ANTICANCER RESEARCH 29: 2273-2284 (2009)

The Characterisation of Flavone-DNA Isoform Interactions as a Basis for Anticancer Drug Development

PATRICIA A. RAGAZZON1†, TRACEY BRADSHAW2, CHARLIE MATTHEWS2, JIM ILEY1 and SOTIRIS MISSAILIDIS1

1Department of Chemistry and Analytical Sciences, The Open University, Milton Keynes, MK7 6AA; 2Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.

Abstract. Background: The interactions of small molecules structures. Triplex structures appear during transcription (2) with nucleic acids are of considerable interest for the design processes, but their stability under physiological conditions of novel anticancer compounds. The physical properties and is low and various approaches have led to the design of small sequence specificity observed in the interactions between a molecules that stabilise this structure for antigene therapy. group of flavonoids with proven antitumour activity and Guanine-rich G-quadruplex structures appear as a cap on the various nucleic acid structures are summarised. Materials telomeres and stabilisation of these can inhibit telomerase and Methods: UV and fluorescence spectroscopy, together activity (3). with competition dialysis, were used to assess the affinity of The interactions of naturally occurring compounds with the drugs for the nucleic acid structures in the presence or these different nucleic acid structures can be studied using a absence of different metal ions. The effect of these battery of physical and biochemical methods in order to compounds on breast and leukaemia cancer cell lines was elucidate binding parameters, such as binding constants, base evaluated using MTS and COMET assays and flow sequence selectivity, intercalation activity and cytometry. Results and Conclusion: The flavonoids studied thermodynamic stability. To gain better understanding as to are weak duplex DNA-binding ligands and the binding of how small molecules can interact selectively with different flavonoids to DNA is affected by metal ions. Baicalein and nucleic acid structures, ideally, drug binding to a diversity of quercetin display stronger affinity for triplex and quadruplex nucleic acids should be compared simultaneously. than for double-stranded DNA and offer interesting scaffolds In the constant search for new medicines, the active for the design of novel, high order DNA-binding agents. ingredients of herbs or herbal extracts from plants of medicinal use have been subject to scrutiny in various drug DNA acts as a repository of genetic information; discovery programs and have formed the basis for the perturbations to the base content, or to the ability of this generation of novel compounds with improved therapeutic information to be identified and read by proteins, result in properties. Baicalein (1) (4, 5) and its derivative baicalin (2) structural and functional changes. If these changes are (6, 7), two flavones extracted from the root of Scutellaria irreparable, the consequence is apoptosis and cell death. baicalensis, are widely used as health supplements and DNA can exist in a variety of conformations and structures, herbal medicines in Asian countries. The cancer preventative the main one being an antiparallel double-stranded (1) helix activity of these flavones may be due to an interaction with though it can also form higher-order triplex and quadruplex drug metabolising enzymes. Interaction with DNA (8) has been proposed on the basis of the almost planar structure of the molecules. Daidzein (3) (9, 10) is an isoflavonoid and its activity seems to be related to oestrogen receptors. †Present address: Peakdale Molecular Ltd, Sheffield Rd, SK230NT. Daidzein induces osteoclasts directly or indirectly from their progenitors and might be a tool for the study of osteoclast Correspondence to: Sotiris Missailidis, Chemistry Department, The differentiation (11, 12). The mode of action chould involve Open University, Milton Keynes, MK7 6AA, U.K. Tel: +44 1908 intercalation into the DNA molecule to form a template- 858382, Fax: +44 1908 858327, e-mail: [email protected] primer and the subsequent inhibition of both activities Key Words: Flavonoids, DNA binding, polynucleotides, triplex indirectly through induction of a conformational change in DNA, quadruplex DNA, cancer cell lines, UV, fluorescence the DNA. Puerarin (4) (13) is the major active ingredient spectroscopy. isolated from the root of Puerariae thomsonii (Benth.). In

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China, it has long been used to treat patients with coronary pairs; poly(dAdT)2, ε260=13100, expressed in phosphate base pairs; atherosclerosis due to its ability to dilate coronary arteries. poly(dGdC)2, ε260=16800, expressed in phosphate base pairs; or Puerarin decreases the myocardial oxygen consumption via a poly(dAdT) ε260=12000, expressed in phosphate base pairs; poly(dGdC) ε =14800, expressed in phosphate base pairs; polydT, receptor blocking effect (14) and improves the 253 ε =8500, expressed in single bases; polydAdTdT, ε =17200, microcirculation by blocking the L-type calcium channels in 264 260 expressed in phosphate triplets; G3A4G3, ε255=11500, expressed in ventricular myocytes (15). Quercetin (5) (16) and rutin (6) bases; C3T4G3, ε271=8300, expressed in bases and (17) are present in Artemisia scoparia (18). They have been AG3T2AG3T2AG3T2AG3, ε260=73000, expressed in strands. found to be potent chemopreventative agents (19) and are used in a wide range of conditions. There is evidence that Thermal denaturation studies. Optical thermal denaturation quercetin can intercalate DNA (20). Quercetin and rutin act experiments were performed in 3 ml stoppered 1 cm pathlength as powerful antioxidants (21), reacting rapidly with reactive quartz cuvettes using a UVIKON XL UV/Visible spectrophotometer (BioTek, Vermont, USA) fitted with a temperature control unit and oxygen species and producing stable phenoxyl radicals. a programmable heated cell holder capable of maintaining the The flavonoid skeleton plays an important role in the temperature to within ±0.1˚C over a range from 25 to 98˚C. interaction of these molecules with DNA, particularly DNA–drug solutions were prepared by the addition of the ligand to through the presence of a planar chromophore, which is give a final drug concentration of 2×10–6 M and a final DNA possibly capable of intercalation between the base pairs (22). concentration of 10–5 M in the appropriate buffer. A fixed DNA:drug The aim of this paper was to study the binding activity of molar ratio of 5:1 was used. Heating was applied at 1˚C/min until compounds 1-6 to a panel of different duplex and higher denaturation was complete, as judged from the increase in optical absorbance at 260 nm. In each case, the temperature of denaturation order DNA structures and confirm their in vitro antitumour of the drug–DNA complex was compared with that for the DNA activity in two cancer cell lines. alone, and the change in thermal denaturation (ΔTm) was calculated complex DNA from ΔTm=Tm – Tm . Materials and Methods Absorption spectroscopy. The absorption spectra were obtained Chemicals. Compounds 1-6 (Figure 1) were purchased from Sigma- using a UVIKON XL UV/Visible spectrophotometer. The Beer- Aldrich (Dorset, UK). Stock solutions (10 mM) were prepared in Lambert law was obeyed across the concentration ranges used for DMSO and stored at 4˚C. Drug solutions were prepared from these both flavonoid and DNA solutions. To study the changes in the UV- stock solutions using filtered pH 7.4 phosphate buffer (10 mM) visible absorption spectra of the compounds upon interaction with containing EDTA (1 mM). The concentrations were determined DNA, the following general method was used; the concentrations of spectrophotometrically, using a 1 cm pathlength quartz cuvette, from compounds 1-6 were adjusted to give an absorbance reading of ca. the following extinction coefficients (M–1 cm–1): baicalein, 0.5. The concentrations of the nucleic acids were increased ε359=14898; baicalin, ε315=15556; daidzein, ε310=15639; puerarin, incrementally by adding 2 μl of the appropriate nucleic acid stock ε340=14572; quercetin, ε375=11806 and rutin, ε365=16956. solution. Using this procedure, the concentration of the compound in the cuvette does not remain constant. Consequently, the Nucleic acid. Salmon testes DNA (STDNA) was purchased from absorbance corrected for dilution was calculated using Equation 1: Sigma-Aldrich and used in filtered phosphate buffer (10 mM), EDTA (1 mM), pH 7.4 solutions. The polynucleotides poly(dAdT)- Acorr=Aorig(1000 + V) / 1000 Equation 1 poly(dAdT) (or poly(dAdT)2), poly(dGdC)-poly(dGdC) (or poly(dGdC)2), poly(dA)-poly(dT) (or poly(dAdT)), poly(dG)- where V is the volume of nucleic acid added. The initial titration poly(dC) (or poly(dGdC)), poly(dT) were purchased from point was the UV spectrum of the free compound, i.e. in the absence Sigma–Aldrich and dissolved in water to generate stock solutions. of nucleic acid, and the final titration point was the absorbance Triplex DNA poly(dA)-poly(dT)-poly(dT) (or polydAdTdT) was recorded for the completely bound ligand, experimentally, the latter prepared using a ratio of 1:1 poly(dAdT):poly(dT) in Na2HPO4 (20 point was attained either when no further change in the absorbance mM), NaH2PO4 (80 mM), NaCl (300 mM) and EDTA (0.1 mM). The was observed on addition of further volumes of nucleic acid or any resulting solution was heated to 95˚C then allowed to cool to room increase in absorbance was due solely to the nucleic acids temperature over 18 h and stored at 4˚C. Triplex purine G-A-G themselves. Generally, the DNA-binding parameters were calculated (G3A4G3:C3T4C3:G3A4G3) was prepared using a ratio of 2:1 from the absorbance values at the maximum wavelength for each (G3A4G3:C3T4C3) and dissolved in 50 mM pH 6.5 cacodylate buffer compound. The experiments were performed at pH 7.4 to enable containing 50 mM MgCl2 and 0.1 mM EDTA; again the resulting comparison under physiological conditions. solution was heated to 95˚C, allowed to cool to room temperature over 18 h then stored at 4˚C. The human quadruplex A-G DNA Fluorescence emission spectroscopy. The binding affinities of the (AG3T2AG3T2AG3T2AG3) and oligonucleotides were purchased drugs to ST DNA were also assessed by fluorescence titrations. from MWG-Biotech Ltd (Milton Keynes, UK). The oligonucleotide Fluorescence emission was measured on a Fluoromax P solution was prepared as for polydAdTdT using Na2HPO4 (6 mM), luminescence spectrophotometer (Horiba JobinYvon, UK). The NaH2PO4 (2 mM), NaCl (185 mM) and EDTA (0.1 mM). spectra of the compounds were obtained using an excitation Molar concentrations of the various DNAs were determined wavelength of 340 nm, excitation and emission slits of 5 and 20 nm, spectrophotometrically, using the following extinction coefficients respectively, and measuring at the following emission wavelengths: –1 –1 (M cm ): STDNA, ε260=13200, expressed as phosphate base baicalein (465 nm), baicalin (440 nm), daidzein (468 nm), puerarin

2274 Ragazzon et al: Flavonoid Interactions with DNA

Figure 1. Structure of test compounds.

(468 nm), quercetin (536 nm) and rutin (532 nm);. The change in bovine serum (GIBCO™, Invitrogen, CA, USA) at 37˚C and 6% the fluorescence emission was monitored throughout the titration in CO2 in air. The cells were sub-cultured twice weekly. a manner identical to that described above for UV absorption. Flow cytometry. The cells were grown in 6-well plates at an initial Competition dialysis. A modification of the original protocol of Ren density of 3×105 cells/ml in the appropriate medium for 24 h at 37˚C and Chaires (23) was used. Briefly, a reservoir containing 200 ml of a with 6% CO2 in air. An incubation period of 24 h using 100 μM of pH 7.4 8 mM sodium phosphate buffer (Na2HPO4 (6 mM), NaH2PO4 the four structurally related flavonoids, 1, 2, 3 and 5 and a similar (2 mM)), containing NaCl (185 mM), EDTA (0.1 mM), and the test concentration of DMSO followed the initial step. The MCF-7 cells compound (1μM) was prepared. Into separate 0.5 ml DispoDialyser® were tripsinised, and both cell lines were collected in FACS tubes. units (1 KDa, Spectrum Europe, Breda, the Netherlands), 0.5 ml After centrifuging for 8 min at 1200 rpm and decantation of the volumes of each nucleic acid solution were pipetted. The concentration supernatant, the cells were kept at 4˚C overnight in Fluorochrome of all the nucleic acids was 75 μM, expressed in terms of the solution (0.1% Triton® X100, 0.1% Na-citrate, 50 μg/ml propidium monomeric unit (base pairs for duplex DNA, triplets for triplex DNA, iodide, 0.1 mg/ml RNAseA). tetrads for tetraplex DNA). These DispoDialyzer® units were placed in ® the reservoir and the reservoir and its contents were covered with MTS cell proliferation assay. The Cell Titer 96 AQueous Non Parafilm and wrapped in foil and allowed to equilibrate with Radioactive Cell Proliferation Assay (Cat# G1112; Promega, UK) continuous stirring at 4˚C for 24h. At the end of the equilibration is a colorimetric method for determining the number of viable cells period, the nucleic acids samples were carefully removed to in proliferation. The tetrazolium compound (3-(4,5-dimethylthiazol- microcentrifuge tubes, and were made up to a final concentration of 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra- 1% (w/v) sodium dodecyl sulfate by the addition of appropriate zolium, inner salt, MTS) was dissolved in PBS and the electron volumes of a 10% (w/v) stock solution. The total concentration of coupling reagent (phenazine methosulfate, PMS) was added each test compound (Ctot) within each dialysis unit was then according to the protocol supplied. The cells were grown in 96-well determined spectrophotometrically, using the appropriate extinction plates at an initial density of 1×104 cells/ml (0.2 ml total volume) in coefficient. The free ligand concentration was determined the appropriate medium for 24 h at 37˚C with 6% CO2 in air. spectrophotometrically using an aliquot of the dialysate solution. Incubation periods of 24, 48 and 72 h with 0, 1, 3, 10, 30, 100, 300 or 1,000 μM of the test compounds followed. Following the Cancer cells. Clonal populations of MCF-7 breast cancer (donation protocol, 40 μl of the MTS–PMS solution were added and the cells of Prof. Malcolm Stevens, University of Nottingham, U.K.) and were incubated at 37˚C with 6% CO2 in air for 2 h prior to CCRFCEM leukemia cells (donation of Dr Thomas Efferth, measuring the absorbance at 490 nm. University of Heidelberg, Germany) were used in these experiments. The cells were routinely grown in RPMI-glutamax (Sigma-Aldrich) COMET assay – alkaline electrophoresis. The protocol was based supplemented with 10% and 5% , respectively, heat-inactivated fetal on the Trevigen Comet Assay protocol (Kit Cat# 4250-050-K;

2275 ANTICANCER RESEARCH 29: 2273-2284 (2009)

Figure 2. Titration of baicalein (50 μM) with STDNA ([DNA]:[1]=0-10:1); the arrow indicates the direction of absorbance change.

Trevigen, Maryland, USA). Briefly, both cell lines were grown The isosbestic points were observed in the titrations as overnight from an initial density of 3×105 cells/ml in the appropriate follows: baicalein (312, 413 nm), daidzein (297, 365 nm), medium at 37˚C with 6% CO2 in air. An incubation period of 24 h quercetin (302, 464 nm) and rutin (311, 420 nm). An in the presence of 100 μM of the test compounds followed. example of a UV titration is shown in Figure 2. Treatments with H2O2 were used as positive control. The cells were suspended in low melting (LM) agarose (Trevigen) (10 μl of The measurement of nucleic acid-compound interactions sample per 70 μl LM agarose) and 75μl of the agarose suspension using spectroscopic methods relies on the change in the were pipetted into each sample well of the Trevigen Comet Slides. electronic absorption spectrum of the compound between its Subsequently, all the slides were refrigerated at 4˚C for 1 h to allow bound and unbound state (24). Moreover, the absorption adherence of the agarose to the slide, then immersed in Lysis spectrum of the compound under study should lie outside the Solution (Trevigen). Following lysis, all the slides were placed in 200-30 nm range where the nucleic acid itself absorbs, which alkali buffer (0.3M NaOH, 200 mM EDTA, pH>13) for 40 min at was the case for compounds 1-6. Since these compounds 4˚C to unwind the DNA. The slides were subjected to electrophoresis at 25 V, 300 mA for 30 min after which the slides also obey the Beer-Lambert law at the concentrations were neutralised in 0.4 M pH 7.5 Tris-HCl buffer, washed with employed for the titrations, it is clear there was no self- deionised water/ethanol and allowed to dry overnight. The slides aggregation, such as self-stacking interactions, of the ligand. were then stained with SYBR green (SYBR green is a cyanine dye The data obtained by the UV titration were fitted to used to stain nucleic acids) in TE (10 mM Tris and 1 mM EDA) Equation 2 (25) to obtain the binding parameters for the buffer, pH 7.5. The samples were viewed using a fluorescent interaction between the compounds under study and the microscope with FITC (fluorescein isothiocyanate) filters various DNA structures. (excitation 494 nm, emission 521 nm).

2 Results and Discussion Ac=nA0D+([Af-A0]{1+nKassD+KassA0–[(1+nKassD+KassA0) 2 ½ –4nKass DA0] }/2nKassD) Equation 2

Binding parameters. Using UV-Vis titration a bathochromic In Equation 2, Ac is the calculated absorbance, Aff is the final shift was observed in all cases, with the exception of absorbance, A0 is the initial absorbance, Kass is the binding baicalein, 1, for which a hypochromic shift was observed. constant, D is the compound concentration and n is the number

2276 Ragazzon et al: Flavonoid Interactions with DNA

Figure 3. Calculated (solid line) and experimental (open circles) data for the binding of 50 μM quercetin to STDNA; data were fitted to Equation 2 (see text).

of base pair binding sites per bound molecule. (For similar From the results in Table I, by comparing baicalein with experiments using fluorescence, fluorescence values F replace baicalin and quercetin with rutin, it was concluded for the absorbance values A). The Ac value was obtained using iterative flavones that the presence of sugar residues decreased the fitting of the n and Kass values with the Origin 6.0 program binding affinity to duplex DNA by up to a factor of 10. For (MicroCal, MA, USA). An example of data fitting by this the isoflavones, daidzein compared with puerarin, the procedure is shown for quercetin 5 and STDNA in Figure 3. presence of a sugar residue was more equivocal, the differences in binding were not so large and in some Duplex binding. The binding site selectivity was considered to instances the parent compound daidzein had the highest be significant only for an A-T/G-C affinity ratio of more than affinity, whereas in others the conjugate puerarin had the 30% (26). In the majority of intercalators, either there is no highest affinity. selectivity, or they demonstrate a random preference. Taking STDNA as our sequence-neutral unit (58% G-C), for the Triplex binding. Baicalein and quercetin, the compounds with group of compounds studied here, it was clear that baicalein the highest affinity for duplex DNA were further investigated and quercetin exhibited the largest association binding with higher-order triplex and quadruplex DNA structures. 4 –1 constants, Kass, of the order of 10 M (Table I), and all the Specific recognition of higher order DNA structures has been flavonoids examined demonstrated little selectivity with regard previously demonstrated for a number of small ligands. At to their preference for specific duplex nucleic acid sequences. least two classes of triplex exist, the purines and pyrimidines, These conclusions were confirmed by the competition dialysis which differ in sequence composition and relative orientation assay (Figure 4), where none of the compounds showed on the phosphodiester backbone (27). Interestingly, our significant specificity for any of the duplex structures, and results showed that baicalein and quercetin showed slightly where no significant change was observed in the melting stronger affinity for both purine and pyrimidine triplexes than temperatures of the various duplex nucleic acid structures for duplex structures (compare the data in Table III with those (ΔT≤1˚C) in the thermal denaturation studies (Table II). in Table I). Thermal denaturation studies confirmed that these

2277 ANTICANCER RESEARCH 29: 2273-2284 (2009)

Table I. Binding association constants, Kass, and n (number of base pair binding sites per bound molecule) for compounds 1-6 and various duplex DNA motifs as determined by UV titration; the r2 correlation coefficent is given in parentheses.

Compound pdApdT pdAdT pdGpdC pdGdC STDNA STDNAa

3 –1 3 –1 3 –1 3 –1 3 –1 3 –1 Kass /10 M Kass /10 M Kass /10 M Kass /10 M Kass /10 M Kass /10 M nn n n n n

Baicalein 13.8 (0.98) 12.5 (0.99) 13.8 (0.99) 12.1 (0.97) 10.0 (0.99) 15.3 (0.97) 2 2 2 2 2.1 7 Baicalin 1.36 (0.97) 1.19 (0.97) 1.18 (0.98) 0.44 (0.94) 1.79 (0.98) 1.29 (0.95) 2.1 2.2 2 1.9 2 2 Daidzein 1.22 (0.98) 5.68 (0.99) 5.51 (0.98) 3.99 (0.99) 1.52 (0.91) 0.95 (0.94) 22 3 2 3 3 Puerarin 6.09 (0.98) 1.26 (0.98) 6.31 (0.93) 6.90 (0.95) 0.96 (0.98) b 2 2 3 2.3 2 Quercetin 23.7 (0.95) 12.3 (0.99) 11.0 (0.99) 14.9 (0.96) 12.1 (0.99) 11.4 (0.92) 12 2 2 2 3 Rutin 5.61 (0.98) 6.59 (0.99) 3.40 (0.90) 3.27 (0.98) 1.18 (0.95) 1.97 (0.95) 3 3 4 4 2.1 2 a: Determined by fluorescence; b: no interaction observed.

two flavonoids had superior affinity for, and stabilisation of, Competition dialysis. In competition dialysis, the relative the triplex DNA structures compared to duplex DNA. Thus, amounts of the bound ligand to the various structures for polydAdT, no significant ΔTm was found for either correlate with the individual compound-DNA affinities. Out compound, but for polydAdTdT, a stabilisation of 3.2˚C for of the six compounds, baicalein and quercetin were baicalein and 7.3˚C for quercetin was observed. In the case demonstrated to have the strongest absolute binding affinity. of G-triplex, stabilisation was so strong that it could not be As the drugs did not present very strong binding activity for measured in an ordinary UV spectrophotometer. Quercetin the different DNA structures, no appreciable differentiation appeared to be the strongest binder, with a Kassociation in the was observed between each class (see Figure 4). order of 2.7×104 M–1 (Table III). From these results it was concluded that the hydroxyl groups in the A and B ring helped to improve binding. G-quadruplex binding. G-quadruplexes are a family of high Furthermore, sugar substitutions, as in the case of baicalin order DNA structures formed in the presence of cations and rutin, decreased the binding activity. Finally, in the case consisting of four guanine residues stabilised in quartets (28). of the isoflavonoids, the B ring situated in the C-2 position The 3’-terminal region of the G-rich strand of human decreased the binding activity. telomeres is single stranded and may adopt a G-quadruplex conformation. This structure has been shown to inhibit Metal ion interactions. In nucleic acids there are two major telomerase elongation activity in vitro (29). Telomerase is classes of metal ion binding sites; the heterocyclic bases and expressed in germinal, but not somatic cells, as well as in the negatively charged phosphate groups. Such binding has the almost 80% of malignancies (30), therefore, the stabilisation potential to stabilise the DNA-ligand-metal ion complex. of the G-quadruplex could interfere with telomerase Quercetin has previously been shown to interact with copper elongation and the replication of cancer cells. Competition and produce DNA damage (32). Thus, the binding of baicalein, dialysis suggested that flavonoids interact preferentially with baicalin, quercetin and rutin to the nucleic acids in the presence tetraplexes. This observation was confirmed from the UV-Vis of six cations that are commonly found in the cell environment: titrations of baicalein and quercetin with the A-G the human Fe2+, part of hemes (33); Fe3+, part of the transferrin family telomeric sequence, an oligonucleotide that mimics the repeats (34); Cu2+, present in drug–DNA interactions (35); Mg2+, the of human telomeric motif and adopts an intramolecular second most abundant intracellular cation modulating many quadruplex structure (31) (Table III). Quercetin exhibited a biological processes (36); Mn2+, role in mitogen-activated 5 –1 2+ Kass of ~10 M for the G-quadruplex, nearly tenfold larger protein (MAP) kinase-dependent pathways (37) and Zn , part than those observed for the duplex and triplex structures and of the zinc finger DNA-binding protein (38) were studied. two orders of magnitude higher than those observed for some For these experiments by a flavone-metal ion complex at of the other compounds in their binding to duplex DNA. two ratios, 1:1 or 1:2 was carried out, then titrated with ST

2278 Ragazzon et al: Flavonoid Interactions with DNA

Table II. ΔTm values for compounds 1-6. Melting point assay and the preliminary set of compounds are shown. For polydAdTdT, some compounds (baicalein and quercetin) stabilised the first transition, while others (baicalin, daidzein and rutin) destabilised this transition. In the case of G-quadruplex, the flavonoids baicalein, baicalin, dadizein, puerarin, quercetin and rutin showed remarkable effects in different points of the tetraplex, by stabilising, destabilising and eliminating some melting transitions, or adding a third one, as in the case of quercetin.

ΔTm/˚C

Nucleic acid Baicalein Baicalin Daidzein Puerarin Quercetin Rutin

STDNA 0.2 0.3 –0.7 –0.3 –1.2 –1.2 polydAdT 0.6 0.1 0.1 0.7 0.7 1.2 poly(dAdT)2 0 0 0 –0.4 –0.1 0.3 polydAdTdT 3.2/–2.4 –6.9/–2.3 –5.8/–2.7 0.6/0.2 7.3/–1.5 –4.3/–1.9 G-quadruplex –-/0 –1.9/-- 0.2/-- 0.2/14.9 –2.9/0.3/-- –0.9/0.5

DNA, with the aim of saturating all the possible hydroxyls Table III. Binding constants determined by UV titration for complex in the ortho positions. The results are presented in Table IV. formation between triplex and G-quadruplex DNA structures and 2 For quercetin, the binding was not improved by the presence baicalein and quercetin (r values in parentheses). of the metal ions, though the n value increased notably for Compound pdAdTpdT Triplex GAG Human 2+ all the metal ions, other than Zn , in the presence of a 1:2 telomere 3 –1 3 –1 3 –1 flavone:metal ion ratio. In the case of rutin, binding was Kass /10 M Kass /10 M Kass /10 M enhanced dramatically by 1:1 ratios of Fe2+, Fe3+ or Zn2+ nn n probably due to the interaction of the metals in position 7, Baicalein 22.7 (0.98) 23.4 (0.94) 19.0 (0.98) or the involvement of the rutinoside in complex formation. 432 In the presence of higher concentrations of metal ions, it is Quercetin 27.1 (0.99) 24.5 (0.98) 85.4 (0.96) possible that the metal ions bound, separately, to both the 3 1.4 2 compound and the DNA thus hindering interaction between the two. Whereas for rutin the sugar residue appeared to n: Number of base pair binding sites per bound molecule. enhance binding, the binding of baicalin in the presence of metal ions remained largely unaffected. In contrast, the binding of the aglycone baicalein decreased markedly in all the cases where a metal ion was present, with the exception MTS cell proliferation assay. This assay measures the cell of excess Fe2+. This might imply that the hydroxyl groups proliferation rate and, conversely, when metabolic events in an ortho disposition in the A ring of baicalein formed lead to apoptosis or necrosis, reduction in cell viability. The metal ion complexes that did not allow the molecule to data as shown in Table VI indicated that the flavonoids with intercalate the double helix. Such complexes of catechols are very similar structures did not produce identical biological well-known (39). responses. After 24 h of incubation, the compounds had 50% Cell studies, flow cytometry. The antitumour potential of inhibition of growth (IG50) values near 100 μM. Prolonged flavonoids has been extensively documented, but the effect incubation to 48 h, resulted in the IG50 values decreasing to on cell cycle distribution remains unclear, although the ca. 65 μM, except for daidzein and quercetin where the IG50 majority of flavonoids appear to arrest the cell cycle during values remained >100 μM. Incubation for 72 h showed that the G2/M-phase, typical of DNA synthesis inhibitors (40, baicalein had activity in the MCF-7, but not the CCRFCEM 41). The chemotherapy-induced apoptosis and cell cycle cells. Baicalin showed activity in both cell lines. Daidzein arrest of the MCF-7 and CCRFCEM cancer cells as promoted growth of the MCF-7 cells, as might be expected demonstrated by flow cytometry are shown in Table V. as these cells express oestrogen receptors and daidzein The treatment with baicalein and baicalin blocked the possesses oestrogen-like structure and activity (42). This MCF-7 cells in the G1-phase, daidzein decreased the cells in compound had little anticancer activity against the pre-G1 and quercetin blocked the cells in the G2 phase. In CCRFCEM cells. After 72 h incubation quercetin exhibited the CCRFCEM cells, baicalein and baicalin acted as blockers antitumour activity against both cell lines. Effective for the G1-phase, daidzein exerted no activity and quercetin anticancer drugs often have IG50 values in the order of 1-5 blocked in the G2-phase. μM(43). Clearly, this group of compounds had low

2279 ANTICANCER RESEARCH 29: 2273-2284 (2009)

Figure 4. Bound concentrations of compounds 1-6 with different types of DNA as determined by competition dialysis; baicalein 1 and quercetin 5 bound more tightly to the different nucleic acid isoforms than did compounds 2-4 and 6.

Figure 5. COMET assay for the MCF7 cell line, (a) negative control, no modification in shape was found, (b) positive control, the presence of hydrogen peroxide produced comet tails, a clear indication of entrance into the cell and DNA damage, (c) baicalein also produced comet tails in the cells, again indicating its entrance into the cell.

anticancer activity in the two cell lines studied, as is the case had no effect over the negative control. The comparison of for many flavonoids (44). these results with the data obtained from the cell proliferation and flow cytometric assays suggested that COMET assay – alkaline electophoresis. While daidzein quercetin exerted its anticancer action in both cell lines by showed no effect in the MCF-7 cells, baicalein (Figure 5) entering the nucleus and affecting the DNA. This may also and quercetin appeared to enter the nucleus and produce have applied to baicalein in the MCF-7, but not CCRFCEM DNA damage. For the CCRFCEM cells, only quercetin cells, implying that this compound binds to a target other appeared to be active, while baicalein, baicalin and daidzein than DNA in the latter cell line. Baicalin had no effect on

2280 Ragazzon et al: Flavonoid Interactions with DNA

Table IV. Binding association constants, Kass, and n (number of base pair binding sites per bound molecule) values for compounds 1, 2, 5 and 6 with STDNA in the presence of metal ions. The ratios of flavone to metal ion are indicated

Baicalein Baicalin Quercetin Rutin

1:1a 1:2 1:1 1:1 1:2 1:1 1:2

3 –1 3 –1 3 –1 3 –1 3 –1 3 –1 3 –1 Cation Kass /10 M Kass /10 M Kass /10 M Kass /10 M Kass /10 M Kass /10 M Kass /10 M nnnnn n n

None 10.0 1.8 12.1 1.2 2.1 2 2 2.1 Cu2+ 2.4 1.4 1.7 12.1 16.2 1.2 b 2.2 2.3 2.1 1.9 6.6 2.7 Fe2+ 1.1 15.4 2.0 1.3 14.4 13.1 1.1 2.7 2.3 2.1 2.5 8.4 2.6 2 Fe3+ 1.2 1.2 2.8 9.5 10.9 75.8 1.2 2.0 2.1 2.8 2.7 7.7 3.2 2.1 Mg2+ 1.1 1.3 1.5 11.0 7.4 1.12 1.1 2.5 2.2 2.6 2.3 4.8 2.5 2.4 Mn2+ 1.2 1.2 1.1 7.9 11.3 10.1 1.1 2.5 2.3 1.9 3.3 6.9 2.0 2.3 Zn2+ 1.6 1.2 1.9 10.7 10.1 46.2 0.9 2.1 2.2 2.1 2.1 3.3 2.5 2.1 a: Ratio of flavone:metal ion; b: no interaction observed.

either cell line in the comet assay, but it exhibited significant Table V. Percentage inhibition obtained by flow cytometry for MCF7 anticancer activity when compared to the other compounds and CCRFCEM cells. (Tables V and VI). It is thus more than likely that baicalin Stages Baicalein Baicalin Daidzein Quercetin DMSO has a cellular target other than DNA.

MCF7 Conclusion Pre G1 3.8% 5.0% 5.7% 6.4% 8.5% G 57.3% 57.2% 48.5% 20.9% 58.3% Baicalein and quercetin were the most potent of the 1 compounds examined. Interaction with DNA is thought to S 21.0% 20.5% 21.9% 10.3% 18.1% involve the planar dihydrobenzpyranone ring system. G2/M 18.3% 17.7% 24.2% 62.6% 15.4% Competition dialysis provided the comparative affinities of the CCRFCEM molecules for triplex, quadruplex and duplex structures. Using Pre G1 21.4% 53.9% 1.3% 29.6% 38.3% UV-Vis titrations, it was confirmed that baicalein binds G1 29.3% 22.0% 49.5% 19.5% 16.6% preferentially to triplexes and quadruplex structures whilst S 35.4% 19.8% 30.1% 33.4% 30.5% quercetin binds preferentially to the telomeric sequence. G2/M 14.9% 4.8% 19.8% 18.2% 15.1% Baicalein showed specificity for the triplex ATT and quadruplex nucleic acid, and was found to be active against the MCF-7 cancer cell line by arresting the cell cycle at the G - 1 Table VI. IG50 values for the MTS proliferation assay for compounds 1, phase. Baicalin and rutin had activity towards AT bases and 2, 3 and 5 against MCF7 and CCRFCEM cells. baicalin arrested the cells during the preG1/G1-phase of the cycle in the CCRFCEM leukaemia cells. In contrast, daidzein IG50 MCF7 /μM IG50 CCRFCEM /μM and puerarin bound weakly, with specificity for GC bases. Compound 24 h 48 h 72 h 24 h 48 h 72 h Daidzein, a phytooestrogen, promoted activity where oestrogen receptors were located, thereby promoting growth Baicalein >100 69.6 33.1 >100 >100 >100 in the MCF-7 breast cancer cells. Baicalin >100 78.8 63.3 >100 64.0 44.6 Quercetin showed preferential interactions with tetraplex Daidzein 93.1 >100 >100 59.7 44.5 >100 and triplex structures and presented activity in the Quercetin >100 >100 83.6 >100 >100 38.2 CCRFCEM cells by arresting the cell cycle in G2-phase. The

2281 ANTICANCER RESEARCH 29: 2273-2284 (2009) hydroxyl groups in positions 5, 7, 3’ and 4’ seem to have a 11 Pugazhendhi D, Watson K, Mills S, Botting N, Pope G and strong influence on the interaction of the molecule with the Darbre P: Effect of sulphation on the oestrogen agonist activity nucleic acids. of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells. J Endocrinol 197: 503-515, 2008. The presence of metal cations significantly affected the 12 Cantero G, Campanella C, Mateos S and Cortés F: Bioflavonoids interaction of compounds with DNA, although their effect as poisons of human topoisomerase II alpha and II beta. varied from metal to metal and between the flavonoid and Biochemistry 46: 6097-6108, 2006. isoflavonoid compounds. The metal ions decreased the binding 13 Guo Q, Rimbach G and Moini H: ESR and cell culture studies association constant of baicalein for STDNA by 10-fold. 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