European Journal of Pharmaceutical Sciences 76 (2015) 192–202

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European Journal of Pharmaceutical Sciences

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Inhibition of DNA topoisomerases I and II and growth inhibition of HL-60cells by novel acridine-based compounds

http://dx.doi.org/10.1016/j.ejps.2015.04.0230928-0987/� 2015 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Department of Biochemistry, Faculty of Science, P. J.Šafárik University in Košice, Moyzesova 11, 040 01 Košice, Slovak Republic.

E-mail address: maria.kozurkova@upjs.sk (M. Kozurková).

Jana Janocková a, Jana Plšíková a, Jana Kašpárková d, Viktor Brabec d, Rastislav Jendzelovsky c,Jaromír Mikeš c, Ján Koval c, Slávka Hamulaková b, Peter Fedorocko c, Kamil Kuca e, Mária Kozurková a,e,⇑a Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, 040 01 Košice, Slovak Republicb Department of Organic Chemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, 040 01 Košice, Slovak Republicc Department of Cellular Biology, Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, 040 01 Košice, Slovak Republicd Department of Biophysics, Faculty of Science, Palacky University in Olomouc, Slechtitelu 11, 78371 Olomouc, Czech Republice Biomedical Research Center, University Hospital Hradec Kralove, Sokolovska 581, Hradec Kralove, Czech Republic

a r t i c l e i n f o

Article history:Received 11 February 2015Received in revised form 4 April 2015Accepted 25 April 2015Available online 8 May 2015

Keywords:Topoisomerases I and IIHL-60 cellsDNA-bindingAcridine derivatives

a b s t r a c t

HL-60 cancer cells were treated with a series of novel acridine derivatives (derivatives 1–4) in order totest the compounds’ ability to inhibit both cancer cell growth and topoisomerase I and II activity.Binding studies of derivatives 1–4 with calf thymus DNA were also performed using a number of tech-niques (UV–Vis and fluorescence spectroscopy, thermal denaturation, linear dichroism and viscometry)to determine the nature of the interaction between the compounds and ctDNA. The binding constantsfor the complexes of the studied acridine derivatives with DNA were calculated from UV–Vis spectro-scopic titrations (K = 3.1 � 104–2.0 � 103 M�1). Some of the compounds showed a strong inhibitory effectagainst Topo II at the relatively low concentration of 5 lM. Topo I/II inhibition mode assays were also per-formed and verified that the novel compounds are topoisomerase suppressors rather than poisons. Thebiological activities of derivatives were studied using MTT assay and flow cytometric methods (detectionof mitochondrial membrane potential, measurement of cell viability) after 24 and 48 h incubation. Theability of derivatives to impair cell proliferation was tested by an analysis of cell cycle distribution.

� 2015 Elsevier B.V. All rights reserved.

1. Introduction (Champoux, 2001; Wang, 2002; Vos et al., 2011). Their activity is

The increasingly high costs involved in developing new drugshave led many scientists to turn their attention to pharmaceuticalagents which have already been the subject of chemicalinvestigation. Acridine derivatives are a very good example of thekind of established and studied agents which are still being foundto offer new and important applications (Kozurková et al., 2014;Plsikova et al., 2012, 2014). One of the best known classes ofmulti-targeted anti-cancer agents, acridine derivatives act byinterfering with DNA synthesis and by inhibiting topoisomerases.The development of DNA topoisomerase inhibitors as cancerchemotherapeutic agents is currently an active area of research(Denny, 2002; Di Giorgio et al., 2008; Oppegard et al., 2009;Goodell et al., 2008; Zhang et al., 2006; Lang et al., 2013;Lafayette et al., 2013; Li et al., 2014).

Topoisomerases (Topo) are responsible for controlling thetopology of DNA and play critical roles in DNA metabolism

an essential element of DNA replication and chromosome segrega-tion. There are two classes of topoisomerases, type I and type II.Topo I acts by making a transient break in one strand of DNA,whereas Topo II introduces transient double-strand breaks intothe DNA structure (Berger et al., 1996). Agents which have beenfound to inhibit the activity of Topo II are divided into two distinctgroups, catalytic inhibitors and poisons. Catalytic inhibitors caninterfere with every step of the topoisomerase catalytic cycle,including DNA binding, ATP binding or hydrolysis, magnesiumbinding, and non-covalent linkages between enzymes and DNA.Indeed, the only step of the process which is not inhibited byTopo II catalytic inhibitors is the stabilization of covalent linksbetween enzymes and the DNA duplex. In contrast, Topo II poisonsact more specifically to stabilize the covalent Topo II-5 phosphoty-rosyl DNA intermediate, which is also known as the cleavable com-plex. Moreover, Topo II poisons can be classified into yet moresubcategories: non-intercalating compounds such as etoposide,and intercalators such as amsacrine and doxorubicin (Webb andEbeler, 2004).

Many clinically useful antineoplastic agents act by inhibitingTopo II, and several analogues which inhibit Topo I are currently

J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202 193

undergoing clinical trials (Moukharskaya and Verschraegen, 2012;Ketron et al., 2012). To date, only a small group of dual acridineinhibitors of topoisomerases I and II have been described in detail(Finlay et al., 1996; Todd et al., 1999; Spicer et al., 1997; Denny,1997), and the development of new topoisomerase poisons andinhibitors with dual effects is still an interesting and active fieldof study (Yao et al., 2015).

Our previous studies have shown that tacrine derivatives canact as dual-effect Topo inhibitors by functioning as both Topo I poi-sons and Topo II catalytic inhibitors, thereby suggesting the poten-tial use of these agents as novel anticancer agents (Janocková et al.,2015). In an effort to further this research and to gain more insightinto the substitution effects on the acridine ring, a series of newacridine derivatives 1–4 (Hamulakova et al., 2014) were synthe-sized with ethylene-piperazine as a linker for benzyl and a tetrahy-droacridine skeleton/scaffold. It was hypothesized that a dualinhibitor of both Topo I and II of this nature would be able toexploit of the ability to act on two distinct enzymatic targets,thereby maximizing the potential therapeutic effects. The biologi-cal activity of the novel compounds were assessed using differenttechniques such as examinations of cell cycle distribution andchanges in mitochondrial membrane potential on HL-60 cell lines.Binding studies of derivatives 1–4 with calf thymus DNA were alsoperformed in order to characterize the mechanism of the agents’effect in more detail.

2. Experimental section

2.1. Acridine derivatives and stock solution preparation

A series of novel synthesized acridine derivatives 1–4 were syn-thesized using techniques which have been published previously(Hamulakova et al., 2014). Substituted acridine derivatives 1–4 wereprepared from a starting base of 6,9-dichloro-2-methoxyacridine(5). 6,9-Dichloro-2-methoxyacridine (5) was first converted to6-chloro-2-methoxy-N-[2-(piperazin-1-yl)ethyl] acridin-9-ylamine(1) through the addition of 1-(2-aminoethyl)piperazine. The reac-tion of compound 1 with methanol and concentrated hydrochloricacid produced compound 2. The compound 3 was then obtainedfollowing the further addition of benzyl chloride. Finally, amine 1reacted with 9-chloro-1,2,3,4-tetrahydroacridine in order toproduce the tacrine/acridine dimer, compound 4 (Scheme 1).

Scheme 1. Synthesis of acridine derivativ

The studied derivatives were dissolved in DMSO to a concentra-tion of 5 � 10�3 M. The chemical structures and characteristics ofthe derivatives are shown in Table 1. Calf thymus sodium saltwas purchased from Sigma Aldrich chemicals (Sigma Aldrich,USA). Calf thymus DNA (ctDNA) stock solution was prepared bydissolving 1 mg/mL in a Tris–EDTA buffer (TE buffer contains1 � 10�2 M Tris–HCl pH 7.4 and 1 � 10�3 M EDTA) at 4 �C over-night. The final concentration of ctDNA was identified spectropho-tometrically at 260 nm. The purity of ctDNA was measured as aratio of the absorption at 260/280 nm and was found to be 1.82,a value which indicates that the ctDNA sample is pure and devoidof protein contamination.

2.2. DNA binding experiments

2.2.1. Absorption spectroscopic studies and calculation of bindingconstant

UV–Vis spectra were measured on a Varian Cary 100 UV–Visspectrophotometer in a quartz cuvette (1 cm path length) in a1 � 10�2 M Tris–HCl buffer (pH 7.4). Fixed concentrations of theacridine derivatives (0.1 � 10�4 M for ligands 1 & 2, 0.3 � 10�4 Mfor ligand 3, and 0.2 � 10�4 M for ligand 4), and gradually increas-ing concentrations of calf thymus DNA (ctDNA) (ranging from 0 to4.05 � 10�7 M) were used in the binding experiments. Eachabsorption spectrum was scanned at a wavelength range of 250–500 nm. The binding constants K of the ligand–DNA complexeswere calculated using the absorption titration data and theMcGhee and von Hippel equation (McGhee and von Hippel, 1974):

rCf¼ Kð1� nrÞ 1� nr

1� ðn� 1Þr

� �n�1

where K is the binding constant, n is the binding site size in the basepairs, Cf is the molar concentration of free ligands and r is the num-ber of ligand molecules that bind to one mol of nucleotide. Thebinding data were fitted using Gnu Octave 2.1.73 software (Busa,2006).

2.2.2. Thermal denaturationThermal denaturation experiments were conducted in a quartz

cuvette (1 cm path length) using a Varian Cary Eclipse spectropho-tometer equipped with a thermostatic cell holder. The absorbanceat 260 nm was monitored for ctDNA (4.25 � 10�5 M) in either the

es 1–3 and acridine/tacrine ligand 4.

Table 1Chemical characteristic of acridine derivatives 1–4.

No Structure Name Molecular weight(g mol�1)

1 6-Chloro-2-methoxy-N-[2-(piperazin-1-yl)ethyl]acridin-9-ylamine 370.88

2 6-Chloro-2-methoxy-N-(2-(piperazin-1-yl)ethyl)acridin-9-amine hydrochloride 407.34

3 N-[2-(4-Benzylpiperazin-1-yl)ethyl]-6-chloro-2-methoxyacridin-9-ylamine 461.00

4 6-Chloro-2-methoxy-N-{2-[4-(1,2,3,4-tetrahydroacridin-9-yl)piperazin-1-yl]ethyl}acridin-9-ylamine

552.11

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presence or absence of derivatives 1–4 (2.47 � 10�5 M) in a BPEbuffer at pH 7.1 (6.0 � 10�3 M Na2HPO4, 2.0 � 10�3 M NaH2PO4,1.0 � 10�3 M EDTA). The temperature was increased at a rate of1 �C/min over a range of 50–110 �C. The thermal melting points(Tm, �C) were determined as the maxima of the first derivative plotsof the melting curves.

2.2.3. Fluorescence studiesFluorescence measurements of the studied acridine derivatives

were performed at room temperature on a Varian Cary Eclipsespectrophotometer in a 10 mm path length quartz cuvette in a1 � 10�2 M Tris–HCl buffer at pH 7.4 using the excitation wave-length of the respective absorption maxima. Emission spectra wererecorded at the region of 400–700 nm. 10 nm slits were used for toobtain excitation and emission beams. Fluorescence intensities areexpressed in arbitrary units. Fluorescence titrations were per-formed by adding increasing amounts of ctDNA directly to thesolutions of acridine derivatives 1–4 (0.1 � 10�4 M). The concen-trations of ctDNA used ranged from 0 to 6.42 � 10�7 M.

2.2.4. LD spectroscopyFlow LD spectra were examined using a flow Couette cell in a

Jasco J-720 spectropolarimeter adapted for LD measurements.The LD spectra of ctDNA at a constant concentration of(3.0 � 10�4 M) were recorded at 25 �C in a 1 � 10�2 M Tris–HClbuffer in the range of 210–600 nm following the addition of thestudied acridine derivatives at concentrations ranging from 0 to7.5 � 10�5 M.

2.2.5. ViscometryViscosity experiments were carried out using a microviscome-

ter (AMVn Automated MicroViscometer, Anton Paar GmbH,Austria) in a 1.6 mm capillary tube at 37 �C in a 1 � 10�2 M Tris–HCl buffer. The viscosity of the solution of ctDNA (at a constantconcentration 3.0 � 10�4 M) was measured in the presence of

increasing amounts of the studied acridine derivatives at concen-trations ranging from 0 to 5.0 � 10�5 M. Viscosity measurementswere taken in triplicate and averaged. This data was then used tocalculate the specific viscosity of the DNA solution in the absenceof the ligands (g0) using the flow times of the buffer both in theabsence (t0) and in the presence (t) of the DNA using the equation

g0 ¼ðt � t0Þ

t0

The specific viscosity of the DNA solution in the presence of thestudied ligands was calculated (g) using the flow time buffer (t0)and the DNA buffer in the presence of the studied ligands (t1) usingthe equation

g ¼ ðt1 � t0Þt0

:

Viscosity data were then plotted (as (g/g0)1/3) against the bind-ing ratio (r) (Dedon, 2001).

2.3. DNA gel electrophoresis assays of topoisomerases

2.3.1. Relaxation assay with topoisomerase IThe effects of studied acridine derivatives 1–4 on the relaxation

of plasmid DNA with topoisomerase I was estimated using a nega-tively supercoiled plasmid pHOT-1 (0.25 lg), which was incubatedfor 45 min at 37 �C with 2 units of calf thymus topoisomerase I(Takara Japan) in both the absence and presence of the studiedacridine derivatives (1, 2.5, 5 and 10 lM, respectively). The reac-tions were carried out in a 3.5 � 10�1 M Tris–HCl buffer (pH 8)containing 7.2 � 10�1 M KCl, 5.0 � 10�2 M MgCl2, 5.0 � 10�2 MDTT, 5.0 � 10�2 M spermicide and 0.1% bovine serum albumin(BSA). The reaction was stopped by addition of 2 lL 10% SDS.Samples were cleaned up by proteinase K digestion (50 lg/mLfor 15 min 37 �C). Gel electrophoresis was performed at 7 V/cmfor 2.5 h in a 0.5 � TBE buffer (8.9 � 10�2 M Tris, 8.9 � 10�2 M

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Fig. 1. UV–Vis absorption spectra of studied acridine derivatives (black upper line,derivatives 1 & 2 at a concentration of 0.1 � 10�4 M; derivative 3 at a concentrationof 0.3 � 10�4 M; derivative 4 at a concentration of 0.2 � 10�4 M) in the presence ofincreasing concentrations of ctDNA (color lines, 0–4.05 � 10�7 M) in a 1 � 10�2 MTris–HCl buffer (pH 7.4; 24 �C). Arrow indicates the absorption change uponincreasing amount of ctDNA. Insert graph shows the plot of [DNA]/versusabsorbance. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202 195

H3BO3, 2.0 � 10�3 M EDTA) on 1% agarose gel which was stainedwith ethidium bromide (1 mg mL�1). DNA bands were visualizedusing UV-light.

2.3.2. Inhibition assay of topoisomerase IIThe inhibition of Topo II was studied by applying 2 units of

human topoisomerase IIa and various concentrations of deriva-tives 1–4 (5, 50 and 100 � 10�6 M) at a final volume of 20 lL to0.16 lg kinetoplast DNA (kDNA) in a fresh assay buffer (mixed10 � topo II incomplete assay Buffer A: 0.5 M Tris–HCl (pH 8),1.50 M NaCl, 0.1 M MgCl2, 5.0 � 10�3 M dithiothreitol, 300 lgBSA/mL and 10 � ATP Buffer B: 2.0 � 10�2 M ATP in water to makea 5 � complete assay buffer). The reaction mixtures were incu-bated at 37 �C for 45 min. The reactions were terminated with a5� stop loading buffer (5% Sarkosyl, 0.125% bromophenol blue,25% glycerol) and were loaded directly onto a 1% agarose gel at7 V/cm in a TAE buffer for 2.5 h. The gel was stained with ethidiumbromide and destained in water before being photographed underUV-light. The known topoisomerase II poisons mAMSA(100 � 10�6 M) was used as positive controls.

2.3.3. Cleavage assay of topoisomerase IITopoisomerase II – catalyzed reactions were carried out in an

assay buffer (mixed 10 � topo II incomplete assay Buffer A: 0.5 MTris–HCl (pH 8), 1.50 M NaCl, 0.1 M MgCl2, 5.0 � 10�3 M dithio-threitol, 300 lg BSA/mL and 10 � ATP Buffer B: 2.0 � 10�2 M ATPin water to make a 5 � complete assay buffer) in the presence of0.2 lg of pHOT-1 supercoiled DNA, 5 units of human topoiso-merase IIa and fixed volumes of the studied acridine derivatives(100 � 10�6 M) to a final volume of 20 lL. The reaction mixtureswere incubated at 37 �C for 30 min, at which point the incubationwas stopped with 10% SDS. The reaction was further incubatedwith proteinase K (50 lg/mL) at 37 �C for 20 min. The sampleswere then cleaned up by extraction using a mixture of chloro-form/isoamyl alcohol (24:1) and run on a 1% agarose gel containingethidium bromide (0.5 lg/mL) at 7 V/cm in a TAE buffer. The poi-soning activity of the tested compounds was determined in boththe presence and absence of linear DNA forms. The known topoiso-merase II poison mAMSA (1–10 � 10�6 M) was used as a positivecontrol.

2.4. Biological studies

2.4.1. Cell culture and experimental designHL-60 cells (human promyelocytic leukemia, ATCC, USA) were

grown in a complete RPMI medium (Gibco, USA) supplementedwith 7.5% NaHCO3 (10 mL/L), penicillin (100 U/mL), streptomycin(100 lg/mL), amphotericin (25 lg/mL, Invitrogen, USA), 10%heat-deactivated fetal calf serum (FCS, PAA Laboratories GmbH,Austria) and maintained at 37 �C, 95% humidity and in a 5% CO2

atmosphere. For the experiments, cells were seeded in Ø 60 mmPetri dishes (TPP, Switzerland), to which the studied acridinederivatives were added. Results were analyzed 24 and 48 h afterthe addition of the compounds.

2.4.2. Detection of mitochondrial membrane potential (MMP)Cells were treated for 24 or 48 h and subsequently harvested by

centrifugation, washed once with Hank’s balanced salt solution(HBSS), resuspended in HBSS supplemented with tetramethylrho-damine ethyl ester perchlorate (TMRE; 0.1 lM/20 min/RT, Sigma–Aldrich, USA), incubated in darkness for 20 minand analyzed usinga BD FACSCalibur flow cytometer (Becton Dickinson, San Jose,USA). Results are presented as a mean ± standard deviation (SD)of three independent experiments.

Table 2DNA binding properties of acridine derivatives 1–4.

Compound kmax Bathochromic shift (nm) Hypochromicity (%) Tma (�C) DTm (�C) K (M�1) DG (kcal mol�1)

Free Bound

1 421 431 10 58.8 99.0 32.0 3.1 � 104 �25.542 421 429 8 34.9 99.0 32.0 6.5 � 104 �27.373 441 441 0 32.7 89.1 22.1 3.5 � 103 �20.164 413 414 1 6.1 96.1 29.1 2.0 � 103 �18.77

a Tm measurements were performed in a BPE buffer, pH 7.1 (6.0 � 10�3 M Na2HPO4, 2.0 � 10�3 M NaH2PO4, 1.0 � 10�3 M EDTA) using derivatives 1–4 (2.47 � 10�5 M) andctDNA (4.25 � 10�5 M) at a heating rate of 1 �C/min. Tm of ctDNA for measurement was 67.01 �C.

1008060

0.03

0.02

0.01

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Temperature [°C]

ctDNA

ctDNA + 1

ctDNA + 2

ctDNA + 3

ctDNA + 4∆A/∆T

Fig. 2. First derivative of the denaturation curves of ctDNA (black lines, ctDNAconcentration was 4.25 � 10�5 M) with studied acridine derivatives (color lines,2.47 � 10�5 M) measured at 260 nm in a BPE buffer, pH 7.1. (For interpretation ofthe references to color in this figure legend, the reader is referred to the web versionof this article.)

196 J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202

2.4.3. Viability and cellular metabolismViability and cellular metabolism was analyzed using fluores-

cein diacetate (FDA, 100 ng/mL; Sigma Aldrich) and propidiumiodide (PI, 25 lg/mL; Sigma Aldrich) double staining. Cells weretreated for 24 or 48 h and subsequently harvested by centrifuga-tion, washed once with HBSS, resuspended in HBSS supplementedwith FDA and incubated in darkness for 20 min prior to measure-ments. The cells were then stained with PI and analyzed using aBD FACSCalibur flow cytometer. Results are presented as amean ± SD of three independent experiments.

2.4.4. Analysis of cell cycle parametersCells were treated for 24 or 48 h and subsequently harvested by

centrifugation, washed in cold phosphate-buffered saline (PBS),fixed in cold 70% ethanol and stored overnight at 4 �C. Prior to anal-ysis, the cells were washed twice in PBS, resuspended in stainingsolution (0.1% Triton X-100, 0.137 mg/mL of ribonuclease A and0.02 mg/mL of PI), incubated in darkness for 30 min at room tem-perature and analyzed using a FACSCalibur flow cytometer. ModFit3.0 (Verity Software House, Topsham, USA) software was used togenerate DNA content frequency histograms and to quantify thenumber of cells in the individual cell cycle phases. Results are pre-sented as a mean ± SD of three independent experiments.

2.4.5. Statistical analysisResults were calculated as a mean ± SD of at least three inde-

pendent experiments. Statistical significance was determined witht-tests and results were deemed significant if p < 0.05.

3. Results and discussion

3.1. Electronic absorption spectroscopic studies

The UV–Vis absorption spectra of the studied acridine deriva-tives show significant absorption in the range of 250–500 nm.Fig. 1 describes the interaction of derivatives 1–4 with increasingamounts of ctDNA and shows that the absorption bands of theligand–DNA complex were affected by increasing concentrationsof ctDNA resulting in a tendency toward hypochromism; a signifi-cant hypochromic shift (58.8–32.7%) was observed for derivatives1–3. A low level of hypochromicity was observed in the case ofligand 4 (6.1%). The spectral absorption characteristics of the stud-ied derivatives are given in Table 2. The binding constants of ligandcomplexes 1–4 with ctDNA was K = 3.1 � 104–2.0 � 103 M�1.

3.2. Thermodynamic parameters

Denaturation of DNA is usually measured photometrically atthe temperature-dependent extinction point at 260 nm. Table 2and Fig. 2 show that Tm of ctDNA was 67 �C and that DTm increasedfrom 22.1 to 32.0 �C in the presence of compounds 1–4.

3.3. Fluorescence emission spectroscopy

The emission spectra of derivatives 1–4 in both the absence andthe presence of ctDNA are shown in Fig. 3, and the results showthat the fluorescence intensity of the ligands decrease withincreasing amounts of ctDNA. The binding parameters obtainedfrom the fluorescence measurements are shown in Table 3.Fluorescence titration was performed by adding varying concen-trations of DNA to a constant concentration of each agent.

3.4. Linear dichroism spectroscopy

The flow LD spectra of acridine derivatives 1–4 in the presenceof double stranded ct DNA, shown in Fig. 4, exhibit an intense neg-ative signal at 260 nm resulting from the DNA bases and acridinederivatives, and weaker negative signals at 300–500 nm. The neg-ative signs of the LD signals which arise in the region of 300–500 nm suggest that the angle of the long axis of the studiedderivatives to the axis of the DNA double helix is greater than 54�.

3.5. Viscosity measurements

The effect of the studied derivatives on the viscosity of ctDNA isshown in Fig. 5 and demonstrates that derivatives 1–4 caused asignificant increase in the viscosity of the DNA solution.

3.6. Topoisomerase inhibition mode

The supercoil relaxation activity of pHOT-1 topoisomerase I inthe presence of varying concentrations (1; 2.5; 5 and 10 lM) of

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Fig. 3. Fluorescence emission spectra of studied acridine derivatives 1–4 (blackupper line, concentration 0.1 � 10�4 M) in 1 � 10�2 M Tris–HCl buffer (pH 7.4;24 �C) with increasing concentrations of ctDNA (color lines, 0–6.42 � 10�7) M.Arrow indicates the fluorescence change upon increasing amounts of ctDNA. (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

Table 3Fluorescence characteristics of studied acridine derivatives 1–4.

Compound kex (nm) kem (nm) Imax F/F0a

1 421 493.92 381.76 0.432 421 493.03 510.76 0.583 441 491.06 274.51 0.314 413 478.95 884.91 1.00

a Fluorescence quantum yields were calculated using compound 4 as standard(Uf = 1).

J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202 197

compounds 1–4 is given in Fig. 6. The results demonstrate thatsupercoiled DNA (line pHOT-1) was fully relaxed by the enzymein the absence of compound (line pHOT-1 + Topo I), and that relax-ation was inhibited to varying and concentration-dependentextents by incubation with derivatives 1–4 (line 1a–4a, 1b–4b).Incubation with compound 3 at a concentration of 5 lM resultedin a lower level of inhibitory activity in comparison with thoserecorded for compounds 1, 2 and 4. Relaxation was partially inhib-ited at 1 lM concentration by addition of all studied acridinederivatives 1–4. Derivatives 1, 2 and 4 fully inhibited relaxationof plasmid DNA at 5 lM concentration. As a positive control wasused camptothecine.

The ability of acridine derivatives 1–4 to inhibit the activity ofhuman Topo II was also examined in this assay. The acridinederivatives were tested at concentrations of 1, 2.5, 5 and 10 lMconcentration. Compounds 1, 3 and 4 were found to inhibit TopoII activity at 10 lM, while compound 2 showed evidence of inhibi-tion at 2.5 lM. mAMSA was used as a positive control (Fig. 7).

As can be seen in Fig. 8, the studied acridine derivatives at con-centrations of 100 lM concentration demonstrated none of the lin-ear DNA bands which are characteristic of topoisomerase poisons.In contrast, a linear DNA band was clearly visible for the positivecontrol mAMSA (100 lM), a known Topo II poison. This findingsuggests that the studied derivatives are topoisomerase catalyticinhibitors rather than poisons.

3.7. Biological studies

The biological effects of derivatives 1–4 were evaluated byapplying different concentrations (1–50 lM) of the derivatives tocancer cells for periods of 24 and 48 h and analyzing the resultsusing various different techniques. Acridine was used as a positivecontrol in the assays. The tested acridine derivatives demonstrateda strong ability to affect mitochondrial physiology and a strongcytotoxic action (>90%). Derivative 3 showed a more pronouncedeffect after 48 h and but derivatives 1, 2 and 4 was found to beactive after just 24 h (Fig. 9). A simultaneous analysis of viabilityand activity of cellular metabolism showed a very similar patternof changes to those observed in the MMP analysis, confirmingthe effectiveness of the four derivatives in killing cancer cells(Figs. 10 and 11). Double staining of the cells was performed withFDA and PI. The data are presented as the percentage of live anddead cells after treatment. A comparison of the calculated IC50 val-ues for both techniques and time points are listed in Table 4.

The ability of the derivatives to impair cell proliferation wastested through an analysis of cell cycle distribution. The most pro-found change was observed after 24 h when compound 1 inducedaccumulation of the cells in G2 phase. In contrast, derivatives 2–4induced accumulation of the cells in S phase (see Table 5).

4. Discussion

UV–Vis absorption spectroscopy is a commonly used techniquewhich monitors changes in the absorption properties of ligand andDNA molecules during binding in order to determine the existence

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Wavelength [mdeg]

LD

ctDNA (3.0x10-4 M)

ctDNA + 2 (1.5x10-5 M)

ctDNA + 2 (3.0x10-5 M)

ctDNA + 2 (4.5x10-5 M)

ctDNA + 2 (6.0x10-5 M)

ctDNA + 2 (7.5x10-5 M)

600500400300

0

-0,02

-0,04

-0,06

-0,08

Wavelength [nm]

LD

ctDNA (3.0x10-4 M)

ctDNA + 3 (1.5x10-5 M)

ctDNA + 3 (3.0x10-5 M)

ctDNA + 3 (4.5x10-5 M)

ctDNA + 3 (6.0x10-5 M)

ctDNA + 3 (7.5x10-5 M)

500400300

0

-0,02

-0,04

-0,06

-0,08

Wavelength [nm]

LD

ctDNA (3.0x10-4 M)

ctDNA + 4 (1.5x10-5 M)

ctDNA + 4 (3.0x10-5 M)

ctDNA + 4 (4.5x10-5 M)

ctDNA + 4 (6.0x10-5 M)

ctDNA + 4 (7.5x10-5 M)

2

600500400

0

-0,002

-0,004

-0,006

Wavelength [nm]

LD

600500400

0

-0,002

-0,004

-0,006

Wavelength [nm]

LD

3

500400

0

-0,002

-0,004

-0,006

Wavelength [nm]

LD

4

600500400300

0

-0,02

-0,04

-0,06

-0,08

Wavelength [nm]

LD

ctDNA (3.0x10-4 M)

ctDNA + 1 (1.5x10-5 M)

ctDNA + 1 (3.0x10-5 M)

ctDNA + 1 (4.5x10-5 M)

ctDNA + 1 (6.0x10-5 M)

ctDNA + 1 (7.5x10-5 M)

600500400

0,002

0

-0,002

-0,004

-0,006

Wavelength [nm]

LD

1

Fig. 4. Linear dichroism spectra of ctDNA (3.0 � 10�4 M, black line) in the presenceof increasing amounts of derivatives 1–4 (c = 0–7.5 � 10�5 M) in 1 � 10�2 M Tris–HCl solution (pH 7.4). Arrow indicates the absorption changes upon increasingamounts of derivatives 1–4.

1

1.1

1.2

1.3

1.4

1.5

1.6

0 0.05 0.1 0.15 0.2 0.25

(ƞ/ƞ

0)1/3

r

1

2

3

4

Fig. 5. Relative specific viscosity of ctDNA (3.0 � 10�4 M) in the presence ofderivatives 1–4 in a 1 � 10�2 M Tris–HCl buffer at 37 �C.

198 J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202

and structure of the interactions (Bhadra and Kumar, 2011).Hypochromism and bathochromism in UV–Vis spectra are gener-ally associated with the binding of complexes to DNA throughintercalation involving a stacking interaction between thearomatic chromophore and the DNA base pairs. The extent ofhypochromism and red shift depend on the strength of the inter-calative interaction (Liu et al., 2002). A significant hypochromicshift (58.8–32.7%) was observed in the case of derivatives 1–3.These spectral characteristic suggest that the compounds interactwith ctDNA through the insertion of the aromatic rings into thedsDNA. A low level of hypochromicity was observed for derivative

4 (6.1%), which indicates that the compound can bind to DNA viaintercalation and groove mode. In order to compare the affinityof the acridine derivatives to DNA, the binding constants K ofligand complexes 1–4 with ctDNA were calculated. The calculatedvalues of the binding constants K ranged from 3.1 � 104 to2.0 � 103 M�1. These results prove the affinity of the acridineligands to DNA and correspond to the range of binding constantswhich are typical for intercalators (104 to 106).

DNA melting is another important tool in the study of the inter-action of small molecules with nucleic acids. When solutions ofDNA are exposed to extreme heat, double strand DNA undergoesa transition into a randomly single-stranded form which is knownas denatured DNA. The interactions between base pairs are inter-rupted during denaturation. This process is a result of the disrup-tion in intermolecular forces such as p-stacking with the flankingbases and hydrogen bonding interactions between DNA base pairs.The denaturation of DNA is usually measured photometrically atthe temperature-dependent extinction point of 260 nm (Wolfet al., 1999). The binding of DNA with intercalators generallyresults in a considerable stabilization of the DNA duplex with acorresponding increase in melting temperature (Tm) (Rajendranand Nair, 2006). Tm of ctDNA was 67 �C and DTm in the presenceof compounds 1–4 increased from 22.1 to 32.0 �C which suggeststhat the studied derivatives stabilized the DNA structure by inter-calation. The observed increase in Tm corresponds to that which istypically recorded for other intercalators (13–14 �C) (Neyhart et al.,1993).

Fluorescence spectroscopy is a modern and highly sensitivetechnique in the study of the interaction between macromoleculesand small molecules. Fluorescence quenching experiments provideadditional information concerning the localization of the drugs andtheir mode of DNA interaction (Gonzalez-Ruiz et al., 2011; Li et al.,1997). The fluorescence emissions from the dyes were excited bywavelengths corresponding to the absorption maxima of the freedyes. The fluorescence of derivatives 1–4 in the presence of DNAis characterized by a remarkable decrease in the intensity of thebands in comparison with those of the free compounds and repre-sents further independent proof of intercalation.

Linear dichroism (LD) spectroscopy measures the difference inthe absorption of light polarized parallel and perpendicular tothe orientation axis of the molecule [LD = A|| � A\]. In general,small molecules do not orient in flow and therefore do not exhibitan LD signal, whereas molecules which are very long and thin (e.g.natural DNA) orient very easily in flow and display strong LD sig-nals. The helical axis orients along the flow axis, with the base pairsorienting approximately perpendicular to this axis (Chou andJohnson, 1993). The p–p⁄ transitions in the DNA base pairs there-fore result in a negative LD signal at 260 nm. The DNA LD bandsat 220–300 nm confirm that the DNA in the presence of derivatives1–4 remained in the B-DNA conformation. Nonetheless, the

Fig. 6. Electrophoresis agarose gels showing inhibition of calf thymus topoisomerase I induced DNA relaxation by derivatives 1–4. Supercoiled DNA (lane pHOT-1) wasincubated at 37 �C for 45 min with topoisomerase I in the absence (lane pHOT-1 + Topo I) or presence of ligands (lane a – 1 lM, lane b – 2.5 lM, lane c –5 lM, lane d – 10 lM).Lane CPT – camptothecine.

Fig. 7. Topoisomerase II inhibition assays – the effect of studied compounds 1–4 (1–10 lM) on the decatenation of kDNA (0.16 lg) by 2U of human Topo II. mAMSA (1–10 lM) was used as a standard.

J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202 199

increase in the amplitude of the DNA negative LD band at 260 nmupon the addition of the acridine derivatives indicate some struc-tural changes, such as DNA stiffening (Rodger et al., 2006; Iharaet al., 2006; Chou and Johnson, 1993). Therefore, the effect ofderivatives 1–4 on the DNA LD signal also indicates that the com-pounds have intercalated with the DNA. Indeed, the LD signals inthe region of 300–500 nm prove unequivocally that derivatives1–4 were bound to DNA, as only bound ligands can be observedin a flow linear dichroism experiment (Rodger and Norden,

1997). In addition, the LD spectra show that the studied acridinederivatives bind to DNA in specific, non-random orientations. Thenegative LD signals that arise in the region of 300–500 nm suggestthat the angle of the long axis of derivatives 1–4 to the axis of theDNA double helix is greater than 54�, as would be expected for anintercalator (Rodger et al., 2006).

Viscosity measurement study was carried out in order to con-firm the mode of DNA binding of derivatives 1–4 which had beeninferred from the LD measurements. The viscosity of a DNA

Fig. 8. Topoisomerase II inhibition mode assay – the effect of acridine derivatives1–4 (100 lM) on the growth of DNA – Topo II cleavage complex. pHOT-1 DNA(0.2 lg) was incubated with 5U of human Topo II in the presence of the studiedderivatives. mAMSA (100 lM) was used as a standard.

200 J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202

solution measured in the presence of DNA binders is regarded asone of the most important tests in establishing the form of DNAbinding (Norden and Tjerneld, 1982; Pilch et al., 1993). A classicalintercalation mode results in the lengthening of the DNA helix,leading to an increase in the viscosity of the DNA (Suh andChaires, 1995). Intercalators are known to increase the length ofDNA and consequently the viscosity, whereas groove binders,which do not lengthen the DNA helix, have no effect on the viscos-ity of the DNA solution (Satyanarayana et al., 1992, 1993). Our vis-cosity data demonstrates that the acridine derivatives caused asignificant increase in the viscosity of the DNA solution. Theseresults are consistent with the lengthening of DNA indicating theintercalation of acridine derivatives, thereby corroborating thefindings of the LD spectra assay.

Plasmid pHOT-1 relaxation was also studied with agarose gelelectrophoresis in order to investigate the effect of the studied acri-dine derivatives on calf thymus Topo I. The aim of the experimentwas to determine whether or not the derivatives could affect theunwinding of supercoiled DNA when the enzyme converts thesupercoiled DNA to a relaxed form. If the natural activity of TopoI were allowed to continue unimpeded, we would expect to seethe supercoiled forms of DNA replaced by relaxed forms of DNA.In contrast, if the enzyme activity were inhibited, no relaxed formsof DNA would be detected. In our experiment, the supercoiled DNAwas fully relaxed by the enzyme in the absence of the studiedderivatives. However, relaxation was inhibited by incubation withderivatives 1–4. Derivative 3 at a concentration of 5 lM exhibited alower level of inhibitory activity in comparison to those ofcompounds 1, 2 and 4. This anomaly suggests that the tested

Fig. 9. Effect of studied acridine derivatives (5 lM) on changes in mitochondrialmembrane potential (MMP). The results are presented as a mean ± SD, statisticalsignificance: p < 0.05 (⁄) for each experimental group compared to untreatedcontrol (C – control, A – acridine).

derivatives may act against human cancer cell lines by means otherthan that of topoisomerase I inhibition.

The ability of derivatives 1–4 to inhibit the activity of humanTopo II was also tested. The specific reaction of Topo II is thedecatenation of catenated kinetoplast DNA (kDNA), a processwhich involves the simultaneous cleavage of two strands ofDNA-helix. If the natural activity of Topo II were allowed to con-tinue unimpeded, we would expect to see catenated kDNAreplaced by closed circular decatenated kDNA. In contrast, if theenzyme activity were inhibited, these bands would not bedetected. In general, the studied compounds caused a completeinhibition of topoisomerase II at a concentration of 10 lM. Thesample 2 showed the strongest inhibitory effect against Topo IIat the relatively low concentration of 2.5 lM. A further experimentshowed that the studied compounds act as topoisomerase catalyticinhibitors.

Cancer cells were also treated with different concentrations ofthe studied derivatives over 24 and 48 h periods in order to estab-lish whether the compounds could demonstrate any biologicallyrelevant effects. The results were tested using a range of differenttechniques (changes in mitochondrial membrane potential,changes in metabolic activity/viability and cell cycle distribution),and acridine was used as a positive control.

The results show that the tested derivatives exerted a strongeffect on mitochondrial physiology and also showed evidence ofpowerful cytotoxic action (>90%). Derivative 3 showed more a pro-nounced effect after 48 h, but the effect of compounds 1, 2 and 4was more immediate and was visible after just 24 h. A simultane-ous analysis of the viability and activity of cellular metabolismshowed a very similar trend to that which was observed in theMMP analysis, and confirmed that the four derivatives were effec-tive in killing cancer cells. The results listed in Figs. 10–12 onlyshow the data for the derivatives at one specific concentration(5 lM), but Table 4 shows a comparison of the calculated IC50 val-ues for both techniques over both time periods. The table clearlyshows that the IC50 values obtained through the two differentmethodologies are consistent and that the classification of thederivatives based on their cytotoxic effects on cancer cells is inthe order 3 < 4 < 1 < 2. The changes in cell proliferation were onlytransient a subsequent equalization of the percentages was laterobserved. However, one exception to this trend was derivative 3,which increased the percentage of cells in the S phase of the cellcycle after a 48 h period.

Fig. 10. Effect of studied acridine derivatives (5 lM) on changes in viability 24 hafter treatment. The results are presented as a mean ± SD, statistical significance:p < 0.05 (⁄) for each experimental group compared to untreated control (C –control).

Fig. 11. Effect of studied acridine derivatives (5 lM) on changes in viability 48 hafter treatment. The results are presented as a mean ± SD, statistical significance:p < 0.05 (⁄) for each experimental group compared to untreated control (C – control,A – acridine).

Table 4IC50 values for the MMP and viability 24 and 48 h after treatment.

TMRE IC50 (lM) 24 h 48 h PI IC50 (lM) 24 h 48 h

1 3.10 1.77 1 3.08 1.772 1.92 1.60 2 1.87 1.573 8.12 4.20 3 5.99 4.204 3.19 2.48 4 3.14 2.05A n.d. n.d. A n.d. n.d.

Table 5The effect of studied acridine derivatives 1–4 (5 lM) on cell cycle distribution.

G1 S G2

24 hC 38.14 ± 1.82 43.72 ± 1.14 18.13 ± 0.91A n.d. n.d. n.d.1 37.08 ± 3.74 22.51 ± 7.84* 40.40 ± 4.09*

2 9.18 ± 1.15* 89.08 ± 2.88* 1.73 ± 1.73*

3 30.87 ± 2.79* 50.59 ± 2.80* 18.53 ± 0.464 16.53 ± 8.51* 73.58 ± 9.67* 14.81 ± 8.77

48 hC 38.08 ± 1.39 44.85 ± 1.77 17.06 ± 1.75A n.d. n.d. n.d.1 41.04 ± 6.17 50.06 ± 11.7 8.89 ± 6.29*

2 36.43 ± 2.82 51.01 ± 4.04 12.56 ± 1.23*

3 24.14 ± 6.97* 58.38 ± 9.06* 17.47 ± 2.094 39.23 ± 6.35 47.54 ± 8.34 13.22 ± 9.75

The results are presented as a mean ± SD.* Statistical significance: p < 0.05 for each experimental group compared to

untreated control (C – control, A – acridine).

J. Janocková et al. / European Journal of Pharmaceutical Sciences 76 (2015) 192–202 201

As can be seen from our results, the studied acridine derivativeswere very effective against HL-60 human acute promyelocytic leu-kaemia cells; rapid MMP dissipation, decreases in cell metabolismand losses of cell viability were all observed at a concentration of5 lM. Researchers have been aware of the anticancer propertiesof acridine derivatives since the 1960s, and extensive studies ofthese agents have continued from that time (Demeunynck et al.,2001; Vispe et al., 2007). For example, sugar moiety acylated acri-dine derivatives have been reported to demonstrate potent in vitrocytotoxic activity against a series human cell lines (Dzierzbicka andKolodziejczyk, 2003), and Antonini et al. (2004) have reported thatcyclization of the acridine derivatives with phosgene leads to an

increase in the DNA binding affinities of these compounds andenhanced in vitro cytotoxic properties (Antonini et al., 2004).Additionally, other new acridine derivatives (Sondhi et al., 2010;Barros et al., 2012, 2013) have been shown to induce cell death,but this occurred at much higher concentrations than we haveobserved for our novel derivatives. From this point of view, wecan confidently state that our novel derivatives can significantlyalter a wide range of cell functions and can also direct cells towardcell death even at low concentrations.

5. Conclusion

In the current study, the interaction of new acridine derivatives1–4 with ctDNA has been studied and analyzed using various tech-niques. The results of the binding studies suggest that these smallmolecules bind to DNA through intercalation. The results of ourUV–Vis absorption spectroscopy prove that the binding of deriva-tives 1–4 has occurred with a binding constant valueK = 3.1 � 104–2.0 � 103 M�1. These findings are indicative of astrong interaction between the derivatives and ctDNA, and this issupported by the results of the fluorescence emission, LD and vis-cometric studies. Our results indicate that the novel derivatives aredual Topo inhibitors, acting both as Topo I inhibitors and as Topo IIcatalytic inhibitors. From a biological point of view, the studiedacridine derivatives 1–4 were very effective against HL-60 humanacute promyelocytic leukaemia cells; rapid MMP dissipation,decreases in cell metabolism and losses of cell viability were allobserved even at the relatively low concentration of 5 lM.

Acknowledgements

This study was supported by Slovak Research and DevelopmentAgency under contract VVCE-0001-07, VEGA Grant No. 1/0001/13,Grant Program of the P. J. Šafárik University in Košice(VVGS-PF-2014-435) and UHHK 00179906. The authors from thePalacky University in Olomouc would like to acknowledge supportfrom grant IGAPrF_2014_029 and acknowledge that their partici-pation in the EU COST Action CM1105 allowed them to exchangethe most recent ideas in the field of compounds of biological signif-icance with many European colleagues on a regular basis.

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