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International Journal of Biological Macromolecules 45 (2009) 174–180

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journa l homepage: www.e lsev ier .com/ locate / i jb iomac

ytotoxic activity of acridin-3,6-diyl dithiourea hydrochlorides in humaneukemia line HL-60 and resistant subline HL-60/ADR

uzana Vantováa, Helena Paulíkováa, Danica Sabolováb, Mária Kozurkováb,∗, Mária Suchánováa,adislav Janovecc, Pavol Kristianc, Ján Imrichc

Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak Technical University, Radlinského 9, SK-81237 Bratislava, Slovak RepublicDepartment of Biochemistry, Institute of Chemistry, Faculty of Science, P.J. Safárik University, Moyzesova 11, SK-04167 Kosice, Slovak RepublicDepartment of Organic Chemistry, Institute of Chemistry, Faculty of Science, P.J. Safárik University, Moyzesova 11, SK-04167 Kosice, Slovak Republic

r t i c l e i n f o

rticle history:eceived 10 February 2009

a b s t r a c t

A series of acridin-3,6-diyl dithiourea hydrochloride derivatives (alkyl-AcrDTU) was prepared and testedagainst sensitive and drug resistant leukemia cell lines for their cytotoxic/cytostatic activity. The prod-

eceived in revised form 20 April 2009ccepted 27 April 2009vailable online 3 May 2009

eywords:cridinesytotoxic activityNA-binding constant

ucts (ethyl-, n-propyl-, n-butyl-, n-pentyl-AcrDTU) showed high DNA binding affinity via intercalation(K = 7.6 − 2.9 × 105 M−1). All derivatives inhibited proliferation of HL-60 cells and its resistant sublineHL-60/ADR, unexpectedly the resistant subline was more sensitive than the parental one (IC50 = 3.5 �M,48-treatment of HL-60/ADR with pentyl-AcrDTU). Cytotoxicity of tested compounds was associated withtheir DNA-binding properties and the level of intracellular thiols has been changed in the presence ofAcrDTU.

© 2009 Elsevier B.V. All rights reserved.

The search for new pharmacologically active drugs has ledo the discovery of many DNA-binding drugs including acridinenticancer and chemotherapeutic agents [1–6]. Acridines haveeen used as antimicrobial [7,8], antiparasitic [9] and anticancergents for many years and binding to DNA considered to be arincipal mode of their bioactivity [10]. The best known acri-ine drug amsacrine [N-(4-(acridin-9-ylamino)-3-methoxyphenyl)ethanesulfonamide] is routinely employed for systemic treat-ent of human cancers [11]. Amsacrine and its analogues have

everal mechanisms of action with topoisomerase II being a mainarget [12]. Blasiak et al. [13] confirmed a possible modulation ofmsacrine cytotoxicity by some free radical scavengers. Acridinenalogues have also been evaluated for their strong DNA-bindingctivity [14] and cytotoxic potency toward sensitive and multidrug-esistant tumor cell lines [15,16]. The planar acridine ring systemtacks well with the base pairs in the intercalation complex. Inter-alation is known to occur without interfering with hydrogenonding of the base and obeys the nearest neighbor exclusion prin-

iple.

Promising biological activities of acridin-3,6-diyl dithiazolidi-ones prepared recently in our laboratory [17] prompted us toxplore anticipated binding activity and proliferative effect of

∗ Corresponding author. Tel.: +421 55 6223582; fax: +421 556222124.E-mail address: maria.kozurkova@upjs.sk (M. Kozurková).

141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2009.04.018

their precursors, acridin-3,6-diyl dithioureas. Due to an instabilityof free bases, we have synthesized hitherto not described sym-metrical 1′,1′′-(acridin-3,6-diyl)-3′,3′′-dialkyldithioureas (AcrDTU)in the form of hydrochlorides, similarly to classical intercala-tors containing a fused-ring aromatic moiety with the positivecharge on the ring system and/or attached side chain. Cytotoxiceffect of title compounds on human myeloid leukemia sensitiveand resistant cell lines HL-60 and HL-60/ADR, respectively, hasbeen investigated by UV–vis spectrophotometry, fluorescence titra-tion, and denaturation transition temperature measurements aswell.

1. Materials and methods

1.1. Materials

Propidium iodide (PI), Hoechst 33342, ethidium bromide, TritonX-100, reduced form of glutathione (GSH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide

(DMSO), and calf thymus DNA were obtained from Sigma–AldrichChemie (Germany). EDTA, RNase A and proteinase K were pur-chased from Serva (Germany), 5,5-dithio-bis(2-nitrobenzoic acid)(DTNB) from Merck (Germany), reduced glutathione and N-acetylcysteine were from Calbiochem (USA) and other chemicalsfrom Lachema (Czech Republic).

Z. Vantová et al. / International Journal of Biolog

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cheme 1. Structure of 1′ ,1′′-(acridin-3,6-diyl)-3′ ,3′′-dialkyldithiourea hydrochlo-ides (AcrDTU), R = ethyl, n-propyl, n-butyl, n-pentyl.

.1.1. Preparation of 1′,1′′-(acridin-3,6-diyl)-3′,3′′-ialkyldithiourea hydrochlorides (AcrDTU)

1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dialkyldithioureas were preparedccording to our previous paper [17]. To a methanol suspen-ion (3 mL) of 3,6-diisothiocyanatoacridine (50 mg, 0.17 mmol), aen-molars excess of appropriate amine was added, the reaction

ixture stirred for 1 h whilst the progress of the reaction was moni-ored by TLC (toluene/acetone 3:1) until completion. The dithiourearoduct was filtered off, dried in vacuo, dissolved in acetone (0.5 mL)nd equimolar amount of HCl in methanol (1 mL of 35% hydrochlo-ic acid in 9 mL of methanol) was added dropwise. The mixture wastirred for 1 h, and then acetone added until beginning of precipi-ation. Resulting hydrochloride product (Scheme 1) was filtered offnd dried in vacuo.

.1.1.1. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-diethyldithiourea hydrochloride.ield: 90%; m.p. 186–188 ◦C, 1H NMR (DMSO, TMS/ppm): ı 11.27bs, 2H, 2× NH), 9.41 (s, 1H, CH-9), 9.14 (s, 2H, CH-4,5), 9.04 (bs, 2H,× NH), 8.23 (d, 2H, CH-1,8, J = 9.2 Hz), 7.72 (d, 2H, CH-2,7, J = 9.2 Hz),.52–3.58 (m, 4H, 2× NCH2), 1.21 (t, 6H, 2× CH3, J = 7.2 Hz); 13CMR (DMSO, TMS/ppm): ı 179.4 (CS), 147.5, 144.8, 141.1, 130.5,21.9, 120.8, 102.5, 38.5 (NCH2), 13.5 (CH3). Anal. calculated for19H21N5S2·HCl (420.00): C, 54.34; H, 5.28; N, 16.67. Found: C,4.18; H, 5.15; N, 16.39.

.1.1.2. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dipropyldithiourea hydrochlo-ide. Yield: 92%; m.p. 191–193 ◦C, 1H NMR (DMSO, TMS/ppm): ı1.38 (bs, 2H, 2× NH), 9.40 (s, 1H, CH-9), 9.21 (s, 2H, CH-4,5), 9.10bs, 2H, 2× NH), 8.23 (d, 2H, CH-1,8, J = 8.6 Hz), 7.72 (d, 2H, CH-2,7,= 8.6 Hz), 3.46–3.51 (m, 4H, 2× NCH2), 1.58–1.67 (m, 4H, 2× CH2),.97 (t, 6H, 2× CH3, J = 7.4 Hz); 13C NMR (DMSO, TMS/ppm): ı 179.5CS), 147.5, 144.8, 141.0, 130.5, 121.8, 120.8, 102.4, 45.4 (NCH2), 21.2CH2), 11.4 (CH3). Anal. calculated for C21H25N5S2·HCl (448.06): C,6.29; H, 5.85; N, 15.63. Found: C, 56.05; H, 5.95: N, 15.48.

.1.1.3. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dibutyldithiourea hydrochloride.ield: 93%; m.p. 213–215 ◦C, 1H NMR (DMSO, TMS/ppm): ı 11.30 (bs,H, 2× NH), 9.40 (s, 1H, CH-9), 9.19 (s, 2H, CH-4,5), 9.03 (bs, 2H, 2×H), 8.30 (d, 2H, CH-1,8, J = 8.8 Hz), 7.71 (d, 2H, CH-2,7, J = 8.80 Hz),.50–3.55 (m, 4H, 2× NCH2), 1.55–1.63 (m, 4H, 2× CH2), 1.35–1.45m, 4H, 2× CH2), 0.94 (t, 6H, 2× CH3, J = 7.4 Hz); 13C NMR (DMSO,MS/ppm): ı 179.5 (CS), 147.5, 144.7, 141.1, 130.5, 121.8, 120.8, 102.4,3.3 (NCH2), 29.9 (CH2), 19.6 (CH2), 13.6 (CH3). Anal. calculated for23H29N5S2·HCl (476.11): C, 58.02; H, 6.35; N, 14.71. Found: C, 58.12;, 6.43; N, 14.85.

.1.1.4. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dipentyldithiourea hydrochlo-ide. Yield: 93%; m.p. 221–223 ◦C, 1H NMR (DMSO, TMS/ppm): ı1.28 (bs, 2H, 2× NH), 9.40 (s, 1H, CH-9), 9.19 (s, 2H, CH-4,5), 9.03bs, 2H, 2× NH), 8.30 (d, 2H, CH-1,8, J = 8.8 Hz), 7.71 (d, 2H, CH-2,7,= 8.8 Hz), 3.49–3.54 (m, 4H, 2× NCH2), 1.57–1.62 (m, 4H, 2× CH2),

13

.33–1.37 (m, 8H, 4× CH2), 0.90 (t, 6H, 2× CH3, J = 6.8 Hz); C NMRDMSO, TMS/ppm): ı 179.4 (CS), 147.5, 144.8, 141.1, 130.5, 121.8,20.8, 102.5, 43.6 (NCH2), 28.5 (CH2), 27.5 (CH2), 21.8 (CH2), 13.8CH3). Anal. calculated for C25H33N5S2·HCl (504.16): C, 59.56; H,.80; N, 13.89. Found: C, 59.41; H, 6.64; N, 13.83.

ical Macromolecules 45 (2009) 174–180 175

1H (400 MHz) and 13C (100 MHz) NMR spectra were measuredon a Varian Mercury Plus NMR spectrometer at room temperaturein DMSO using TMS as an internal standard (0 ppm for both nuclei).Melting points were determined with a Boetius hot-stage appara-tus and are uncorrected. Elemental analyses were performed on aPerkin-Elmer CHN 2400 analyzer.

1.1.2. Cell culture: human HL-60 leukemia cells and resistantsubline HL-60/ADR

Human myeloid leukemia cell line HL-60 and its multidrug-resistant subline HL-60/ADR were obtained from Dr. P. Ujhazy,Roswell Park Cancer Institute, Buffalo. The cell lines were grownin RPMI 1640 medium supplemented with 10% fetal calf serum(FCS, Grand Island Biological Co., Grand Island, NY, USA), penicillin(100 units/mL), streptomycin (100 �g/mL) and 2 mM glutamine.The cells were maintained at 37 ◦C in a humidified 5% CO2 atmo-sphere.

1.2. Methods

1.2.1. UV–vis absorption measurementsUV–vis spectra were measured on a Varian Cary 100 UV–vis

spectrophotometer in 0.01 M Tris buffer (pH 7.4). A solution ofthe calf thymus DNA (CT DNA) in TE (Tris–EDTA) buffer was son-icated for 5 min and the DNA concentration was determined fromits absorbance at 260 nm. The purity of DNA was determined bymonitoring the value A260/A280. DNA concentrations measured at260 nm are expressed as micromolar equivalents of base pairs andranged from 0 to 45 �M bp. The derivatives AcrDTU were all dis-solved in DMSO from which working solutions were prepared bydilution using a 0.01 M Tris buffer to concentration 25 �M. All mea-surements were performed at 25 ◦C.

1.2.2. Fluorescence measurementsFluorescence measurements were scanned on a Varian Cary

Eclipse spectrofluorimeter with a 10 nm slit width for excitationand emission beams. Emission spectra were recorded in the region412–443 nm using an excitation wavelength 393 nm. Fluorescenceintensities are expressed in arbitrary units. Fluorescence titrationswere conducted by adding increasing amounts of CT DNA directlyinto the cell containing the solution of AcrDTU (c = 12.7 × 10−6 M,0.01 M Tris buffer, pH 7.4). The concentration range of DNA was0–100 �M bp. All measurements were performed at 25 ◦C.

1.2.3. Tm measurementsThermal denaturation studies were conducted using a Varian

Cary Eclipse spectrophotometer equipped with a thermostatic cellholder. The temperature was controlled by a thermostatic bath(±0.1 ◦C). The absorbance at 260 nm was monitored for eitherctDNA (10 �M) or a mixture of DNA with AcrDTU (5 �M) in BPEbuffer, pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA), witha heating rate 1 ◦C min−1. The melting temperatures were deter-mined as the maximum of the first derivative plots of meltingcurves.

1.2.4. Equilibrium binding titrationThe binding affinities were calculated from absorbance spec-

tra according to the method of McGhee and vonHippel using datapoints from the Scatchard plot [18–20]. The binding data were fittedusing a GNU Octave 2.1.73 software.

1.2.5. Cytotoxic studiesCell viability in the presence or absence of tested sub-

stances was determined using a MTT (3-(4,5 dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide; Sigma) microculture tetra-zolium assay as described previously [21]. Cells harvested in the log

1 Biological Macromolecules 45 (2009) 174–180

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1

tLP(Hwcacws4d

1

tAdlcwetd

2

2

UibFai

cTwopci

Fig. 1. Spectrophotometric titration of propyl-AcrDTU (25 �M) in 0.01 M Tris buffer(pH 7.4, 25 ◦C) by the concentration increase of CT DNA (from top to bottom, 0–45 �Mbp, at 5 �M intervals).

Table 1UV–vis absorption data of AcrDTU derivatives.

Compound �max (nm) Hypochromism (%)

Free Bound �� (nm)

Ethyl-AcrDTU 380 381 1 55

The representative fluorescence spectra of propyl-AcrDTU solu-tion titrated by the growing concentration of CT DNA showed afluorescence decrease which proved, similarly as before, the bind-ing of our proflavine probes to DNA (Fig. 3). Fluorescence quenching

Table 2DNA-binding properties of AcrDTU derivatives.

Compound K × 105 (M−1) n aTm (◦C)

Ethyl-AcrDTU 7.6 1.9 80Propyl-AcrDTU 6.8 2.4 78

76 Z. Vantová et al. / International Journal of

hase of growth were counted and seeded (2.5 × 104 cells/100 �Lell) in 96-well microtitre plates. After 24 h of incubation at 37 ◦C

nd 5% CO2, cultures were treated with varying concentrations ofhe drug. After 45 or 69 h exposure to the compound, MTT (50 �L,mg/mL) was added to each well. After 3 h, the cell cultures wereentrifuged, the supernatant discarded and the resulting pelletshoroughly extracted into 200 �L of DMSO. Absorption at 540 nmas recorded using a MicroPlate Reader (Labsystem Multiscan,ultisoft, Finland). The MTT assay was performed three times for

ach compound.Cell proliferation, growth curves and cytotoxic potential of com-

ounds were determined by a trypan blue dye exclusion test. Forhree-day experiments; cells were seeded in 1 × 105 cells/mL inetri dishes and tested substances (or DMSO—control) were addedfter 24 h, then cell proliferation was checked after 24, 48 and 72 h.rowth curves were obtained and inhibition concentrations, IC50,etermined. All dye exclusion tests were done three times.

.2.6. Analysis of cell morphologyApoptosis was evaluated by a direct counting of cells with apop-

otic morphological features after Hoechst 33342 and PI staining.eukemia cells were stained with 10 �M of Hoechst 33342 andI for 10 min and then counted under a fluorescent microscopeJENA LUMAR, Carl Zeiss, Germany). Having been stained withoechst 33342 and PI, necrotic cells showed red-stained nuclei,hereas viable cells showed green and round nuclei. Apoptotic

ells exhibited green or red nuclear fragmentation, apoptotic bodiesnd plasma membrane blebbing. AcrDTU derivatives are fluores-ent substances, therefore morphological changes of leukemia cellsere examined without Hoechst 33342, using fluorescent micro-

copic examination with a 378–456 nm excitation block filter and20–450 nm emission filters. Images of cells were captured using aigital camera (Olympus Camedia C-4000).

.2.7. Determination of LMWTThe level of LMWT (low-molecular weight thiols or non-protein

hiols) was measured according to the method described bynderson [22]. Cells were washed twice with a PBS solution andeproteinized with TCA (5% final concentration). Cellular NPSH

evel was determined with an Ellman’s reagent by measuring thehange of UV absorbance at the wavelength 412 nm. Values of NPSHere calculated from a standard curve (reduced glutathione) and

xpressed in nmol/106 cells. All experiments were performed inriplicate and results shown in graphs represent the mean and stan-ard deviation.

. Results and discussion

.1. DNA binding studies

To determine binding constants of synthesized derivatives, theirV–vis absorption spectra have been taken. Spectra showed signif-

cant absorption in the range 350–450 nm, typical for transitionsetween the �-electronic energy levels of the proflavine ring.ig. 1 provides an example illustrating characteristic changes in thebsorption spectra during titration of propyl-AcrDTU with increas-ng amounts of CT DNA.

The binding of drugs to DNA resulted in noticeable spectralhanges characterized by bathochromic shifts and hypochromism.he red shift of the drug absorption band was pronounced

ith propyl-AcrDTU and butyl-AcrDTU whereas only mild shifts

ccurred for ethyl-AcrDTU and pentyl-AcrDTU (Table 1). Theresence of isosbestic points indicated spectroscopically distincthromophores, viz. free and bound species. Such spectral behaviors generally associated with intercalation.

Propyl-AcrDTU 396 402 6 23Butyl-AcrDTU 399 406 7 36Pentyl-AcrDTU 400 402 2 35

Additional evidence for intercalation into DNA was obtainedfrom thermal denaturation studies. Binding of a small molecule toDNA is assumed to stabilize the helix against its thermal denat-uration and therefore it is accompanied by the rise of transitiontemperature, Tm, for the double- to single-stranded form morphingof DNA. Experimentally, temperatures Tm for DNA in the solutionwithout and with the intercalator are mutually compared whilstmonitoring some property dependent on the DNA helix. Due to thedifference between the extinction coefficients of DNA bases in thedouble-stranded form vs. the single-stranded form at 260 nm, theabsorbance increases sharply at Tm as the DNA strands separate.The helix denaturation of DNA was thus monitored as a function oftemperature by recording absorbance at 260 nm.

As shown in Fig. 2, the DNA melting experiment revealed that Tm

of the calf thymus DNA was 73.0 ◦C and in the presence of AcrDTUincreased to 77.0–80.0 ◦C (Table 2), thus emphatically confirmingheightened helix stability as the result of intercalation of proflavinederivatives into DNA. These findings are consistent with our previ-ous results [14,17].

Butyl-AcrDTU 4.9 2.5 77Pentyl-AcrDTU 2.9 2.8 77

a Tm measurements were performed in BPE buffer, pH 7.1 (6 mM Na2HPO4, 2 mMNaH2PO4, 1 mM EDTA) using 10 �M drug and 20 �M bp CT DNA with a heating rate1 ◦C min−1. Tm of CT DNA is 73 ◦C.

Z. Vantová et al. / International Journal of Biological Macromolecules 45 (2009) 174–180 177

Fig. 2. First derivative of the helix denaturation curves of CT DNA (black line) withethyl-AcrDTU (red line), propyl-AcrDTU (blue line), butyl-AcrDTU (violet line) andptw

ui

DMesbaEbutpr(i[ta[

FTa

entyl-AcrDTU (green line) measured at 260 nm in BPE buffer, pH 7.1. (For interpre-ation of the references to colour in this figure legend, the reader is referred to theeb version of the article.)

pon the DNA titration was observed for all derivatives investigatedn this study.

To determine binding constants of studied compounds withNA, UV–vis titration data from spectrophotometric analysis andcGhee and vonHippel plots [18–20] were used. Binding param-

ters for the interaction of AcrDTUs with calf thymus DNA areummarized in Table 2. Calculated binding constants, K, and neigh-or exclusion parameters, n, indicated a direct dependence betweenn intercalation capability and structural modifications of AcrDTUs.stimations for n in the range 1.9–2.8 confirmed that multiple siteinding affected the interaction at neighboring sites. The large val-es of binding constants K, determined by spectrophotometry inhe range 2.9–7.6 × 105 M−1, were indicative of high affinity of theroflavine chromophore for the DNA-base pairs. They are compa-able to typical binding constants for intercalation of dyes into DNA104–106 M−1) [23] and in good agreement with the binding affin-ty of standard proflavine hemisulphate (K = 7.95 × 105 M−1, n = 1.2)24]. Compared to another standards, Ks found are approximately

en times lower than that of doxorubicin (1.27 × 106 M−1) [25] butbout ten times higher than the value for amsacrine (4.0 × 104 M−1)26].

ig. 3. Changes in the emission fluorescence spectra of propyl-AcrDTU (12.7 �M) inris buffer (0.01 M, pH 7.4, 25 ◦C) in the absence and presence of CT DNA (0–100 �M,t 10 �M steps), �ex = 393 nm.

Fig. 4. Correlation of the chain length (Å) of alkyl substituents in AcrDTU with theDNA binding constants (K × 105 M−1) obtained from spectrophotometry (k = −0.717,r = 0.992; S = 0.066).

A linear correlation found between the DNA binding constantsand the length of AcrDTU alkyl substituents showed that the bind-ing affinity increased with the decreasing length of the chain inthe order: pentyl-AcrDTU < butyl-AcrDTU < propyl-AcrDTU < ethyl-AcrDTU (Fig. 4).

2.2. Cytotoxic effect on human leukemia cells

Another evidence of the sensitivity towards AcrDTU wasobtained from IC50 values determined by the MTT assay (Table 3).Results demonstrated that human leukemia HL-60 cells were moresensitive to ethyl- and propyl-AcrDTU than to butyl- and pentyl-AcrDTU.

Unexpectedly, the resistant cell line HL-60/ADR appeared tobe more sensitive to butyl- and pentyl-AcrDTU than its parentalline HL-60. IC50 values for these derivatives were in the range3.5–10.0 �M, pentyl-AcrDTU being the most effective compoundagainst HL-60/ADR. Ethyl- and propyl-AcrDTU were cytotoxicagainst parental and resistant cell lines in the same extent.

The MTT reduction is generally attributed to the mitochon-drial activity, but it has also been related to non-mitochondrialenzymes, endosomes and lysosomes [27,28]. Because MDR1 andMRP proteins interfere with the MTT assay [29,30], the cytotoxi-city of propyl-AcrDTU, which is able to act as a substrate and/orinhibitor for MDR1 and MRP proteins, was estimated here by adirect cell counting. After 72 h treatment, propyl-AcrDTU decreased

the proliferation rate of HL-60 and HL-60/ADR cells in compar-ison with the control (Fig. 5a and b). In the case of the HL-60cells, 1.5 �M propyl-AcrDTU had the cytostatic effect already after24 h incubation. Increasing the concentration to 25 �M resulted

Table 3The IC50 values of AcrDTU derivatives determined by MTT assay.

Compound IC50 (�M)

HL-60 HL-60/ADR

48 h 72 h 48 h 72 h

Ethyl-AcrDTU 8.9 ± 2.4 7.4 ± 1.3 7.5 ± 0.5 10.0 ± 0.7Propyl-AcrDTU 7.2 ± 0.7 6.7 ± 2.3 8.0 ± 1.2 9.0 ± 1.1Butyl-AcrDTU 34 ± 3.4 20 ± 2.7 7.0 ± 1.5 7.5 ± 1.5Pentyl-AcrDTU 32 ± 2.5 14 ± 1.7 3.5 ± 1.6 4.5 ± 1.6

Cells were incubated for 0–72 h without (control) or with 0–100 �M of the com-pound. IC50 are the concentrations that produced 50% inhibition of the cell viability.Results are expressed as a mean ± S.D. (n = 3).

178 Z. Vantová et al. / International Journal of Biological Macromolecules 45 (2009) 174–180

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radical based genotoxic potential of the drug can be modulated bysome free-radical scavengers, e.g. vitamin E or thiols. Our resultsalso suggest that free radicals could contribute to the propyl-AcrDTU cytotoxicity (Fig. 9).

ig. 5. (a) In vitro growth inhibition of HL-60 cells by propyl-AcrDTU. Concentrationxclusion test using trypan blue. Three-day cell growth is shown. (b) In vitro growthange 1.5–12.5 �M. Viability of HL-60/ADR cells was measured by dye exclusion tes

n the strong cytotoxicity of this substance. The growth inhibi-ion activity of propyl-AcrDTU was evaluated as a function ofhe drug concentration, IC50 = 6.7 ± 2.3 �M (for 72 h). The effect ofropyl-AcrDTU on a HL-60/ADR cell growth showed that resistantubline was more sensitive to this compound than the parental line,C50 = 7.5 ± 1.5 �M (for 72 h).

The MTT assay showed that the cytotoxicity of AcrDTUs againsteukemia cells, in contrast to the DNA-binding activity, could not beorrelated with the size of alkyl chains. In accord with our previousndings [17] we assume that geometry of intercalators more thanhe binding affinity can contribute to their antitumor properties.

Changes in the cell morphology of HL-60 and HL-60/ADR cellsnd intracellular accumulation of the drug were examined using auorescent microscopy. 10 �M propyl-AcrDTU was incubated withL-60 and HL-60/ADR cells for 1 and 12 h (Figs. 6 and 7). As shown

n Fig. 6A and 7A, propyl-AcrDTU was internalized already after 1 hncubation with the sensitive and the resistant cells. The treatedL-60 cells had an intact membrane and the condensed greenucleus was clearly visible. If PI was used, slightly orange nucleiL-60 cells were observed, especially after 12 h incubation (Fig. 6Bnd D). Similarly, propyl-AcrDTU was accumulated by HL-60/ADRlready after a short-time incubation but the substance appeared inhe plasmatic membrane of HL-60/ADR cells after 12 h incubationFig. 7B). In spite of intracellular accumulation of propyl-AcrDTUnd its high DNA-binding activity, the substance did not inducentranucleosomal fragmentation of DNA usually observed as a typ-cal “DNA-ladder” (results not shown).

The HL-60 cell line, sensitive to DNA intercalators, is deficient inp53 protein, which plays a central role in the cellular response to

he DNA damage [31]. Indicated high sensitivity of HL-60 leukemiaells to AcrDTU is predominantly due to their DNA-binding activity,ut other modes of action in principle cannot be excluded and havelso to be taken into account, e.g. interactions with DNA- or non-NA-binding proteins and induction of oxidative stress. Oxidativeamage to DNA by metal complexes was confirmed when elec-ron transfer was studied by Núnez et al. [32]. Amsacrine interactsith DNA and topoisomerase II enhancing the formation of a cleav-

ble DNA–enzyme complex, thus leading to apoptosis of cancerells. In addition, participation of this anticancer drug in an oxida-ive metabolism has been suggested to account for its cytotoxicity

32].

Several researchers have pointed to the electron transfer mech-nism as possible factor that could hold the intercalator insidehe DNA and induce the damage of DNA [33,34]. Baguley et al.33] showed that amsacrine acts as an electron-donor in electron-

bstance were in the range 1.5–25 �M. Viability of HL-60 cells was measured by dyetion of HL-60/ADR cells by propyl-AcrDTU. Concentrations of substance were in thetrypan blue. Three-days cell growth is shown.

transfer reactions in DNA, whilst N-[2-(dimethylamino)ethyl]acridin-4-carboxamide (DACA) behaves as an electron-acceptor.Based on a structural analogy to amsacrine we suppose that ourcompounds could also participate in the electron transfer reactionswith DNA. Though acridinium dithioureas can theoretically adopta thione or thiol tautomeric structure [35], the latter can be reli-ably excluded according to an energy calculation [36]. Therefore,a possible factor for electron transfer reactions of our compoundsmight be sought in the interaction of DNA with the thiourea systemsconjugated with the charged acridine �-orbital (Scheme 2).

Amsacrine was proved to induce the oxidative stress in cellsas well as react with glutathione [37], important intracellularantioxidant and the main low-molecular cellular thiol (LMWT). Ifpropyl-AcrDTU, analogously to amsacrine, induced the oxidativestress in the cell, the level of LMWT should change. Therefore wehave estimated the level of LMWT in cells after short- and long-term treatment with propyl-AcrDTU. After short, 1 h, treatment, thelevel of LMWT decreased in both, sensitive and resistant, HL-60 cells(Fig. 8a). The effect was dose-dependent only for the resistant cellline. Both lines, HL-60 and HL-60/ADR, evidenced a regenerationof the LMWT levels within a 24 h incubation period (Fig. 8b). Thelevel of LMWT was completely recovered only when the cell linewas treated with 5 �M propyl-AcrDTU.

The return of the intracellular LMWT level after 24 h resultedprobably from the induction of expression of enzymes catalyzingthe GSH-synthesis [38]. A decrease of propyl-AcrDTU cytotoxic-ity observed upon addition to HL-60 of N-acetylcysteine (NAC), aknown antioxidant and the precursor of the GSH synthesis, is sup-portive for such presumption (Fig. 9).

Blasiak et al. [13] showed that free radicals could be involvedin the formation of DNA lesions induced by amsacrine and a free-

Scheme 2. Scheme is shoving the interaction possibility of free electron pair onnitrogen thiourea moiety with �-orbital of the aromatic ring.

Z. Vantová et al. / International Journal of Biological Macromolecules 45 (2009) 174–180 179

Fig. 6. HL-60 cells treated without (control) and with 10 �M propyl-AcrDTU for 1 h (A and B) and 12 h (C and D). HL-60 cells were stained with PI (B and D) the control wasstained with PI and Hoechst 33342. Images are representative of three independent experiments.

Fig. 7. HL-60/ADR cells treated with 10 �M propyl-AcrDTU. Cells were incubated with the derivative for 1 h (A), and 12 h (B). HL-60/ADR cells incubated without propyl-AcrDTU(control) were stained with PI and Hoechst 33342. Images are representative of three independent experiments.

Fig. 8. (a) LMWT concentration in HL-60 treated with propyl-AcrDTU. (b) LMWT concentration in HL-60/ADR treated with propyl-AcrDTU.

180 Z. Vantová et al. / International Journal of Biolog

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ig. 9. The protective effect of NAC on HL-60 cells treated with propyl-AcrDTU10 �M) and propyl-AcrDTU (10 �M) + NAC (500 �M).

. Conclusion

1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dialkyldithiourea hydrochloriderobes have been proved to possess a strong DNA-binding activityK = 2.9–7.6 × 105 M−1) that decreased with an increasing length ofide alkyl substituents. The MTT assay showed that the cytotoxicityf AcrDTUs against leukemia cells was moderate (IC50 = 4.5–10 �M)nd, in contrast to the DNA-binding activity, was not the functionf the alkyl length. Based on a structural analogy to amsacrine weuppose, that AcrDTUs are engaged also in the electron transfereactions via the interaction of the charged, thiourea substituted,cridine �-orbital with DNA. Depletion of glutathione levelshortly after the AcrDTU administration that have been restoredfter 24 h confirmed oxidative stress associated with the formationf free radicals. Protection of cells has been achieved by theddition of N-acetylcysteine. The sensitivity of resistant cell lineL-60/ADR to alkyl-AcrDTU was noteworthy and presumablyould be associated with the interaction of AcrDTU with MRP1roteins.

The ability of AcrDTU to inhibit the cell proliferation and otherforementioned properties of these drugs including the bindingnto DNA make them promising compounds to induce the leukemiaells apoptosis.

cknowledgments

Financial support for this work from the Slovak Grant AgencyEGA (1/4305/07, 1/0053/08 and 1/0476/08) and the State NMR

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ical Macromolecules 45 (2009) 174–180

Programme (Grant No. 2003SP200280203) is gratefully acknowl-edged.

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