce cleavable complexes in HT29 in concentrations up to 100 µM (Figure 4B). Thus, the results of the ICE assay support the hypothesis that within intact cells DEL acts as a catalytic inhibitor of topoisomerase IIR without stabilizing the cleavable complex. The Catalytic Topoisomerase Inhibitors MER and DEL Suppress the Stabilization of the Cleavable Complex Induced by DOX in HT29 Cells. The above-mentioned results indicate that DEL targets topoisomerases prior to their binding to the DNA. To extend this hypothesis, the ICE assay was used for competition studies in cultured HT29 cells (Figure 5). Addressing the question of whether the potential interference of DEL is based on the inhibition of the catalytic topoisomerase activity, the topoisomerase inhibitor MER was tested in comparison. HT29 cells were pretreated with DEL or MER for 30 min and then coincubated with DEL or MER and DOX in

combination for 1 h. MER at a concentration of 400 µM significantly diminished the amount of topoisomerase IIR complexes induced by 10 µM DOX. In comparison, the polyphenol DEL significantly suppressed the level of DOX (1 and 10 µM)-stabilized topoisomerase IIR/DNA intermediates at concentrations g10 µM (Figure 4). In contrast, the cleavable complex stabilized by ETO (10 µM) was not affected by the coincubation with DEL (data not shown), indicating a further mechanism of action with regard to the decrease of ETO-induced DNA strand breaks by DEL contributes. DEL Modulates the Transcript Level of Genes Involved in DNA Repair and Apoptosis. We addressed the question of whether DEL modulates the transcription of key genes of DNA repair and apoptosis induction. After incubation of HT29 cells with DEL (10 and 50 µM) for 90 min, the RNA was isolated and analyzed by the RT2 Profiler PCR Array System “Human DNA Damage Signaling Pathway”. This array comprises 84 genes involved in DNA damage signaling pathways. The genes featured are those associated with the ATR/ ATM signaling network and transcriptional targets of DNA damage response. DEL at a concentration of 10 µM enhanced the relative transcript level of the proapoptotic gene CIDEA (4-fold) and NBN (3.3-fold), involved in the repair of doublestrand breaks, whereas the proapoptotic genes BTG2 (3-fold) and PCBP4 (3-fold) were only affected at 50 µM DEL.


Figure 3. Impact of DEL on the DNA strand-breaking properties of (A) ETO or (B) DOX and (C) effect of DEL on DOX-induced oxidative DNA damage in HT29 cells in the comet assay. For the coincubation experiments, HT29 cells were preincubated for 30 min with the solvent control (0.2% DMSO) or the respective anthocyanidin (DEL), followed by 1 h of coincubation of DEL with (A) 10 µM ETO or (B, C) 10 µM DOX. The redox cycler menadione (20 µM) was included in the testing as a positive control for oxidative DNA damage. The data presented are the means ( SD of at least three independent experiments, each performed in duplicate. Significances indicated refer to the significance level compared to the respective control (Student’s t test: *, p < 0.05; **, p < 0.01; ***, p < 0.001) or to the respective topoisomerase II poison (ETO or DOX; Student’s t test: ###, p < 0.001).

Discussion Anthocyanidins with vicinal hydroxy groups at the B-ring (DEL, cyanidin) have been identified as potent inhibitors of the catalytic activity of human topoisomerases (1). Many flavonoids of different structural classes, e.g., genistein, myricetin, quercetin, or EGCG, have been reported to target human topoisomerases (29-35, 45-48). Most of the classical topoisomerase poisons, e.g., ETO, interact with topoisomerase activity in a redox-independent mechanism (34, 49). Within the class of


polyphenols several act as classical topoisomerase inhibitors like the soy isoflavone genistein or exhibit a redox-dependent mechanism of action, e.g., the green tea catechin EGCG (34, 35, 45). Like EGCG, DEL bears three vicinal hydroxy groups at the B-ring (Scheme 1). Therefore, we addressed the question of whether DEL influences topoisomerase IIR activity in a redox-dependent manner. As previously reported, the ability of redox-dependent topoisomerase inhibitors to affect topoisomerase activity is abrogated by the presence of the reducing agent DTT (50, 51). In the present study, we showed that the inhibitory properties of DEL toward topoisomerases are independent of the presence of DTT (Figure 1), thus indicating a redox-independent mechanism of action. The majority of polyphenols have been described to act as topoisomerase poisons (34, 45, 52), whereas recent studies indicated that DEL does not affect the stability but the formation of the covalent enzyme/DNA complex (1). Furthermore, we previously showed that DEL significantly diminishes the DNA strand-breaking properties of the topoisomerase I poison camptothecin (1). In the present study we investigated whether the protective effect of DEL against topoisomerase poisons is limited to type I topoisomerases or is also of relevance for type II topoisomerases. Both topoisomerase poisons DOX and ETO stabilize the cleavable complex, consisting of the two topoisomerase II subunits covalently bound to DNA via a phosphotyrosine linkage. However, in contrast to the nonintercalating epipodophyllotoxin ETO, DOX affects topoisomerase II activity by intercalation between the DNA base pairs at -1 and +1 positions of the single-strand regions and with the CAP-like domain of the enzyme (53-55). In a cell-free test system using recombinant topoisomerase IIR, DEL in concentrations g10 µM effectively decreased the level of ETO (100 µM)- and DOX (10 µM)-stabilized covalent enzyme/DNA intermediates (Figure 2), supporting the hypothesis that DEL also interferes with the effects of topoisomerase II poisons. Bisdioxopiperazines, a novel class of topoisomerase poisons, unable to stabilize the cleavable complex, have also been reported to suppress the formation of cleavable complexes by ETO (56). This class of topoisomerase poisons avoids the reopening of the closed clamp, a step of the catalytic topoisomerase II cycle, and blocks ATP hydrolysis (57). The accumulation of this closed clamp conformation might affect transcription, replication, and chromatin assembly or disassembly (58). These results indicate that for a deeper insight into the mechanism of action of DEL further investigations have to be considered. In previous studies using recombinant topoisomerases IIR and IIβ DEL showed no preference for one isoform, at least in the cell-free test system (1). Thus, in the present study the experiments were performed exemplarily for one isoform, topoisomerase R. Of course, it cannot be excluded so far that within intact cells some preference of DEL might occur. We furthermore investigated whether the potential protective effect versus the topoisomerase II poison ETO or DOX is also of relevance within intact cells, affecting the DNA-damaging properties of these topoisomerase II poisons. Indeed, in HT29 cells DEL (10 µM) significantly decreased the DNA strandbreaking properties of ETO in equimolar concentrations (Figure 3A). This protective effect was observed to a comparable extent for DOX (Figure 3B). In accordance with the results of the combination of the topoisomerase I poison camptothecin with DEL (1), also the combination with topoisomerase II poisons ETO and DOX resulted in an apparent maximum of preventive activity of DEL at 10 µM, followed by the recurrence or even

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Figure 4. Detection of the covalent topoisomerase IIR/DNA intermediate in HT29 cells in the ICE assay. The cells were treated with the respective test compound for 1 h. (A) DNA content measured as absorbance at 260 nm. (B) Topoisomerase IIR (170 kDA) levels in the ICE assay fractions. Representative gels of four independent experiments.

enhancement of DNA-damaging properties. However, in line with previous studies (1), DEL itself induced DNA strand breaks in the HT29 cells at concentrations g50 µM (Figure 3A,B). Thus, the loss of protective properties against the topoisomerasemediated DNA strand breaks of DOX and ETO at higher concentrations of DEL might result from the overlay of the protective effects of DEL on the level of topoisomerase catalysis with the DNA-damaging properties of this anthocyanidin. The results of the present study clearly show that the DNA-damaging properties of DEL are not due to the stabilization of the cleavable complex (Figure 4B). However, it has to be considered that DEL represents indeed an inhibitor of the catalytic activity of

topoisomerases I and II. It is tempting to speculate that the DNAdamaging properties of DEL might be due to increasing torsion stress resulting from the blocked catalytic activity of the topoisomerases. Thus, depending on the respective concentration range, DEL might reduce or (in the case of ETO) even enhance the DNA-damaging properties of topoisomerase II poisons. The epipodophyllotoxin F11782 has been reported to affect eukaryotic topoisomerases and to inhibit the binding of the enzyme to DNA (59). In comparison to DEL, F11782 did not stabilize the covalent topoisomerase/DNA intermediate, but the incubation of cells resulted in DNA strand breaks (60). Jensen and coworkers (59) showed that F11782 can also induce an ATP-



Figure 5. Impact of DEL or MER on the DOX-stabilized topoisomerase IIR/DNA intermediates in the ICE assay. HT29 cells were pretreated with DEL or MER for 30 min followed by coincubation with DEL or MER and DOX (1 or 10 µM) for 1 h. A representative immunoblot is shown. The level of topoisomerase IIR/DNA intermediate was calculated as coincubated cells over control cells (treated with DOX) with respect to the DNA content × 100 (T/C, %). The data presented are the means ( SD of four independent experiments. Significances indicated refer to the significance level compared to the respective control (Student’s t test: *, p < 0.05; **, p < 0.01; ***, p < 0.001).

independent noncovalent salt-stable complex of human topoisomerase II with DNA that does not depend on the ability to cleave DNA. Such a mode of action for DEL might be speculated and demands further investigations. Several mechanisms have been proposed to contribute to the DNA-damaging effects of DOX, e.g., the induction of apoptosis or impact on the glutathione redox pathway (41), molecular targets which have also been reported to be affected by anthocyanins (61-63). DOX has also been shown to enhance oxidative DNA damage via the formation of hydrogen peroxide (39-41). However, on the other hand, DEL has been described to act as an antioxidant in vitro and in vivo (3, 4, 61-63). Therefore, the question was addressed of whether and to which extent oxidative stress is involved in the DNA damage induced by DOX, and whether the extent of oxidative DNA damage is affected by DEL. DOX (10 µM) significantly enhanced the level of fpg-sensitive sites in HT29 cells, indicative of the induction of oxidative stress. However, the results demonstrated that DEL modulates the presumably topoisomerase-mediated basic DNA damage but not the prooxidative effects of DOX (Figure 3C). In accordance with these results, we showed previously that anthocyanidins lacking topoisomerase inhibitory properties do not affect the stabilization of the cleavable complex by the topoisomerase I poison camptothecin (1). These results underline the hypothesis that targeting topoisomerase is crucial for the protective effect of DEL. The competition experiments in the ICE assay demonstrated that DEL diminished the cleavable complex stabilizing effect of DOX (Figure 5). These results are in line with the cell-free cleavage assay (Figure 2). To support

our hypothesis that DEL acts as a catalytic inhibitor targeting enzyme activity prior to binding to DNA, we included MER for comparison in our testing. The catalytic topoisomerase II inhibitor MER affects either enzyme DNA binding or ATP hydrolysis and does not intercalate (43, 44). In a cell-free system the level of ETO-induced cleavable complexes was suppressed by coincubation with MER (43). We found that the pre- and coincubation of HT29 cells with MER at a concentration of 400 µM led to a highly significant decrease of DOX-induced topoisomerase IIR/DNA intermediates (Figure 5). In contrast, whereas DEL effectively diminished the ETOinduced cleavable complex formation in a cell-free test system (Figure 2), it was unable to suppress the level of ETO-related topoisomerase IIR complexes in HT29 cells in the ICE assay (data not shown). Previous studies showed that the covalent complexes formed by topoisomerase in the presence of ETO have an increased stability compared with those formed in the presence of intercalating drugs (64), an effect which might play a role in the lack of effectiveness of DEL in coincubation experiments with ETO in the ICE assay. Furthermore, the discrepancy between the effect of DEL on ETO-mediated DNA damage (Figure 3A) and the stabilization of DNA/topoisomerase intermediate levels indicate a further mechanism of action of DEL contributes. Nonhomologous end-joining pathways have been described to play a crucial role in cell survival following the treatment with topoisomerase inhibitors (59, 65). In the present study, we showed that DEL increases the level of gene transcripts of four genes involved in DNA damage signaling (Figure 6). NBN is a member of the MRE11/NBN/RAD50

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Figure 6. Effects of DEL on the relative transcript level of genes involved in apoptosis signaling. HT29 cells were incubated with DEL (10 and 50 µM) for 90 min. The RNA was extracted as described in the Experimental Procedures. Reverse transcription was carried out using the RT2 PCR Array First Strand Kit. A 1 µg portion of total RNA per sample was used. Real-time PCR was performed with a BioRAD CFX96 with the RT2 Real-Time SYBR Green PCR Master Mix. The level of the relative transcripts was calculated using the ∆∆Ct method. The data presented are the means ( SD of three independent experiments.

complex (MRN), which contributes to telomere maintenance and surveillance of the progressing replication fork and has a crucial role in the response to DNA double-strand breaks (66, 67). In addition, MRN influences the induction of cell cycle checkpoints in response to DNA damage away from replication forks. This checkpoint function has been linked to MRN’s role as an upstream activator and a downstream target of the ataxia telangiectasia mutated kinase (68). The impact of DEL on the transcription of this enzyme involved in the repair of DNA double-strand breaks might contribute to the observed preventive effects with regard to the ETO-induced DNA damage. Furthermore, the transcript levels of the proapoptotic genes PCBP4 (69), CIDEA (70), and BTG2 (71) were found to be enhanced (Figure 6). Previous studies showed that DEL induces apoptosis via the activation of caspases 3, 8, and 9 and PARP cleavage in the human colon carcinoma cell line HCT116 after 48 h of incubation (72). Considering the short incubation time of 90 min, it appears unlikely that apoptosis plays a major role in the observed cellular effects of DEL; however, a certain contribution cannot be excluded. Several flavonoids naturally occurring in the diet have been characterized as topoisomerase II poisons in vitro, possessing DNA strand-breaking properties (73, 74). It is tempting to speculate that DEL might also modulate the DNA-damaging effect of these food constituents. The potent bioactive properties of DEL elucidated so far raise the question as to whether in vivo relevant substance concentrations might be achieved. However, conclusive studies on the bioavailability of anthocyanidins are quite limited. After oral administration of anthocyanins from berries, anthocyanins were determined in the plasma within minutes, reaching a maximum at 2 h (75). The bioavailability of anthocyanins appears to be quite low, with probably much less than 0.1% of the intake (76-78). However, depending on the sugar moiety and the degree of methoxylation, blueberry anthocyanins were reported to reach the ileostomy bags of respective patients to a substantial amount, indicating that under physiological conditions a certain portion of anthocyanins reach the colon under physiological circumstances undegraded (79). Thus, although the systemic bioavailability appears to be low, locally in the gastrointestinal tract bioactive concentrations might be achieved especially by the consumption of enriched functional food or food supplements.

Esselen et al.

In summary, DEL was demonstrated to inhibit topoisomerase II without affecting the stability of the cleavable complex, not only under cell-free conditions but also in intact human colon carcinoma cells. Depending on the concentration range, DEL was found to suppress the DNA strand-breaking properties of topoisomerase II poisons as exemplified for ETO and DOX, without affecting the prooxidative effects of DOX. Furthermore, DEL modulates gene transcript levels involved in the repair of DNA double-strand breaks and the onset of apoptosis. Thus, in an appropriate concentration range, DEL might represent a protective factor against topoisomerase poisons. This effect appears to be desirable with respect to healthy individuals being exposed to diet-related topoisomerase poisons. However, it might be speculated that, in cancer patients receiving treatment with therapeutic topoisomerase poisons, high intake of potentially protecting compounds may counteract the therapeutic effectiveness. Acknowledgment. This study was performed as a part of FlavoNet, supported by Grant MA1659/4-1/2 from the Deutsche Forschungsgemeinschaft (DFG). The recombinant topoisomerase IIR was a generous gift from Fritz Boege, Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich Heine University, Duesseldorf, Germany.

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