DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives

Spectrochimica Acta Part A 87 (2012) 232–240

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives M.B. Gholivand a,∗ , S. Kashanian a , H. Peyman b a b

Faculty of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and Nanotechnology Research Center (NNRC), Razi University, Kermanshah, Iran Department of Chemistry, Ilam Branch, Islamic Azad University, Ilam, Iran

a r t i c l e

i n f o

Article history: Received 2 September 2011 Received in revised form 1 November 2011 Accepted 13 November 2011 Keywords: Quinizarin Danthron DNA groove binding Cytotoxic activity DNA cleavage

a b s t r a c t The interaction of native calf thymus DNA (CT-DNA) with two anthraquinones including quinizarin (1,4-dihydroxy anthraquinone) and danthron (1,8-dihydroxy anthraquinone) in a mixture of 0.04 M Brittone–Robinson buffer and 50% of ethanol were studied at physiological pH by spectrofluorometric and cyclic voltammetry techniques. The former technique was used to calculate the binding constants of anthraquinones–DNA complexes at different temperatures. Thermodynamic study indicated that the reactions of both anthraquinone–DNA systems are predominantly entropically driven. Furthermore, the binding mechanisms on the reaction of the two anthraquinones with DNA and the effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the experiments indicated that the binding modes of quinizarin and danthron with DNA were evaluated to be groove binding. Moreover, the cytotoxic activity of both compounds against human chronic myelogenous leukemia K562 cell line and DNA cleavage were investigated. The results indicated that these compounds slightly cleavage pUC18 plasmid DNA and showed minor antitumor activity against K562 (human chronic myeloid leukemia) cell line. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Small molecules can react with DNA via covalent or noncovalent interactions, with interest generally focusing on the latter. There are several types of sites in the DNA molecule where such binding can occur: (i) between two base pairs (full intercalation), (ii) in the minor groove, (iii) in the major groove and (iv) on the outside of the helix [1]. Lerman [2,3] was the first to propose that planar organic compounds can bind to DNA by intercalation. Since that groundbreaking work, there has been a significant interest in the study of the binding of small molecules to DNA [4,5]. Anthraquinone dihydroxy derivatives (DHAQs) are interesting from several points of view. They belong to the group of coloring pigments exploited in art and industry since ancient times. More recently they have been shown to possess antimicrobial, cytotoxic, and antiviral properties [6]. Their structural motif is found in a wide group of photosensitive compounds considered for treatment of tumors and viruses (e.g. Daunomycin, Adriamycin, and Hypericin). These properties, together with relatively small size, opens the possibility to enjoy widespread usage in various areas of chemistry, biochemistry and pharmacology [7]. Quinizarin (1,4-dihydroxyanthraquinone; Fig. 1)

∗ Corresponding author at: P.O. Box 67149, Iran. Tel.: +98 831 4274559; fax: +98 831 4274559. E-mail address: [email protected] (M.B. Gholivand). 1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2011.11.045

and danthron (1,8-dihydroxyanthraquinone; Fig. 1) are the simplest molecules showing the chromophore framework peculiar to several compounds of biological and pharmaceutical interest. The former is present in some antitumor drugs as aclacinomycin, emodin, and hypericin and the latter in doxorubicin, daunorubicin, and adriamycin [6,8–11]. Anthraquinone and its derivatives are well-known intercalators [12,13]. They are a frequently found motif in DNA targeting drugs [12,14–18]. Not surprisingly, conjugation of anthraquinone to oligonucleotides has served as a common strategy for the development of high affinity oligonucleotides [19–27]. Furthermore, the low reduction potential of anthraquinone derivatives opens possibilities for charge transport through DNA [28–32] and electrochemical DNA sensing [33–35]. Since DNA is an important cellular receptor, many chemicals through binding to DNA exert their antitumor effects. Therefore, the changing of DNA replication and the inhibiting growth of the tumor cells, which is the basis of designing new and more efficient antitumor drugs and their effectiveness depend on the mode and affinity of the binding [36]. From this point of view, the hydroxy-derivatives of 9,10anthraquinones as intercalator and fluorescent dye are suitable for such an investigation in order to obtain detailed information on the drug/biomolecule interaction. In this work, the binding modes of the interaction of anthraquinones with DNA were investigated by the electrochemistry and fluorescence quenching technique. The results indicated that the binding modes of quinizarin and danthron with DNA were

M.B. Gholivand et al. / Spectrochimica Acta Part A 87 (2012) 232–240

Fig. 1. Schematic structure of quinizarin and danthron.

evaluated to be groove binding. The thermodynamic study also indicated that the reaction of quinizarin and danthron with DNA were predominantly entropically driven. Moreover, the cytotoxic activity of both compounds against human chronic myelogenous leukemia K562 cell line, and DNA cleavage were investigated. 2. Experimental 2.1. Materials The highly polymerized of calf thymus DNA (CT-DNA) was purchased from Sigma Corp and used as received. Quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8dihydroxyanthraquinone) were purchased from Fluka and were used without any purification. Sodium hydroxide, potassium iodide, sodium dodecyl sulfate (SDS), potassium acetate, chloroform, isoamylalcohol, sucrose, agarose, acetic acid, boric acid and orthophosphoric acid were purchased from Merck. RNase was purchased from Roche, Germany. Deionized water was used throughout. Plasmid DNA (pUC18) was extracted from Escherichia coli [37]. K562 (human chronic myeloid leukemia) cell line was kindly provided from Medical Biology Research Center, Kermanshah University of Medical Sciences. 2.2. Methods and instrumentation The stock solution of DNA was prepared in accordance with our previous work [38]. Solutions of DNA gave the ratio of UV absorbance at 260 and 280 nm, A260/A280; of 1.9 indicating that the DNA was sufficiently free of protein. Concentrated stock solutions of DNA were prepared in buffer. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient of 6600 M−1 cm−1 at 260 nm after dilutions. Stock solutions were stored at 4 ◦ C and were used in no more than 4 days. The stock solution of quinizarin and danthron (2.8 mM) were prepared by dissolving appropriate amounts of each compound in small volume ethanol and then diluted with Britton–Robinson (BR) buffer. BR buffer 0.04 M is a mixture of 0.04 M acetic acid, 0.04 M boric acid and 0.04 M of orthophosphoric acid. The pH of the buffer was adjusted to 7.0 with 0.1 M sodium hydroxide. The cyclic voltammetric analyses were performed using an Autolab PGSTAT-30 interfaced to a computer supplied by the general purpose electrochemical system (GPES) software. A conventional three-electrode system comprising a glassy carbon electrode as the working electrode, a saturated Ag/AgCl (inside a Luggin capillary containing 3.0 mol L−1 KCl) as the reference electrode and a platinum wire as the counter electrode were used. Electrochemical experiments were carried out in a 25 mL voltammetric cell at room temperature. The surface of the working electrode was polished using a 0.05 mm alumina prior to each experiment and was rinsed with double distilled water before usage. The supporting electrolyte was 40 mM of BR buffer solution (pH 7.0) which was prepared with double distilled water. Cyclic

233

voltammograms were recorded by progressive addition of DNA solutions from 0 to 2 mM to quinones at a constant concentration (0.5 ␮M) [5,39]. Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200) by progressive addition of DNA solutions from 0 to 0.1 mM to quinones at a constant concentration (50 ␮M), (Ri = [DNA]/[quinones] = 0, 0.3, 0.7, 1, 1.5, and 2) at three temperature levels (298, 306, 318 K). Comparison of the quenching effects of ss-DNA and ds-DNA was performed by adding small aliquots (10 ␮L) of a concentrated ss-DNA or ds-DNA solution respectively to quinones solution at a constant concentration (0.5 ␮M). All solutions were allowed to equilibrate for 5 min before measurements were made [40]. Iodide quenching experiments were conducted by adding stoichiometric small aliquots of potassium iodide stock solution (0.1 M) to quinones (50 ␮M) and quinones–DNA ([quinone] = 50 ␮M, [DNA] = 0.1 mM) solutions. The fluorescence intensity was recorded, and then the quenching constants were calculated [40,41]. Four series of assay solutions were prepared to measure the fluorescence intensity and each series of solutions contained various amounts of CT-DNA and a fixed amount of quinones and NaCl [40]. Reactions of two quinones with pUC18 (0.99 mg mL−1 ) were performed in a TE (Tris–HCl, EDTA) buffer, pH 8.0, and the contents were incubated for 2 h at 37 ◦ C. Concentration dependence studies on CT-DNA cleavage were performed using different ratios of quinones (Ri = [quinones]/[DNA] = 0.00, 0.05, 0.3 and 0.5). Electrophoresis was conducted at 80 V for 1 h in TBE (Tris–HCl, boric acid and EDTA) buffer solution using the 0.8% agarose gel to analyze the CT-DNA reactions. The gel was stained with ethidium bromide and photographed using UV illumination [40]. 2.3. Cell cultures and treatments The human chronic myeloid leukemia-K562 cell line, used for treatment with the quinones, was provided. The K562 cell line was maintained in RPMI 1640, supplemented with 10% fetal bovine serum (FBS), 10 U/mL penicillin, 100 ␮g/mL streptomycin, 0.2 mg mL−1 l-glutamine, 0.002 g/mL NaHCO3 and 0.0011 g/mL sodium pyruvate in an atmosphere of 96% air/5% CO2 , with 96% humidity, at 37 ◦ C. Cells were seeded at a density of 4 × 105 cells/mL, and incubated at 37 ◦ C. After 24 h they were treated with quinones, while in exponential growth phase, for the indicated time periods. Quinones solutions were prepared as a stock immediately before use. The quinones were added to the cell medium where they remained constantly for the indicated time periods [40]. 2.4. Cytotoxicity assay by trypan blue staining A trypan blue (TB) exclusion assay [42] was used to detect the cytotoxicity induced by two quinones. 100 ␮L aliquots of the exponentially growing were incubated with various volumes of the stock solution of quinones that were diluted with cell medium to reach various final concentrations ranging from 0, 1.28, 3.2, 8, 20, 50 ␮M. After 24, 48 and 72 h, 20 ␮L of samples was added to 20 ␮L TB (0.4%) in a test tube. The total viable cell number was determined with a hemocytomer chamber. The cell exposure time to trypan blue did not exceed 5 min, because extensive exposure is possible to cause an increase in the dead cell population (trypan blue positive) due to the trypan blue toxicity. The total number of cells and the number of blue-stained cells (dead cells) were counted on a haemocytometer by observing under a microscope. In order to calculate the concentration of each drug that produces a 50% inhibition of cell growth (IC50), the concentration of viable cells was calculated and results were finally expressed as the % cell viability versus quinones concentration. As the reactivity of trypan blue is based on the fact that this chromophore is negatively charged and does not interact with the cell unless the membrane is

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Qinizarin 2

a

Current A×10-5

1

1

0

b -1

-1

c -2 -1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Potential V

Danthron 12

a

Current A×10-6

8

4

0

-4

b c

-8 -0.6

-0.4

-0.2

0

0.2

0.4

0.6

Potential V Fig. 2. Cyclic voltammograms for quinizarin (A) and danthron (B) in the absent and presence of DNA at different concentrations. [quinone] = 0.5 mM; [DNA] = 0.0–2.0 mM.

damaged, all cells that exclude the dye are viable. The well-known dye penetrates the cellular membrane only in the case of damage of the structural integrity of the cells (necrosis or secondary necrosis), and it enters in the interior and becomes attached to intracellular proteins, giving to the cell a blue pigmentation. As a consequence, dead cells stain blue, while live cells exclude trypan blue. However, it cannot distinguish necrotic versus apoptotic cells [43].

2.5. Cell proliferation The study of the proliferative capacity of the leukemia cells comprises determination of the number of cells that were divided in the culture in the presence/absence of the compounds under study, as well as creation of the growth curves. To achieve this purpose, in the same manner as previously described exponentially growing human leukemia cells were seeded at a density of 4 × 105 cells/mL, in duplicate, and 24 h later they were treated with quinones, at 37 ◦ C in a humidified 5% CO2 atmosphere. After 24, 48 and 72 h, each sample was mixed with TB (0.4%) thoroughly and total cell number was counted on a haemocytometer by observing under a microscope [40].

3. Results 3.1. Cyclic voltammetric study Cyclic voltammetric measurements were carried out for 0.5 ␮M of qunizarin and or danthron in the absence or presence of CT-DNA at a glassy carbon electrode in aqueous media with 0.04 mM of BR buffer solution (pH 7.0) as a supporting electrolyte in the range of −0.8 to 0.8 V versus Ag/AgCl. The results are shown in Fig. 2. The voltammograms show two reduction peaks and one oxidation peak. The peaks current were decreased when CT-DNA was added to the solution of each quinone. 3.2. Fluorescence quenching studies To investigate the interaction mode between the two quinones and CT-DNA, fluorescence titration experiments were performed. Preliminary experiments showed that the two titled quinones emit luminescence in the BR buffer with maximum wavelengths 535 and 560 nm for the quinizarin and 526 and 558 nm for the danthron. When CT-DNA was added to the quinones solution, their fluorescence intensity decreased with increasing the concentration of CT-DNA without any change in the shape and position

M.B. Gholivand et al. / Spectrochimica Acta Part A 87 (2012) 232–240 Table 1 Stern–Volmer quenching constants for the interaction of quinizarin and danthron with DNA at different temperatures.

Quinizarin

Danthron

−1

Temperature (K)

Ksv × 10

298 306 318 298 306 318

1.8257(±0.0010) 1.5033(±0.0005) 1.2918(±0.0007) 1.5389(±0.0010) 1.2098(±0.0004) 1.0407(±0.0005)

L mol

−13

Kq × 10

L mol

−1

1.8257(±0.0010) 1.5033(±0.0005) 1.2918(±0.0007) 1.5389(±0.0010) 1.2098(±0.0004) 1.0407(±0.0005)

−1

s

A

6

298 K

5

306 K

2

R

4

0.98 0.99 0.99 0.98 0.98 0.99

F0/F

Quinone

−5

235

318 K

3 2 1 0 5

0

F0 = 1 + Kq [DNA] = 1 + KSV [DNA] F

Fluorescence Intensity

25

298 K

4

306 K

3

318 K

2 1

A

0 0

5

10

15

20

25

[DNA] × 10-5 M Fig. 4. Stern–Volmer plots of the quenching of fluorescence of quinizarin (A) and danthron (B) with DNA at different temperatures (298, 306 and 318 K).

3.3. The composition of the binary complex and thermodynamic studies If the binding reaction between the CT-DNA with each quinone happens with a similar and independent binding sites (n) in the CTDNA, and if the binding capability of CT-DNA at each binding site is equal, then the composition of the binary complex can be deduced from the following formula [45]: DNA + nL → DNALn

250 200

log

150 100 50 0 500

520

540 560 Wavelenghth (nm)

580

600

B

40 Fluorescence Intensity

20

B

5

(1)

where F0 and F are the fluorescence intensities in the absence and presence of CT-DNA, respectively, Kq is the bimolecular quenching constant,  is the lifetime of the fluorophore and KSV is the Stern–Volmer quenching constant which can be considered as a measure for efficiency of fluorescence quenching by CT-DNA. It is well known that there are two quenching processes: static and dynamic quenching [44]. They can be distinguished by their differing dependence on temperature. Therefore, the effect of temperature was investigated and the quenching constants of the quinizarin and danthron in the presence of different amounts of CTDNA at different temperatures (298, 306, and 318 K) were obtained. These experiments were carried out three times and the results are shown in Fig. 4 and Table 1. These results show that, the KSV decreases by temperature rising.

300

15

[DNA] × 10-5 M

F0/F

of the fluorescence peaks. The results are shown in Fig. 3, which confirm the interaction between the quinones and CT-DNA. Such a decrease in the intensity is called fluorescence quenching. The Stern–Volmer KSV is used to evaluate the fluorescence quenching efficiency. According to the classical Stern–Volmer equation (Eq. (1)):

10

30 20 10 0 450

500

550 Wavelenghth (nm)

600

650

Fig. 3. Emission spectra of the quinizarin (A) and danthron (B) in the absent and presence of the increasing DNA concentrations in BR buffer (pH 7.2), [quinine] = 50 ␮M, [DNA] = 0–0.1 mM.

(F0 − F) = log Kf + n log[DNA] F

(2)

where CT-DNA is the quencher, and L is the quinone (qunizarin and or danthron) with a fluorophore, DNALn is the complex, whose resultant formation constant is Kf . F0 and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of CT-DNA, respectively. A plot log(F0 − F)/F versus log[DNA] will give a straight line with a slope of n and a y-axis intercept log Kf . Therefore, the fluorescence titration data were fitted using Eq. (2) and the results were used to determine the binding constant (Kf ) and the binding stoichiometry (n) for the complex formation of each quinone with CT-DNA. Evaluation of the formation constants for the quinizarin–DNA and danthron–DNA at three different temperatures (298, 306, and 318 K) were carried out and the results are presented in Table 2 (n = 3). In order to have a better understanding of the thermodynamic of the interaction between each quinone and CT-DNA, contributions of the enthalpy and entropy should be determined in the reaction. The thermodynamic parameters of quinones–DNA formation were calculated via Van’t Hoff equation (Eq. (3)) [46]: Ln Kf =

−H S + RT R

(3)

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Table 2 Formation constant and N for quinizarin and danthron with DNA at different temperatures.

Quinizarin

Danthron

Temperature (K)

Kf × 105

n

R2

298 306 318 298 306 318

0.6792(±0.0005) 1.0814(±0.0002) 1.3772(±0.0010) 0.1671(±0.0009) 0.2432(±0.0002) 0.2582(±0.0007)

1.208 1.234 1.308 1.012 1.035 1.081

0.98 0.99 0.99 0.99 0.99 0.98

2

F0/F

Quinone

A

2.5

1.5

[NaCl]=0 M [NaCl]=0.2 M

1

[NaCl]=0.4 M

0.5

[NaCl]=0.6 M

0 0

10

15

20

25

[DNA] ×10-5 M

13

Quinizarin

B

1.5

Danthron

12 11

F0/F

lnKf

5

10

[NaCl]=0 M

1

[NaCl]=0.2 M [NaCl]=0.4 M

9

[NaCl]=0.6 M 0.5

8 0.0031

0.0032

0.0033

0

0.0034

5

1/T

by plotting ln Kf versus 1/T (Fig. 5), H and S were determined. With knowing these two values, G was calculated from the following standard equation (Eq. (4)) [47]: (4)

The results for the quinizarin and danthron are shown in Table 3.

3.4. The effect of ionic strength on the fluorescence quenching The study of the ionic strength effect is an efficient method to distinguish the binding mode between molecules and CT-DNA. Increasing the cation concentration will increase the combination probability between cation and CT-DNA phosphate backbone. Due to a competition for phosphate anion, the addition of cations will weaken the surface-binding mode of interaction between CT-DNA and molecules [48]. In order to study the effect of ionic strength on the fluorescence quenching, different concentrations of NaCl were used to adjust the ionic strength of the quinones solutions at room temperature (298 K). The results showed that the fluorescence intensity ratio had no significant change when the concentration of NaCl was changed in the range of 0–0.6 M (Fig. 6). Such data indicated that the interaction between the quinones and CT-DNA were not surface-binding mode.

Table 3 Thermodynamic parameters for binding of quinizarin and danthrone with DNA. Quinizarin H (kJ mol−1 ) S (kJ mol−1 K−1 ) G (kJ mol−1 ) 298 K 306 K 318 K

27.095(±0.0008) 0.183(±0.0010) −27.708(±0.002) −29.179(±0.004) −31.386(±0.006)

15

20

25

Fig. 6. Effect of NaCl on the fluorescence intensity of quinizarin (A) and danthron (B), [quinine] = 50 ␮M, [DNA] = 0.1 mM.

Fig. 5. Van’t Hoff plot for quinizarin–DNA and danthron–DNA.

G = H − TS

10

[DNA] ×10-5 M

Danthron 16.287(±0.0003) 0.136(±0.0007) −24.270(±0.0010) −25.359(±0.0005) −26.992(±0.0008)

3.5. Iodide quenching studies Iodide ions are dynamic, or collision fluorescence quencher for the small fluorescent molecule. They can effectively quench the fluorescence of a small molecule. The binding mode of the small molecule with CT-DNA can be deduced from the variation of the fluorescence in the absence and the presence of CT-DNA. In order to study the quenching activity of iodide ion on the fluorescence of the quinones in the absence and presence of CT-DNA, the effect of the presence of I− were investigated. The results are presented in Fig. 7. In aqueous solutions, iodide quenched the fluorescence of both quinones effectively. The KSV values between the quinizarin and danthron and iodide anions in the presence of CT-DNA decreased slightly, which indicated that quinones could be partly protected. Therefore, the binding mode of the quinones with CT-DNA were found to be groove binding. All of experiments were carried out at room temperature (298 K).

3.6. Comparison of the effects of ssDNA and dsDNA The ssDNA solution was prepared by heating native dsDNA solution in a boiling water bath for 8 min and then rapidly cooling it in an ice water bath. When it gradually up to 298 K, dsDNA turns to be ssDNA [38]. As shown in Fig. 8, both dsDNA and ssDNA can quench the fluorescence of both quinines at room temperature (298 K). These results indicated that the quinones react with the base pairs of duplex DNA in groove binding. Furthermore, ssDNA has a stronger effect. If the binding mode was intercalation, the quenching effect of ssDNA would be weaker than that of dsDNA, for the release of the double strands of CT-DNA. In addition, the maximum emission wavelength of the quinones did not change with adding the calf thymus DNA, which also indicated that the interaction is a groove binding, not an intercalative binding mode [50].

M.B. Gholivand et al. / Spectrochimica Acta Part A 87 (2012) 232–240

A

3

F0/F

Table 4 IC50 values for quinizarin and danthron.

KI-Quinizarin

2.5

237

KI-Quinizarin-DNA

2 1.5

Quinone

24 h

8h

72 h

Quinizarin Danthron

47 >50

19 46

7 12

1 0.5 0 0

0.001

0.002

0.003

0.004

0.005

[KI] M

B

3

KI-Danthron

F0/F

2.5

KI-Danthron-DNA

2 1.5 1 0.5 0 0

0.001

0.002

0.003

0.004

0.005

[KI] M Fig. 7. Fluorescence quenching plot of quinizarin (A) and danthron (B) by KI in the absence and presence of DNA, [quinine] = 50 ␮M, [DNA] = 0.1 mM.

3.7. Cleavage of pUC18 DNA by two quinines For cleavage studies, first, plasmid DNA was isolated as follows: pure culture of E. coli containing plasmid DNA was incubated at 37 ◦ C for 12 h in a nutrient broth containing ampicillin. The broth

A

2

Quinizarin+ds Quinizarin+ss

F0/F

1.5

1

3.8. Effect on leukemia cells: cell viability, cell proliferation

0.5

0 0

5

10

15

[DNA] ×10-5 M

B

2.5

Danthron+ds

2

F0/F

was harvested after 12 h and centrifuged at 4000 rpm for 5 min and decanted and the entire medium was drained. The pellet was resuspended in 1 mL of SET (sucrose, EDTA, Tris–HCl) buffer by vortexing. Appropriate concentrations of NaOH and sodium dodecyl sulfate buffer were added to make a final concentration of 10% (v/v), mixed well (without vortexing), and then incubated on ice for 5 min. Then, 1.5 mL of potassium acetate solution (5 M) was added and mixed immediately. Following 5 min of incubation on ice, 4.5 mL chloroform:isoamylalcohol (24:1) was added as a extraction mixture and the resulting solution was centrifuged for 10 min at 8000 rpm at 48 ◦ C. The supernatant was collected in a new tube, 10 mL of pure EtOH (kept at room temperature (RT) was added, and centrifuged for 5 min at 10,000 rpm. The pellet was washed with 5 mL of 70% EtOH (kept at RT), centrifuged for 5 min at 5000 rpm, and dried. The pellet was dissolved in TE (Tris–HCl, EDTA) buffer (nearly 200 ␮L), 50 ␮L RNase A (1 mg mL−1 in TE) was added, and then incubated at 37 ◦ C for 1–2 h. The homogeneity of plasmid DNA was confirmed by gel electrophoresis. DNA was stored at −20 ◦ C until used [37]. Reactions of the two the quinones with pUC18 (0.99 mg mL−1 ) were performed in TE buffer (pH 8). The contents were incubated for 2 h at 37 ◦ C. Concentration dependence studies were performed using the different quinone ratios (Ri = [quinone]/[DNA] = 0.00, 0.05, 0.30, and 0.50). Electrophoresis was conducted at 80 V for 1 h in TBE buffer solution using 0.8% agarose gel to analyze the DNA reactions. The gel was stained with ethidium bromide and photographed using UV illumination. Gel electrophoresis pattern for the cleavage of super coiled pUC18 DNA is shown in Fig. 9. Lane a is the control DNA. In the presence of the quinones, DNA cleavage was observed. It could be seen that DNA cleavage depends strongly on the concentration of the quinones. At the low quinones concentration (Lanes b and e), the super coiled DNA (Form I) can be converted into nicked form (II). When the concentration ratio of quinines to DNA reached 0.5, remarkable DNA cleavage was observed.

Danthron+ss

K562 cells were exposed to increasing doses of the both quinones (0.00, 1.28, 3.20, 8.00, 20.00 and 50 ␮M) and the resulting viability and proliferation were measured by the TB assay at 24, 48 and 72 h. The results are shown in Figs. 10 and 11 respectively. As it can be seen from Figs. 10 and 11, the decrease in the percent of cell viability and cell proliferation are dose- and time dependent. In other words, the percents of cell viability and cell proliferation were decreased with increasing the concentration of each quinone at each time period. IC50 values for the quinizarin and danthron are reported in Table 4. It was observed that IC50 values were decreased with time rising, too.

1.5 1 0.5 0 0

2

4

6

8

10

12

14

[DNA] ×10-5 M Fig. 8. Effect of dsDNA and ssDNA on the quinizarin (A) and danthron (B) fluorescence intensity, [quinine] = 50 ␮M.

Fig. 9. Cleavage of pUC18 DNA in the presence of increasing amounts of quinine. Line a is DNA alone, lines b–d are DNA in present of quinizarin (Ri = [quinizarin]/[DNA] = 0.05, 0.3 and 0.5) and lines e–g are DNA in present of danthron (Ri = [danthron]/[DNA] = 0.05, 0.3 and 0.5).

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A

Cell Viability%

100

complement to the optical techniques. In the present study this technique was employed to understand the nature of CT-DNA binding of the quinones, and the results are shown in Fig. 2. As shown in Fig. 2, the quinizarin and danthron present a well-defined oxidation peaks at about 0.5 and 0.35 V, respectively. Each of these anodic peaks can be attributed to the oxidation of the phenolic hydroxyl group presented in the dye marker to its quinone derivative after two electrons’ transfer [49]. On the reversed scan, the occurrence of two cathodic peaks, labeled as b and c were observed, respectively, due to the reduction of species generated after secondary reaction involving the oxidized form [49]. In the presence of CT-DNA, the cyclic voltammograms of the quinones exhibited small shifts in the anodic and cathodic peak potentials followed by decrease in both peak currents, indicating the interaction existing between the quinones and CT-DNA. The decreases in peak currents can be explained in terms of the slow diffusion of the quinones bound to the large CT-DNA molecule [50].

24h 48h

80

72h

60 40 20 0 0

10

20

30

40

50

[Quinizarin] μM

B

120

24h 48h

Cell Viability%

100

72h

80 60

4.2. Fluorescence quenching studies 40 20 0 10

0

20

30

40

50

[Danthron] μM Fig. 10. Plot of cell viability% of K562 versus increasing of quinizarin and danthron concentration (0.0, 1.28, 3.2, 8.0, 20, 50 ␮M) displayed after 24 h, 48 h and 72 h.

4. Discussion 4.1. Cyclic voltammetry The application of electrochemical technique to study the interaction of electroactive compounds with CT-DNA provides a useful

A

120

24h

Proliferation%

100

48h

80

72h

60 40 20 0 0

10

20

30

40

50

[Quinizarin] μM

B

Proliferation%

120

24h

100

48h

80

72h

60 40 20 0 0

10

20 30 [Danthron] μM

40

50

Fig. 11. Plot of proliferation% of K562 versus increasing of quinizarin and danthron concentration (0.0, 1.28, 3.2, 8.0, 20, 50 ␮M) displayed after 24 h, 48 h and 72 h.

The influence of quenching efficiency of CT-DNA concentrations on the quinones emission were investigated. As seen in Fig. 3, CT-DNA quenches the quinones emission intensity. Fluorescence quenching can occur by dynamic quenching (collisional quenching), static quenching, or by combined dynamic and static quenching mechanisms. Fluorescence quenching data can be described by the Stern–Volmer equation. A Stern–Volmer plot can be linear or non-linear (upward or downward curving) depending on the quenching mechanism. Dynamic quenching or collisional quenching requires contact between the excited lumophore and the quenching species (quencher). The rate of quenching is diffusion controlled and depends on temperature and viscosity of the solution. The probability of collision between the analyte and quencher is significant during the lifetime of the excited species if the quencher concentration to be enough high. As mentioned above the other form of quenching is static quenching in which the quencher and the fluorophore in ground state form a stable complex. Fluorescence is only observed from the unbound fluorophore. The lifetime is not affected in this case; measurement of the lifetime provide a means of distinguishing between dynamic and static quenching [51]. Dynamic and static quenching can also be distinguished by their differing dependence on temperature [52] (Fig. 3a). Dynamic quenching depends upon diffusion. Since higher temperatures lead to larger diffusion coefficients, the KSV can be increased by rising the temperature. In contrast, increased temperature is likely the result of decrease in complexes stability, and thus lower values of the static quenching constants were resulted. By using Eq. (1), the KSV of both quinones in the presence of CTDNA at different temperatures (298, 306, and 318 K) were obtained. The obtained results (Fig. 4 and Table 1) show that the probable quenching mechanism of both quinones fluorescence by CT-DNA are static quenching procedures, because the KSV were decreased by temperature rising. As can be seen from Table 1 the KSV values of the quinizarin are bigger than those obtained for danthron at all tested temperature. The small values of KSV for the quinizarin with respect to the danthron can be related to the higher affinity of the former for CT-DNA binding. According to literature [2] for  ≈ 10−8 s, the bimolecular quenching constants (Kq ) were calculated and the results are summarized in Table 1. The Kq values are larger than the limiting diffusion rate constant of biomolecule (2.00 × 1010 ), which indicates the static quenching occurred in the quinones quenching by CT-DNA [2,53]. Similar to other techniques [38], this technique also confirms that, the intensity of interaction for the quinizarin is stronger than the danthron. In the emission spectra of both quinones, with increasing CT-DNA concentrations the emission intensities were decreased. The quenching of the

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luminescence by CT-DNA is consistent with a photoelectron transfer from the guanine base of CT-DNA to the excited state of the quinones [54–57]. It was found that the interaction between groove binders and ds-DNA causes a strong fluorescence quenching [58]. This decrease in emission intensity of the quinones also agrees with findings obtained with other groove binders [59]. 4.3. Thermodynamic studies The binding constants of the two quinones with CT-DNA were determined at 298, 306, and 318 K. It is seen from Table 2 that the binding constants of the two quinones increase along with temperature. These results indicate that the binding action of the quinones and CT-DNA is endothermal reaction. There are several acting forces between small molecular and biomacromolecule, such as hydrogen bond, van der Waals, electrostatic force, hydrophobic force. Evaluation of the formation constants for the quinones at different temperatures allows thermodynamic parameters of quinones–DNA formation to be determined via Van’t Hoff equation (Eq. (3)). The results are presented in Table 3. It can be seen from the results obtained, that for the binding systems of the quinizarin and danthron to CT-DNA, H > 0, binding action is endothermal. When H < 0 or H ≈ 0, S > 0, the mainly acting force is electrostatic force; when H < 0, S < 0, the mainly acting force is van der Waals or hydrogen bond and when H > 0, S > 0, the mainly force is hydrophobic [60]. For all the binding system of the quinizarin and danthron with CT-DNA, H > 0, S > 0. Therefore, the hydrophobic forces are the main acting force in the binding of the two quinines and CT-DNA. The more negative values of G for the quinizarin with respect to the danthron can be related to the higher affinity of the former for CT-DNA binding. The positive H and S values of the quinones–DNA (Table 4) indicate that the quinones–DNA binding is entropy-favored but enthalpy-disfavored. The favorable binding entropy mainly arises from hydration changes. In contrast to a large and positive change in hydrational entropy, the bindinginduced change in configurational entropy is insignificant [61]. The free energy changes (G) for these interactions are negatively large due to their strong association (Table 3). As stated above the reaction proceeds with a favorable change in enthalpy. It can be deduced that the positive value of H in the interaction signifies the contribution of the positive S results in a more negative G and the favoring binding process. It appears that the major contributing factor in the quinones–DNA complex stability is entropic in origin. Consequently, the release of water molecules or counterions results in positive enthalpy and entropy values in mentioned interactions [62].

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Line 2 was the fluorescence quenching of I− on the 50 ␮M quinizarin and or danthron in the presence of 0.1 mM CT-DNA. The fluorescence quenching efficiency of I− on both quinones in the presence of CT-DNA is smaller than that obtained in the absence of CT-DNA. The quenching constants (KSV ) are calculated from the Stern–Volmer equation. KSV of the free quinizarin and or danthron by I− ion are 313 and 357 M−1 respectively and in the presence of CT-DNA, KSV are 289 and 326 M−1 respectively (shown in Fig. 7). It is apparent that a very little change in iodide quenching interactions can be observed when the quinones are bound to CT-DNA, which indicated that the bound quinone molecules have not intercalated into the base pairs of CT-DNA. If the interaction between each quinone and CT-DNA is intercalative binding, the quinizarin and or danthron molecules would be embedded in the CT-DNA double strand and be well protected by the CT-DNA double strand, with the result that the fluorescence of each quinone could not be quenched by I− . The fluorescence spectrometry indicated that intercalative binding did not exist. If the hydrophobic interaction (concluded from the effect of ionic strength on the quenching of quinones by CTDNA and thermodynamic study) was the only mode, the quinones would be all on the outside of the CT-DNA and they would not be protected by CT-DNA. As a result, the quenching effect of I− on each quinone in quinone–DNA system was the same as that in the free quinone system. Therefore, it could be concluded that groove binding surely coexisted with the hydrophobic interaction because each quinone would be protected partly by the double strand structure of CT-DNA in groove binding. Iodide quenching results provide direct evidence for the groove binding of each quinone with CT-DNA [63].

4.6. Comparison of the effects of ss-DNA with ds-DNA Fig. 8 shows the interactions of the native DNA and the denatured DNA with the quinones. As can be seen, the native and denatured DNA could quench the fluorescence intensity of both quinone systems. On the other hand, the results of interaction between the quinones with ds- and ss-DNA (Fig. 8), suggest that, both quinones react with the base pairs of duplex DNA in grooves through the hydrophobic force. If the binding mode was intercalation, the quenching effect of ss-DNA would be weaker than that of ds-DNA. Furthermore, the maximum emission wavelengths of two quinones do not change by adding calf thymus DNA, whereas the shift in maximum emission wavelengths is associated with the intercalative mode. This finding also suggests that the interaction is a groove binding rather than to be an intercalative binding mode [51].

4.4. Effect of the ionic strength on the fluorescence properties In order to prove whether each quinone provides an electrostatic binding or other kinds of binding with CT-DNA, the effect of different concentrations of NaCl were investigated. It is apparent that intercalative binding and groove binding are related to the groove in the CT-DNA double helix, but the electrostatic binding can take place out of the groove. The results showed that the fluorescence intensity ratio had no significant change when the concentration of NaCl was within 0–0.6 M range (Fig. 6). It was concluded that, NaCl has no effect on CT-DNA binding with quinones. Therefore, the results indicated that the quinones do not give an electrostatic or outside binding with CT-DNA. 4.5. Iodide quenching studies The experimental data are plotted in Fig. 7 according to the Stern–Volmer equation. Line 1 was the fluorescence quenching of I− on the 50 ␮M quinizarin and or danthron in the absence of CT-DNA.

4.7. Cleavage of pUC18 DNA The ability of each quinone to perform DNA cleavage is generally monitored by agarose gel electrophoresis. Generally on electrophoresis, a relatively fast migration of intact super coiled form (Form I) is observed. On the other hand, the cleavage of both strands, generates a slower moving open circular form (Form II) and linear form (Form III) with intermediate mobility. Fig. 9 shows the electrophoretic pattern of plasmid DNA treated with varying concentrations of the quinizarin and danthron. Both tested quinones showed remarkable cleavage. As shown in Fig. 9, with increasing concentration of the quinizarin and danthron, the amount of Form I of pUC18 plasmid DNA was diminished gradually, whereas Form II increased. Also, under comparable conditions, the quinizarin exhibits a higher DNA-nicking efficiency than the danthron. This finding confirms that the insertion of the quinizarin between DNA bases is more than the danthron [64].

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4.8. Effect on leukemia cells: cell viability, cell proliferation It was shown in Figs. 10 and 11 that in each time period with increasing concentration of the quinones, the percentage of cell viability and cell proliferation were decreased. In addition, the value of these decreases was increased with time rising. These means, the cytotoxicity effect of the quinones increases in a dose- and timedependent manner. Also, the IC50 values for the quinizarin and danthron (Table 4) were decreased with time rising. Along with the other findings, here, it was observed that the cytotoxicity effect of the qinizarin is more considerable than that of the danthron. 5. Conclusions Quinizarin and danthron exhibit a high binding affinity for CTDNA. Different instrumental methods were used to investigate the interaction mechanism. The following results support that the mentioned quinones can bind to CT-DNA via groove binding. 1. In voltammetric studies, in the presence of CT-DNA, the cyclic voltammograms of the quinones exhibited small shifts in the anodic and cathodic peak potentials followed by decrease in both peak currents, indicating the interaction existing between the quinones and CT-DNA. 2. The study of temperature effect on the fluorescence quenching of the quinones by CT-DNA show that the probable quenching mechanisms are static quenching procedure, because the KSV was decreased by temperature rising for both cases. 3. Thermodynamic studies indicate that the binding action of the quinones and CT-DNA are endothermal reaction. Also, for binding of the quinizarin and danthron with CT-DNA, H > 0, S > 0, which indicate the hydrophobic forces are the main acting force in the binding of both quinones and CT-DNA. 4. The study of the effect of the ionic strength on the fluorescence properties shows that the fluorescence intensity ratio has no significant change when the concentration of NaCl was increased. Therefore, quinones do not give an electrostatic or outside binding with CT-DNA. 5. Iodide quenching studies show groove binding surely coexisted with the hydrophobic interaction because each quinone would be protected partly by the double strand structure of CT-DNA in groove binding. Iodide quenching results provide direct evidence for the groove binding of each quinone with CT-DNA. 6. ssDNA and dsDNA can quench the fluorescence of quinones, and ssDNA has a stronger effect, which suggests that quinones react with the base pairs of duplex DNA in a groove through hydrogen binding and van der Waals force. 7. The ability of each quinone to perform DNA cleavage was studied. It was indicated quinizarin exhibits a higher DNA-nicking efficiency than danthron. 8. The percentage of cell viability and cell proliferation were decreased in each time period with increasing concentration of quinones. In addition, cytotoxicity effect of quinones increases in a dose- and time-dependent manner. Also, IC50 values for quinizarin and danthron were decreased with time rising. References [1] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 111 (1989) 3051–3058. [2] L. Lerman, J. Mol. Biol. 3 (1961) 18–30. [3] L.S. Lerman, Proc. Natl. Acad. Sci. U.S.A. 49 (1963) 94–102. [4] I. Fukuda, A. Kaneko, S. Nishiumi, M. Kawase, R. Nishikiori, N. Fujitake, H. Ashida, J. Biosci. Bioeng. 107 (2009) 296–300. [5] Y. Ni, D. Lin, S. Kokat, Anal. Biochem. 352 (2006) 231–242. [6] W.A. Remers, The Chemistry of Antitumour Antibiotics, Wiley, New York, 1981. [7] M. Shamsipur, A. Siroueinejad, B. Hemmateenejad, A. Abaspour, H. Sharghi, K. Alizadeh, J. Electroanal. Chem. 600 (2007) 345–358.

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