Chemico-Biological Interactions 233 (2015) 65–70
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Probing the interaction of anthraquinone with DNA by spectroscopy, molecular modeling and cancer cell imaging technique Lei Yang a,b, Zheng Fu a, Xiaoqing Niu a, Guisheng Zhang a, Fengling Cui a,⇑, Chunwu Zhou b,⇑ a b
School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang 453007, China Cancer Institute & Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100021, China
a r t i c l e
i n f o
Article history: Received 30 December 2014 Received in revised form 16 March 2015 Accepted 23 March 2015 Available online 31 March 2015 Keywords: AODGlc DNA Melting Intercalation Molecular modeling Cell imaging
a b s t r a c t A new anthraquinone derivative, (E)-2-(1-(4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2yloxyimino)ethyl)-1,4-dihydroxyanthracene-9,10-dione (AODGlc), was synthesized and its binding properties towards DNA were explored under physiological conditions by fluorescence spectroscopy, DNA melting as well as docking techniques. The experimental results revealed that AODGlc could bind to calf thymus DNA (ctDNA) through intercalation between DNA base pairs. The values of thermodynamic parameters at different temperatures including DG, DH, and DS and the molecular modeling study implied that hydrophobic interactions and hydrogen bonds were the main interactions in the AODGlc– ctDNA system. Cervical cancer cells (HepG2 cells) were used in cell viability assay and cell imaging experiment. AODGlc could interact with HepG2 cells and kill HepG2 cells under high concentration with nice curative effect, indicating its potential bioapplication in the future. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In recent years, considerable attention has been paid to the study of interactions between small molecules and deoxyribonucleic acids (DNA) [1,2]. It is well known that some small molecules can not only be used as biological probes but also as anticancer drugs targeting DNA molecules [3–5]. Many anticancer drugs are known to interact with DNA to exert their biological activities by interacting with DNA through non-covalent bond thus changing the molecular configuration of DNA and damage their template effects [6,7]. As the development of anticancer drugs is slow and costly, one approach to accelerate the availability of new drugs is to reposition drugs approved for other indications as anticancer agents [8]. Although the anthracyclines are widely used in cancer therapy [9,10], there are two major limitations in their clinical use: cardiotoxicity and drug resistance [11,12]. To improve the drug resistance, researchers are trying to synthesize new anticancer drugs through modifying existing drugs with different functional groups. On account of this, AODGlc (Fig. 1) was synthesized via changing glycosyl on anthracyclines. This study was to be aimed at investigating the binding mechanism of AODGlc with ctDNA by spectroscopic methods and DNA ⇑ Corresponding authors. Tel./fax: +86 373 3326336 (F. Cui), +86 010 87788553 (C. Zhou). E-mail addresses: [email protected] (F. Cui), [email protected]. com (C. Zhou). http://dx.doi.org/10.1016/j.cbi.2015.03.026 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
melting techniques, as well as molecular modeling. Besides, fluorescence imaging technique is employed to probing the efficacy of AODGlc on the cervical cancer cells. This work will contribute to elucidating the binding mechanism of anthraquinones with DNA and designing new and efficient drug molecules.
2. Materials and methods 2.1. Materials Calf thymus DNA was obtained from Sigma Chemical Co. (St. Louis, MO). The ratio of the UV absorbance at 260/280 nm was larger than 1.8, which indicated that ctDNA was sufficiently free from any contamination [13]. AODGlc was synthesized and characterized by NMR. 1H NMR (400 MHz, DMSO-d6) (ppm): 13.26 (1H, s), 12.56 (1H, s), 8.27 (2H, s), 8.00 (2H, s), 7.35 (1H, s), 5.58 (1H, s), 4.95 (2H, dd, J1 = 4.8, J2 = 5.6), 4.50 (1H, t, J = 6.0), 3.53–3.71 (3H, m), 3.14–3.20 (2H, m), 2.11–2.18 (1H, m), 1.59–1.66 (1H, m), 1.20 (2H, d), 0.76–0.85 (1H, m), 13C NMR (100 MHz, DMSO-d6) (ppm): 187.5, 186.7, 164.6, 156.0, 155.4, 155.3, 137.0, 135.7, 135.6, 133.3, 128.2, 127.2, 127.1, 113.7, 107.0, 74.6, 71.7, 68.4, 64.2, 67.4, 15.5. The concentration of DNA stock solution was determined according to the absorbance at 260 nm by using the extinction coefficient of 6600 L mol1 cm1 [14]. Other chemicals and reagents were of analytical grade and used without further purification.
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2.4. Iodide quenching experiment Iodide quenching experiments were carried out by adding different proportion iodide stock solutions to the AODGlc and the AODGlc–ctDNA mixture. 2.5. Melting studies Melting temperature of ctDNA was determined in the absence and presence of complexes by monitoring the absorption of DNA at 260 nm with temperature ranging from 30 to 100 °C, using TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China). 2.6. Molecular modeling
Fig. 1. Molecular structure of AODGlc.
2.2. Fluorescence measurements Fluorescence measurements were carried out with a Varian Cary Eclipse spectrofluorimeter. Different concentrations of ctDNA were added to the fixed complex solutions and the fluorescence spectra were simultaneously recorded from 520 to 750 nm with an excitation wavelength of 254 nm. The quenching type was determined by the Stern–Volmer quenching method. The plots from the fluorescence titration data at three different temperatures were investigated according to the Stern–Volmer equation [15,16]:
I0 ¼ 1 þ K q s0 ½Q ¼ 1 þ K SV ½Q I
ð1Þ
Here, I0 and I are the fluorescence intensities in the absence and presence of the quencher, respectively. [Q] is the concentration of quencher, s0 is the average lifetime of the fluorescence molecule in the absence of quenching reagent and its value is about 108 s [17], Kq is the quenching rate constant of biomacromolecule. KSV is the Stern–Volmer dynamic quenching constant. The binding constant K of complexes to ctDNA at various temperature was obtained using the following equation [18]:
log
I0 I ¼ log K þ n log½Q I
ð2Þ
Here I0 and I are the fluorescence intensities of the fluorophore in the absence and in the presence of different concentrations of [Q], respectively. The thermodynamic parameters, enthalpy change (DH), entropy change (DS) and free energy change (DG) are very important for confirming binding modes. These parameters can be calculated as van’t Hoff equation [19]:
log K ¼
DH DS þ 2:303 RT 2:303 R
DG ¼ DH T DS ¼ RT ln K
ð3Þ ð4Þ
The crystal structure of ctDNA used for docking was extracted from the Protein Data Bank [20] with identifier 453D. The structure of AODGlc generated by Chemdraw was energy-minimized with the help of the Tripos force field using Gasteiger–Marsili charges in the Sybyl 6.9 suite. Before docking run, the macromolecule file was prepared for docking by adding polar hydrogen atoms and Gasteiger charges, and a maximum of 10 conformers was considered for the ligand. During the docking process, rotatable bonds in the ligand were assigned with FlexX. The radius around the solvent molecules was set to 12 Å. PyMol [21] was used to visualize the docked conformations and calculate the distances between possible hydrogen bonding partners. 2.7. Cell culture, cell viability assay and cell imaging The cytotoxicity experiment was performed as literatures [22,23]. Briefly, HepG2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U mL1 penicillin, and 100 U mL1 of streptomycin. Cell culture was maintained at 37 °C in a humidified condition of 95% air and 5% CO2 in culture medium. The cytotoxicity of AODGlc was tested by CCK-8 cell viability assay. Specifically, HepG2 cells were seeded in 96-well plates at a density of 5 104 cells mL1. After 24 h attachment, the cells were incubated with 0, 2.436 105, 4.872 106, 2.436 106, 9.744 107, 1.949 107 mol L1 AODGlc for 48 h. Then, AODGlc was removed and 10 lL of CCK-8 dye and 100 lL of DMEM cell culture media was added to each well and incubated for 1 h at 37 °C. The 96-well plates were then analyzed with a microplate reader (VictorI, Perkin-Elmer) at the wavelength of 450 nm, with the reference wavelength at 620 nm. Three replicate wells were used for each control and test concentrations per microplate, and results are presented as mean ± SD. HepG2 cells were seeded in confocal 35 mm clear cover-glass petri-dishes before imaging. After cell attachment, AODGlc was added to each dish at the concentration of AODGlc diluting 100 times. After 3 h exposure, AODGlc was removed and cells were subsequently washed by PBS for 3 times. Cell images were taken with a Laser Scanning Confocal Microscope (LCSM) Zesis 710 3channels (Zesis, Germany) with the excitation wavelength of 458 nm.
where K is the binding constant at the corresponding temperature and R is gas constant.
3. Results and discussion
2.3. Effect of ionic strength
3.1. Fluorescence studies
The experiments on salt effect were conducted by adding different aliquots (0–0.25 M) of the NaCl stock solutions to the complexes-ctDNA mixture.
Fluorescence technique is a powerful tool in the study of the interactions [24], provided that one of the interacting species exhibits an intrinsic fluorescence. Fig. 2 displays the variation
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Fig. 2. The influence of ctDNA concentration on the fluorescence emission spectra of AODGlc with the excitation of 254 nm. 1–7: CctDNA = 0, 1.52, 3.04, 4.56, 6.08, 7.60 and 9.12 105 mol L1; CAODGlc = 6.10 106 mol L1.
Fig. 3. Effect of ionic strength on the fluorescence intensity of AODGlc in the absence and presence of ctDNA. CAODGlc = 2.44 106 mol L1, CctDNA = 0.76 105 mol L1.
in the emission spectra of the AODGlc upon addition of increasing concentrations of ctDNA. The fluorescence of the AODGlc was efficiently quenched by ctDNA, revealing that the interaction between AODGlc and ctDNA occurred. Fluorescence quenching could proceed via different mechanism, usually classified as dynamic quenching and static quenching [25]. For the dynamic quenching, the quenching constant values will increase with increasing temperature, but the reverse effect would be observed for static quenching [26]. The binding constant (K) and the binding stoichiometry (n) for the formation of ctDNA–AODGlc were summarized in Table 1. The results indicated that the probable quenching mechanism of complexes by ctDNA involved static quenching, because KSV decreased with increasing temperature.
3.2. Effect of the ionic strength on the fluorescence properties The effects of different concentrations of NaCl on the fluorescence intensity of AODGlc–ctDNA were studied, and the results are shown in Fig. 3. As known to all, a metal ion may interact with the phosphate groups, sugar moiety or the base residue of a nucleotide, leading to the decrease of binding sites of ctDNA, which influences the interaction of small molecular with ctDNA [27]. If the compound intercalates between the adjacent base pairs of DNA, it will be not susceptible to the surrounding change because the intercalator is under the protection of base pairs above and below [28]. From Fig. 3, no obvious changes happened to the fluorescence emission intensities when adding NaCl into the AODGlc– ctDNA system, indicating that the interaction between the AODGlc and ctDNA was neither electrostatic binding nor groove binding, but intercalative binding.
Fig. 4. Fluorescence quenching plot of AODGlc by KI in the absence and presence of ctDNA. CAODGlc = 2.44 106 mol L1, CctDNA = 0.76 105 mol L1.
3.3. Iodide quenching studies Generally, iodide anion is a dynamic, or collision, fluorescence quencher for the small fluorescent molecules [29]. The binding mode between the small molecule and DNA can be deduced from the fluorescence change in the absence and presence of DNA. When the interaction mode belongs to groove binding, the fluorescence probe should be partly protected by DNA, and iodide anions can partly quench its fluorescence. Different from groove binding, intercalation provides much less protection for the bound
Table 1 The quenching constants, binding constants, number of binding sites and thermodynamic parameters of the interaction of AODGlc with ctDNA at different temperatures. T (K) 301 310 320
KSV (L mol1) 4
1.377 10 1.066 104 8.145 103
R 0.996 0.995 0.991
K (L mol1) 4
2.359 10 2.932 105 4.162 105
n
R
DG (kJ mol1)
DH (kJ mol1)
DS (J mol1 K1)
1.059 1.341 1.404
0.997 0.998 0.998
26.16 30.53 35.40
120.3
486.5
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presence of the compound was displayed in Fig. 5. An increase of 6 °C for AODGlc was observed in the Tm profile of the compound as compared to free ctDNA. The increase in the helix melting temperature indicated the enhanced stability of the double helix when AODGlc binding to ctDNA, which was a clear evidence for intercalative or phosphate binding of AODGlc with ctDNA.
3.5. Molecular modeling studies
Fig. 5. Melting curves of ctDNA in the presence and absence of AODGlc.
Generally, molecular modeling is widely used in the design of new drugs, as it may provide some insight into the interactions between the ligands and macromolecules [32]. The structure of AODGlc was optimized using Tripos force field with Gasteiger– Marsili charges by molecular modeling software Sybyl 6.9. FlexX program was applied to calculate the possible conformation of the ligands that binds to the ctDNA. As shown in Fig. 6, the docked structure exhibits the optimal energy ranking results of AODGlc interaction with ctDNA. It suggested that the chromophore of AODGlc could slide into the G–C rich region of DNA. As for AODGlc, the N2’ H21 atom of G-23 was at a distance of 2.4 and 2.5 Å from methoxyl O and N atoms, respectively. Similarly, the N3 of G-23 was 2.0 Å from the hydroxy H atom, atom O of carbonyl with N2’ H21 atom of G-22 was 2.1 Å. The results of molecular modeling indicated that the interaction between AODGlc and ctDNA was dominated by intercalation and hydrogen bonding forces might play an important role in the binding.
3.6. Thermodynamic parameters and binding force It was found that the binding constants of to AODGlc to ctDNA increased with the temperature, suggesting that the binding reaction of the AODGlc with ctDNA was endothermic [6]. To confirm this point of view, the thermodynamic parameters was calculated according to Eqs. (3) and (4). The values of DH, DG and DS obtained were listed in Table 1. There are several acting forces between small molecular and biomacromolecule, such as hydrophobic force, hydrogen bond, van der Waals, electrostatic force and so on [33]. When DH < 0 or DH 0, DS > 0, the mainly acting force is electrostatic force; when DH < 0, DS < 0, the mainly acting force is Van der Waals or hydrogen bond and when DH > 0, DS > 0, the mainly force is hydrophobic [34]. The results indicated that the hydrophobic force played a main role in the binding of AODGlc with ctDNA besides the hydrogen bonding, which supplemented the binding force results from the molecular modeling docking.
3.7. Cell culture, cell viability assay and cell imaging Fig. 6. Molecular modeling of AODGlc showing intercalation binding to ctDNA with preference of the G–C rich region.
molecule, owing to the electrostatic repelling [30]. In this study, the values of KSV for AODGlc, and AODGlc–ctDNA were 1560.5 and 1078.3 L/mol, respectively from Stern–Volmer equation (Fig. 4). The results demonstrated that iodide quenching effect decreased when AODGlc was bound to ctDNA, suggesting that the binding mode of AODGlc with ctDNA was intercalation.
3.4. DNA melting studies DNA melting studies [31] can provide another evidence for the interaction of the AODGlc with ctDNA. Interaction of drugs with double stranded DNA can influence the melting temperature Tm. The thermal denaturation profile of DNA in the absence and
The experiment of CCK-8 cells viability assay was used for studying the cytotoxicity of AODGlc. The results were listed in Table 2. When the concentration of AODGlc was 2.436 105 mol L1, the viability of HepG2 cells was low. While the concentration of AODGlc was 1.949 107 mol L1, AODGlc almost had no influence on HepG2 cells. Using AODGlc as a probe, the bioimaging experiments in vitro were conducted by LCSM. HepG2 cells were used for cell images. As shown in Fig. 7A and D were the bright field, C and F were the fluorescence of AODGlc. As expected, HepG2 cells treated by the AODGlc (1.218 107 mol L1) became quite bright (Fig. 7B and E), showing green color by exciting at the wavelength of 458 nm, while no visible fluorescence was detected in the untreated control group under the same conditions (Fig. 7A and D). From Fig. 7B and E, it was found that AODGlc could interact with HepG2 cells and form cell imaging.
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Fig. 7. CLSM images of HepG2 cells incubated with 1.218 107 mol L1 of AODGlc for 3 h. (a) bright field (A, D), (b) fluorescence (C, F) excited with 458 nm, (c) bright field + fluorescence (B, E). The magnification was 10 (A, B, C) and 40 (D, E, F), respectively.
Table 2 The cytotoxicity of AODGlc for CCK-8 cells at different concentration after incubating 48 h.
Transparency Document
The concentration of AODGlc (mol L1)
Viability (%)
The Transparency document associated with this article can be found in the online version.
2.436 105 4.872 106 2.436 106 0.974 106 1.949 107
24.52 ± 0.32 33.11 ± 0.27 53.57 ± 3.14 92.30 ± 3.74 103.9 ± 2.99
Acknowledgements
4. Conclusion In this work, AODGlc was synthesized with the aim to get more favorable ctDNA binding properties. The study of AODGlc interaction with ctDNA was performed by spectral analysis and docking method. Experimental results suggested that AODGlc binds to ctDNA with an intercalation mode, which was due to the large coplanar aromatic rings in two complexes that facilitate their intercalation to the base pairs of double helical ctDNA. The thermodynamic parameters showed that hydrophobic interaction might play a predominant role in the binding of AODGlc with ctDNA. Besides, the calculation of binding energy from docking studies indicated the importance of hydrogen bonding. It is also found that AODGlc is effective in the treatment of cancer cells under a certain concentration and could interact with HepG2 cells to achieve cell imaging. From the standpoint of new drug discovery, such strategies may provide a platform for the development of new therapeutic reagents for diseases on the molecular level warrant further in vivo experiments and pharmacological assays. Conflict of interest The authors declare that there are no conflicts of interest.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Probing the interaction of anthraquinone with DNA by spectroscopy, molecular modeling and cancer cell imaging technique Lei Yang a,b, Zheng Fu a, Xiaoqing Niu a, Guisheng Zhang a, Fengling Cui a,⇑, Chunwu Zhou b,⇑ a b
School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang 453007, China Cancer Institute & Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100021, China
a r t i c l e
i n f o
Article history: Received 30 December 2014 Received in revised form 16 March 2015 Accepted 23 March 2015 Available online 31 March 2015 Keywords: AODGlc DNA Melting Intercalation Molecular modeling Cell imaging
a b s t r a c t A new anthraquinone derivative, (E)-2-(1-(4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2yloxyimino)ethyl)-1,4-dihydroxyanthracene-9,10-dione (AODGlc), was synthesized and its binding properties towards DNA were explored under physiological conditions by fluorescence spectroscopy, DNA melting as well as docking techniques. The experimental results revealed that AODGlc could bind to calf thymus DNA (ctDNA) through intercalation between DNA base pairs. The values of thermodynamic parameters at different temperatures including DG, DH, and DS and the molecular modeling study implied that hydrophobic interactions and hydrogen bonds were the main interactions in the AODGlc– ctDNA system. Cervical cancer cells (HepG2 cells) were used in cell viability assay and cell imaging experiment. AODGlc could interact with HepG2 cells and kill HepG2 cells under high concentration with nice curative effect, indicating its potential bioapplication in the future. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In recent years, considerable attention has been paid to the study of interactions between small molecules and deoxyribonucleic acids (DNA) [1,2]. It is well known that some small molecules can not only be used as biological probes but also as anticancer drugs targeting DNA molecules [3–5]. Many anticancer drugs are known to interact with DNA to exert their biological activities by interacting with DNA through non-covalent bond thus changing the molecular configuration of DNA and damage their template effects [6,7]. As the development of anticancer drugs is slow and costly, one approach to accelerate the availability of new drugs is to reposition drugs approved for other indications as anticancer agents [8]. Although the anthracyclines are widely used in cancer therapy [9,10], there are two major limitations in their clinical use: cardiotoxicity and drug resistance [11,12]. To improve the drug resistance, researchers are trying to synthesize new anticancer drugs through modifying existing drugs with different functional groups. On account of this, AODGlc (Fig. 1) was synthesized via changing glycosyl on anthracyclines. This study was to be aimed at investigating the binding mechanism of AODGlc with ctDNA by spectroscopic methods and DNA ⇑ Corresponding authors. Tel./fax: +86 373 3326336 (F. Cui), +86 010 87788553 (C. Zhou). E-mail addresses: [email protected] (F. Cui), [email protected]. com (C. Zhou). http://dx.doi.org/10.1016/j.cbi.2015.03.026 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
melting techniques, as well as molecular modeling. Besides, fluorescence imaging technique is employed to probing the efficacy of AODGlc on the cervical cancer cells. This work will contribute to elucidating the binding mechanism of anthraquinones with DNA and designing new and efficient drug molecules.
2. Materials and methods 2.1. Materials Calf thymus DNA was obtained from Sigma Chemical Co. (St. Louis, MO). The ratio of the UV absorbance at 260/280 nm was larger than 1.8, which indicated that ctDNA was sufficiently free from any contamination [13]. AODGlc was synthesized and characterized by NMR. 1H NMR (400 MHz, DMSO-d6) (ppm): 13.26 (1H, s), 12.56 (1H, s), 8.27 (2H, s), 8.00 (2H, s), 7.35 (1H, s), 5.58 (1H, s), 4.95 (2H, dd, J1 = 4.8, J2 = 5.6), 4.50 (1H, t, J = 6.0), 3.53–3.71 (3H, m), 3.14–3.20 (2H, m), 2.11–2.18 (1H, m), 1.59–1.66 (1H, m), 1.20 (2H, d), 0.76–0.85 (1H, m), 13C NMR (100 MHz, DMSO-d6) (ppm): 187.5, 186.7, 164.6, 156.0, 155.4, 155.3, 137.0, 135.7, 135.6, 133.3, 128.2, 127.2, 127.1, 113.7, 107.0, 74.6, 71.7, 68.4, 64.2, 67.4, 15.5. The concentration of DNA stock solution was determined according to the absorbance at 260 nm by using the extinction coefficient of 6600 L mol1 cm1 [14]. Other chemicals and reagents were of analytical grade and used without further purification.
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2.4. Iodide quenching experiment Iodide quenching experiments were carried out by adding different proportion iodide stock solutions to the AODGlc and the AODGlc–ctDNA mixture. 2.5. Melting studies Melting temperature of ctDNA was determined in the absence and presence of complexes by monitoring the absorption of DNA at 260 nm with temperature ranging from 30 to 100 °C, using TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China). 2.6. Molecular modeling
Fig. 1. Molecular structure of AODGlc.
2.2. Fluorescence measurements Fluorescence measurements were carried out with a Varian Cary Eclipse spectrofluorimeter. Different concentrations of ctDNA were added to the fixed complex solutions and the fluorescence spectra were simultaneously recorded from 520 to 750 nm with an excitation wavelength of 254 nm. The quenching type was determined by the Stern–Volmer quenching method. The plots from the fluorescence titration data at three different temperatures were investigated according to the Stern–Volmer equation [15,16]:
I0 ¼ 1 þ K q s0 ½Q ¼ 1 þ K SV ½Q I
ð1Þ
Here, I0 and I are the fluorescence intensities in the absence and presence of the quencher, respectively. [Q] is the concentration of quencher, s0 is the average lifetime of the fluorescence molecule in the absence of quenching reagent and its value is about 108 s [17], Kq is the quenching rate constant of biomacromolecule. KSV is the Stern–Volmer dynamic quenching constant. The binding constant K of complexes to ctDNA at various temperature was obtained using the following equation [18]:
log
I0 I ¼ log K þ n log½Q I
ð2Þ
Here I0 and I are the fluorescence intensities of the fluorophore in the absence and in the presence of different concentrations of [Q], respectively. The thermodynamic parameters, enthalpy change (DH), entropy change (DS) and free energy change (DG) are very important for confirming binding modes. These parameters can be calculated as van’t Hoff equation [19]:
log K ¼
DH DS þ 2:303 RT 2:303 R
DG ¼ DH T DS ¼ RT ln K
ð3Þ ð4Þ
The crystal structure of ctDNA used for docking was extracted from the Protein Data Bank [20] with identifier 453D. The structure of AODGlc generated by Chemdraw was energy-minimized with the help of the Tripos force field using Gasteiger–Marsili charges in the Sybyl 6.9 suite. Before docking run, the macromolecule file was prepared for docking by adding polar hydrogen atoms and Gasteiger charges, and a maximum of 10 conformers was considered for the ligand. During the docking process, rotatable bonds in the ligand were assigned with FlexX. The radius around the solvent molecules was set to 12 Å. PyMol [21] was used to visualize the docked conformations and calculate the distances between possible hydrogen bonding partners. 2.7. Cell culture, cell viability assay and cell imaging The cytotoxicity experiment was performed as literatures [22,23]. Briefly, HepG2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U mL1 penicillin, and 100 U mL1 of streptomycin. Cell culture was maintained at 37 °C in a humidified condition of 95% air and 5% CO2 in culture medium. The cytotoxicity of AODGlc was tested by CCK-8 cell viability assay. Specifically, HepG2 cells were seeded in 96-well plates at a density of 5 104 cells mL1. After 24 h attachment, the cells were incubated with 0, 2.436 105, 4.872 106, 2.436 106, 9.744 107, 1.949 107 mol L1 AODGlc for 48 h. Then, AODGlc was removed and 10 lL of CCK-8 dye and 100 lL of DMEM cell culture media was added to each well and incubated for 1 h at 37 °C. The 96-well plates were then analyzed with a microplate reader (VictorI, Perkin-Elmer) at the wavelength of 450 nm, with the reference wavelength at 620 nm. Three replicate wells were used for each control and test concentrations per microplate, and results are presented as mean ± SD. HepG2 cells were seeded in confocal 35 mm clear cover-glass petri-dishes before imaging. After cell attachment, AODGlc was added to each dish at the concentration of AODGlc diluting 100 times. After 3 h exposure, AODGlc was removed and cells were subsequently washed by PBS for 3 times. Cell images were taken with a Laser Scanning Confocal Microscope (LCSM) Zesis 710 3channels (Zesis, Germany) with the excitation wavelength of 458 nm.
where K is the binding constant at the corresponding temperature and R is gas constant.
3. Results and discussion
2.3. Effect of ionic strength
3.1. Fluorescence studies
The experiments on salt effect were conducted by adding different aliquots (0–0.25 M) of the NaCl stock solutions to the complexes-ctDNA mixture.
Fluorescence technique is a powerful tool in the study of the interactions [24], provided that one of the interacting species exhibits an intrinsic fluorescence. Fig. 2 displays the variation
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Fig. 2. The influence of ctDNA concentration on the fluorescence emission spectra of AODGlc with the excitation of 254 nm. 1–7: CctDNA = 0, 1.52, 3.04, 4.56, 6.08, 7.60 and 9.12 105 mol L1; CAODGlc = 6.10 106 mol L1.
Fig. 3. Effect of ionic strength on the fluorescence intensity of AODGlc in the absence and presence of ctDNA. CAODGlc = 2.44 106 mol L1, CctDNA = 0.76 105 mol L1.
in the emission spectra of the AODGlc upon addition of increasing concentrations of ctDNA. The fluorescence of the AODGlc was efficiently quenched by ctDNA, revealing that the interaction between AODGlc and ctDNA occurred. Fluorescence quenching could proceed via different mechanism, usually classified as dynamic quenching and static quenching [25]. For the dynamic quenching, the quenching constant values will increase with increasing temperature, but the reverse effect would be observed for static quenching [26]. The binding constant (K) and the binding stoichiometry (n) for the formation of ctDNA–AODGlc were summarized in Table 1. The results indicated that the probable quenching mechanism of complexes by ctDNA involved static quenching, because KSV decreased with increasing temperature.
3.2. Effect of the ionic strength on the fluorescence properties The effects of different concentrations of NaCl on the fluorescence intensity of AODGlc–ctDNA were studied, and the results are shown in Fig. 3. As known to all, a metal ion may interact with the phosphate groups, sugar moiety or the base residue of a nucleotide, leading to the decrease of binding sites of ctDNA, which influences the interaction of small molecular with ctDNA [27]. If the compound intercalates between the adjacent base pairs of DNA, it will be not susceptible to the surrounding change because the intercalator is under the protection of base pairs above and below [28]. From Fig. 3, no obvious changes happened to the fluorescence emission intensities when adding NaCl into the AODGlc– ctDNA system, indicating that the interaction between the AODGlc and ctDNA was neither electrostatic binding nor groove binding, but intercalative binding.
Fig. 4. Fluorescence quenching plot of AODGlc by KI in the absence and presence of ctDNA. CAODGlc = 2.44 106 mol L1, CctDNA = 0.76 105 mol L1.
3.3. Iodide quenching studies Generally, iodide anion is a dynamic, or collision, fluorescence quencher for the small fluorescent molecules [29]. The binding mode between the small molecule and DNA can be deduced from the fluorescence change in the absence and presence of DNA. When the interaction mode belongs to groove binding, the fluorescence probe should be partly protected by DNA, and iodide anions can partly quench its fluorescence. Different from groove binding, intercalation provides much less protection for the bound
Table 1 The quenching constants, binding constants, number of binding sites and thermodynamic parameters of the interaction of AODGlc with ctDNA at different temperatures. T (K) 301 310 320
KSV (L mol1) 4
1.377 10 1.066 104 8.145 103
R 0.996 0.995 0.991
K (L mol1) 4
2.359 10 2.932 105 4.162 105
n
R
DG (kJ mol1)
DH (kJ mol1)
DS (J mol1 K1)
1.059 1.341 1.404
0.997 0.998 0.998
26.16 30.53 35.40
120.3
486.5
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presence of the compound was displayed in Fig. 5. An increase of 6 °C for AODGlc was observed in the Tm profile of the compound as compared to free ctDNA. The increase in the helix melting temperature indicated the enhanced stability of the double helix when AODGlc binding to ctDNA, which was a clear evidence for intercalative or phosphate binding of AODGlc with ctDNA.
3.5. Molecular modeling studies
Fig. 5. Melting curves of ctDNA in the presence and absence of AODGlc.
Generally, molecular modeling is widely used in the design of new drugs, as it may provide some insight into the interactions between the ligands and macromolecules [32]. The structure of AODGlc was optimized using Tripos force field with Gasteiger– Marsili charges by molecular modeling software Sybyl 6.9. FlexX program was applied to calculate the possible conformation of the ligands that binds to the ctDNA. As shown in Fig. 6, the docked structure exhibits the optimal energy ranking results of AODGlc interaction with ctDNA. It suggested that the chromophore of AODGlc could slide into the G–C rich region of DNA. As for AODGlc, the N2’ H21 atom of G-23 was at a distance of 2.4 and 2.5 Å from methoxyl O and N atoms, respectively. Similarly, the N3 of G-23 was 2.0 Å from the hydroxy H atom, atom O of carbonyl with N2’ H21 atom of G-22 was 2.1 Å. The results of molecular modeling indicated that the interaction between AODGlc and ctDNA was dominated by intercalation and hydrogen bonding forces might play an important role in the binding.
3.6. Thermodynamic parameters and binding force It was found that the binding constants of to AODGlc to ctDNA increased with the temperature, suggesting that the binding reaction of the AODGlc with ctDNA was endothermic [6]. To confirm this point of view, the thermodynamic parameters was calculated according to Eqs. (3) and (4). The values of DH, DG and DS obtained were listed in Table 1. There are several acting forces between small molecular and biomacromolecule, such as hydrophobic force, hydrogen bond, van der Waals, electrostatic force and so on [33]. When DH < 0 or DH 0, DS > 0, the mainly acting force is electrostatic force; when DH < 0, DS < 0, the mainly acting force is Van der Waals or hydrogen bond and when DH > 0, DS > 0, the mainly force is hydrophobic [34]. The results indicated that the hydrophobic force played a main role in the binding of AODGlc with ctDNA besides the hydrogen bonding, which supplemented the binding force results from the molecular modeling docking.
3.7. Cell culture, cell viability assay and cell imaging Fig. 6. Molecular modeling of AODGlc showing intercalation binding to ctDNA with preference of the G–C rich region.
molecule, owing to the electrostatic repelling [30]. In this study, the values of KSV for AODGlc, and AODGlc–ctDNA were 1560.5 and 1078.3 L/mol, respectively from Stern–Volmer equation (Fig. 4). The results demonstrated that iodide quenching effect decreased when AODGlc was bound to ctDNA, suggesting that the binding mode of AODGlc with ctDNA was intercalation.
3.4. DNA melting studies DNA melting studies [31] can provide another evidence for the interaction of the AODGlc with ctDNA. Interaction of drugs with double stranded DNA can influence the melting temperature Tm. The thermal denaturation profile of DNA in the absence and
The experiment of CCK-8 cells viability assay was used for studying the cytotoxicity of AODGlc. The results were listed in Table 2. When the concentration of AODGlc was 2.436 105 mol L1, the viability of HepG2 cells was low. While the concentration of AODGlc was 1.949 107 mol L1, AODGlc almost had no influence on HepG2 cells. Using AODGlc as a probe, the bioimaging experiments in vitro were conducted by LCSM. HepG2 cells were used for cell images. As shown in Fig. 7A and D were the bright field, C and F were the fluorescence of AODGlc. As expected, HepG2 cells treated by the AODGlc (1.218 107 mol L1) became quite bright (Fig. 7B and E), showing green color by exciting at the wavelength of 458 nm, while no visible fluorescence was detected in the untreated control group under the same conditions (Fig. 7A and D). From Fig. 7B and E, it was found that AODGlc could interact with HepG2 cells and form cell imaging.
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Fig. 7. CLSM images of HepG2 cells incubated with 1.218 107 mol L1 of AODGlc for 3 h. (a) bright field (A, D), (b) fluorescence (C, F) excited with 458 nm, (c) bright field + fluorescence (B, E). The magnification was 10 (A, B, C) and 40 (D, E, F), respectively.
Table 2 The cytotoxicity of AODGlc for CCK-8 cells at different concentration after incubating 48 h.
Transparency Document
The concentration of AODGlc (mol L1)
Viability (%)
The Transparency document associated with this article can be found in the online version.
2.436 105 4.872 106 2.436 106 0.974 106 1.949 107
24.52 ± 0.32 33.11 ± 0.27 53.57 ± 3.14 92.30 ± 3.74 103.9 ± 2.99
Acknowledgements
4. Conclusion In this work, AODGlc was synthesized with the aim to get more favorable ctDNA binding properties. The study of AODGlc interaction with ctDNA was performed by spectral analysis and docking method. Experimental results suggested that AODGlc binds to ctDNA with an intercalation mode, which was due to the large coplanar aromatic rings in two complexes that facilitate their intercalation to the base pairs of double helical ctDNA. The thermodynamic parameters showed that hydrophobic interaction might play a predominant role in the binding of AODGlc with ctDNA. Besides, the calculation of binding energy from docking studies indicated the importance of hydrogen bonding. It is also found that AODGlc is effective in the treatment of cancer cells under a certain concentration and could interact with HepG2 cells to achieve cell imaging. From the standpoint of new drug discovery, such strategies may provide a platform for the development of new therapeutic reagents for diseases on the molecular level warrant further in vivo experiments and pharmacological assays. Conflict of interest The authors declare that there are no conflicts of interest.
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