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Intercalation of antitumor drug doxorubicin and its analogue by DNA duplex: Structural features and biological implications
International Journal of Biological Macromolecules 66 (2014) 144–150
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Intercalation of antitumor drug doxorubicin and its analogue by DNA duplex: Structural features and biological implications Daniel Agudelo, Philippe Bourassa, Gervais Bérubé, Heidar-Ali Tajmir-Riahi ∗ Department of Chemistry, Biochemistry and Physics, University of Québec at Trois-Rivières, C.P. 500, Trois-Rivières G9A 5H7 Québec, Canada
a r t i c l e
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Article history: Received 12 November 2013 Received in revised form 10 February 2014 Accepted 13 February 2014 Available online 20 February 2014 Keywords: Doxorubicin DNA Intercalation Conformation Molecular modeling
a b s t r a c t The intercalation of antitumor drug doxorubicin (DOX) and its analogue N-(trifluoroacetyl) doxorubicin (FDOX) with DNA duplex was investigated, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Both DOX and FDOX were intercalated into DNA duplex with the free binding energy of −4.99 kcal for DOX–DNA and −4.92 kcal for FDOX–DNA adducts and the presence of H-bonding network between doxorubicin NH2 group and cytosine-19. Spectroscopic results showed FDOX forms more stable complexes than DOX with KDOX-DNA = 2.5(±0.5) × 104 M−1 and KFDOX-DNA = 3.4(±0.7) × 104 M−1 . The number of drug molecules bound per DNA (n) was 1.2 for DOX and 0.6 for FDOX. Major alterations of DNA structure were observed by DOX intercalation with a partial B to A-DNA transition, while no DNA conformational changes occurred upon FDOX interaction. This study further confirms the importance of unmodified daunosamine amino group for optimal interactions with DNA. The results of in vitro MTT assay carried out on SKC01 colon carcinoma corroborate the observed DNA interactions. Such DNA structural changes can be related to doxorubicin antitumor activity, which prevents DNA duplication. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Doxorubicin (Scheme 1) remains as one of the most effective chemotherapeutic anticancer drugs of the past 50 years and is crucial to the treatment of a range of neoplasms including acute leukemia, malignant lymphoma, and breast cancer [1,3]. However, like all the other anticancer drugs, the efficacy of DOX is associated with high systemic toxicity to healthy tissue [3]. In particular, the dose-dependent cardiotoxicity induced by DOX is cumulative and life-threatening, making the development of targeted DOX delivery systems of particular importance [1,4]. Furthermore, the development of multiple drug resistance to anthracyclines is also an impediment for its long term use [1]. Others type of secondary effects includes hair loss, gastrointestinal discomfort, anemia, leucopenia, etc. [1,3]. DOX is an anticancer drug that inhibits topoisomerase II, interfering with the topoisomerase IIDNA complex, leading to the formation of double-stranded breaks of DNA or direct intercalation with DNA, which in turn inhibits DNA duplication and transcription to mRNA [5,6]. Production of reactive oxygen species (ROS) is also thought to be a cytotoxic effect of DOX on cancer cells [7]. Despite the fact that much work has
been done, the antitumor antibiotic DOX still remains to this day a drug of choice for the treatment leukemia, Hodgkin’s disease, breast carcinoma, ovarian carcinoma and other cancers and is still a subject of intense research [1]. In this study, we sought to verify the impact of a relatively subtle molecular modification on the daunosamine moiety of doxorubicin towards its interaction with DNA. Hence, using drug analogue FDOX (Scheme 1) with blocked NH2 group helps us to understand the role of NH2 in drug efficacy and drug-DNA complexation. This comprehensive structural analysis of doxorubicin-DNA adducts could have major biological and biomedical implications. We now report the structural analysis of calf-thymus-DNA complexes with DOX and FDOX by FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Structural information regarding the drug intercalation into DNA duplex and the effect of drug on DNA stability and secondary structure are provided. This is the first spectroscopic and structural analysis of DNA interaction with DOX and FDOX at molecular level, which can contribute to further clarifying the mechanism of action of antitumor activity of doxorubicin. 2. Experimental
Abbreviations: DOX, doxorubicin; FDOX, N-(trifluoroacetyl) doxorubicin; FTIR, Fourier transform infrared; CD, Circular dichroism. ∗ Corresponding author. Tel.: +1 819 376 5011x3310; fax: +1 819 376 5084. E-mail address: [email protected] (H.-A. Tajmir-Riahi). http://dx.doi.org/10.1016/j.ijbiomac.2014.02.028 0141-8130/© 2014 Elsevier B.V. All rights reserved.
2.1. Materials Doxorubicin hydrochloride was generously provided by Pharmacia/Farmitalia Carlos Erba, Italy and N-(trifluoroacetyl)
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Scheme 1. Chemical structures of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX).
doxorubicin was synthesized according to the published methods [8,9]. Highly polymerised type I calf-thymus DNA sodium salt (7% Na content) was purchased from Sigma Chemical Co., and was deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. In order to check the protein content of DNA solution, the absorbance at 260 and 280 nm was recorded. The A260 /A280 ratio was 1.85, showing that the DNA was sufficiently free from protein [10]. Other chemicals were of reagent grade and used without further purification. 2.2. Preparation of stock solutions Sodium-DNA was dissolved to 1% (w/w) (10 mg/ml) in Tris–HCl (pH 7.3) at 5 ◦ C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the stock DNA solution was determined spectrophotometrically at 260 nm by using molar extinction coefficient of 6600 cm−1 M−1 (expressed as molarity of phosphate groups) [11,12]. The UV absorbance at 260 nm of a diluted solution (40 M) of calf-thymus DNA used in our experiments was measured to be 0.25 (path length was 1 cm) and the final concentration of the stock DNA solution was calculated to be 25 mM in DNA phosphate. The average length of the DNA molecules, estimated by gel electrophoresis was 9000 base pairs (molecular weight ∼ 6 × 106 Da). The solutions of DOX and FDOX (0.15 M to 1 mM) were prepared in water for DOX and in ethanol/water 25/75% for FDOX and diluted in Tris–HCl (pH 7.4). The drug solution was added drop-wise to DNA solution with constant stirring to ensure the formation of homogeneous solution. 2.3. FTIR spectroscopic measurements Infrared spectra were recorded on a BOMEM DA3-0.02 Fourier transform infrared spectrometer, equipped with a nitrogen cooled HgCdTe detector and a KBr beam splitter. Solution spectra were recorded in solution on AgBr windows with resolution of 2 cm−1 and 100 scans with drug concentrations 0.125, 0.25 and 0.5 mM and a final DNA concentration of 12.5 mM (P). The water subtraction was carried out with 0.1 M NaCl solution used as a reference at pH 7.3 [13]. A good water subtraction was achieved as shown by a flat baseline around 2200 cm−1 where the water combination mode is located. This method is a rough estimate, but removes the water content in a satisfactory way. The difference spectra [(DNA solution + drug) − (DNA solution)] were obtained, using the sharp DNA band at 968 cm−1 as internal reference. This band, which is due to deoxyribose C-C stretching vibrations, exhibits no spectral
changes (shifting or intensity variation) upon drug-DNA complexation, and cancelled out upon spectral subtraction. The spectra are smoothed with Savitzky-Golay procedure [13]. 2.4. CD spectroscopy Spectra of DNA and its drug complexes were recorded at pH 7.4 with a Jasco J-720 spectropolarimeter. For measurements in the Far-UV region (200–320 nm), a quartz cell with a path length of 0.01 cm was used. Three scans were accumulated at a scan speed of 50 nm per minute, with data being collected at every nm from 200 to 320 nm. Sample temperature was maintained at 25 ◦ C using a Neslab RTE-111 circulating water bath connected to the waterjacketed quartz cuvettes. Spectra were corrected for buffer signal and conversion to the Mol CD (ε) was performed with the Jasco Standard Analysis software. The drug concentrations used in our experiment varied from 125 M to 500 M with the final DNA concentration of 2.5 mM. 2.5. Fluorescence spectroscopy Fluorimetric experiments were carried out on a Perkin-Elmer LS55 Spectrometer. Stock solution of drug (30 М) in Tris–HCl (pH 7.4) was also prepared at 24 ± 1 ◦ C. Various solutions of DNA (1 to 200 M) were prepared from the above stock solutions by successive dilutions at 24 ± 1 ◦ C. Samples containing 0.06 ml of the above drug solution and various DNA solutions were mixed to obtain final DNA concentrations ranging from 1 to 100 М with constant drug content (30 М)· The fluorescence spectra were recorded at ex = 480 nm and em from 500 to 750 nm. The intensity of the band at 592 nm from doxorubicin and its analogue [14] was used to calculate the binding constant (K) as reported [15–20]. Results from fluorescence measurements can be used to estimate the binding constant of drug-DNA complex. From eq. (1): log [(F0 − F)] = log KA + n log [Q ]
(1)
The accessible fluorophore fraction (f) can be calculated by modified Stern–Volmer equation: 1 F0 1 = + F0 − F fK [Q ] f
(2)
where, F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting molecule). K is the Stern–Volmer quenching constant, [Q] is the molar concentration of quencher and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional
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Fig. 1. FTIR spectra and difference spectra [(DNA solution + drug solution) − (DNA solution)] in the region of 1800–600 cm−1 for the free calf-thymus DNA and its DOX (A) and FDOX (B) complexes in aqueous solution at pH 7.4 with various drug concentrations and constant DNA content (12.5 mM).
fluorescence contribution of the total emission for an interaction with a hydrophobic quencher [21,22]. The K will be calculated from F0 /F = K[Q] + 1. 2.6. Molecular modeling The docking studies were performed with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, Wa, http://www.arguslab.com). DNA structure were obtained from the PDB (ID: 6TNA) [23] and the three dimensional structures of DOX and FDOX were generated from PM3 semi-empirical calculations using Chem3D Ultra 6.0. The docking runs were performed on the ArgusDock docking engine using high precision with a maximum
of 150 candidate poses. The conformations were ranked using the Ascore scoring function, which estimates the free binding energy. Upon docking of drug to DNA, the current configurations were optimized using a steepest decent algorithm until convergence, within 40 iterations and nucleobase residues within a distance of 3.5 A˚ relative to drug were involved in the complexation. 3. Results 3.1. FTIR spectra of drug-DNA complexes Drug-DNA intercalation causes major alterations of DNA in-plane vibrational frequencies that are presented in Fig. 1.
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(0.125 and 0.25 mM), no major shifting of CD bands was observed, while major intensity changes occurred, which is indicative of no alterations of B-DNA conformation (Fig. 2). However, at high DOX content (0.5 mM), major shifting of the bands at 211 to 214 and 246 to 240 nm was observed and the band at 280 was split to 270 and 258 nm in the spectra of DOX–DNA complexes (Fig. 2A). The spectral shifting was accompanied with major intensity increase of these bands upon DOX intercalation (Fig. 2A). These spectral shifting are due to a partial B to A-DNA transition upon drug interaction, whereas the major intensity changes observed are due to base destacking as DOX intercalation occurs. Similar spectral changes observed when B to A-DNA conformational changes occurred [29,31]. These spectral shifting was not observed here in the CD spectra of FDOX–DNA complexes and therefore DNA remains in B-family structure upon FDOX complexation (Fig. 2B). The reason for the differences can be due to the blockage of the drug NH2 group in DOX by trifluoroacetyl moiety in FDOX. This emphasizes the important role of free NH2 group in drug-DNA interaction. 3.3. Fluorescence spectra and stability of drug-DNA complexes
Fig. 2. Circular dichroism of the free calf-thymus DNA and its DOX (A) and FDOX complexes (B) in aqueous solution with 2.5 mM DNA concentration and 0.125 to 0.5 mM drug concentrations at pH 7.4.
Intercalation of DOX and FDOX into DNA duplex brought major spectral changes for DNA in-plane vibrational frequencies [11,24–28]. Spectral shifting and intensity increase were observed for the guanine band at 1710, thymine band at 1661 and adenine band at 1610 cm−1 , upon drug complexation. The guanine band at 1710 shifted toward a lower frequency at 1707 (DOX) and 1708 (FDOX), while thymine band at 1661 appeared at 1653 in the spectra of both DOX and FDOX–DNA complexes (Fig. 1, complex 0.5 mM)). The shifting was accompanied by a major intensity increase for these vibrations as shown by positive spectral features at 1700–1683 (guanine), 1653–1649 (thymine), 1512–1513 and 1458 cm−1 (cytosine) in the difference spectra of DOX and FDOX–DNA adducts (Fig. 1 diffs, 0.125 and 0.5 mM). Drug intercalation also induced spectral changes for DNA phosphate group as the band at 1225 (PO2 asymmetric) gained intensity, while the band at 1088 cm−1 (PO2 symmetric) shifted to a lower frequency at 1084 cm−1 , in the spectra of drug-DNA complexes (Fig. 1, complex 0.5 mM). These spectral changes are due to drug intercalation into DNA duplex, which alters the nucleobase vibrational frequencies). Similar infrared spectral changes were observed for DNA when well known intercalating agents ethidium bromide, acridine orange and methylene blue were intercalated into DNA duplex [28]. 3.2. CD spectra and DNA conformation The CD spectra of the free calf-thymus DNA and DOX and FDOX complexes with different drug concentrations are shown in Fig. 2. The CD spectrum of the free DNA composed of four major peaks at 211 (negative), 222 (positive), 246 (negative) and 280 nm (positive) (Fig. 2). This is consistent with CD spectra of double helical DNA in B conformation [29–31]. Upon DOX complexation
Since DNA is a weak fluorophore, the titrations of DOX and FDOX were done against various DNA concentrations, using drug excitation at 480 nm and emission at 500–750 nm [12]. When drug interacts with DNA, fluorescence may change depending on the impact of such interaction on the drug conformation, or via direct quenching effect. The decrease of fluorescence intensity of DOX or FDOX has been monitored at 592 nm for drug-DNA systems (Fig. 3A and B). The plot of F0 /(F0 − F) vs. 1/[DNA] is shown in Fig. 3A and B . Assuming that the observed changes in fluorescence come from the interaction between the drug and DNA, the quenching constant can be taken as the binding constant of the complex formation. The K value obtained is the averages of four and sixreplicate run for drug-polymer systems. Each run involves several different concentrations of DNA (Fig. 3A and B). The overall binding constants KDOX-DNA = 2.5 (±0.5) × 104 M−1 and KFDOX-DNA = 3.4 (±0.7) × 104 M−1 (Fig. 3A and B ) showing more stable complexes formed with FDOX than DOX. The f value calculated from Eq. (7) represents the mole fraction of the accessible population of fluorophore to quencher. The f values were from 0.20 to 0.60 for these drug-DNA complexes indicating a large portion of fluorophore was exposed to quencher. The number of drug molecules bound per polymer (n) is calculated from log [(F0 − F)/F] = logKS + nlog[DNA] for the static quenching [32–35]. The n values from the slope of the straight line plot showed 1.2 (DOX) and 0.6 (FDOX) drug molecules that are bound per DNA molecule (Fig. 4). The results indicate some degree of cooperativity for drug–DNA interaction. To calculate the fluorescence quenching coefficient constant (KQ) for drug-DNA complexes we have plotted F0 /F against Q and the results are show in Fig. 5. The plot of F0 /F versus Q is a straight line for drug-DNA adducts indicating that the quenching is mainly static in these drug-biopolymer complexes (Fig. 5). The quenching constant KQ was estimated according to the Stern–Volmer equation: F0 = 1 + kQ t0 [Q ] = 1 + Ksv [Q ] F
(3)
where F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration and Ksv is the Stern–Volmer quenching constant [36,37], which can be written as Ksv = kQ t0 ; where kQ is the drug quenching rate constant and t0 is the lifetime of the fluorophore in the absence of quencher about 1.1 ns for free DOX and FDOX around neutral pH [14]. The quenching constants (KQ ) are 3.4 × 1013 M−1 /s for DOX–DNA and 1.2 × 1012 M−1 /s for FDOX–DNA complexes (Fig. 5). Since
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Fig. 4. The plot of log(F0 − F)/F as a function of log [DNA] for calculation of number of bound drug molecule (n) per DNA (A) DOX–DNA and (B) FDOX–DNA complexes.
Fig. 3. Fluorescence emission spectra of drug-DNA systems in 10 mM Tris-HCl buffer pH 7.4 at 25 ◦ C for (A) DOX–DNA: (a) free DOX (20 M), (b–j) with DOX–DNA complexes at 5, 10, 15, 20, 30, 40, 50, 60 and 70 M; (B) FDOX–DNA: (a) free FDOX (20 M), (b–j) with FDOX–DNA complexes at 5, 10, 20, 30, 40, 60, 80, 90 and 100 M. Inset: F0 /(F0 − F) vs. 1/[DNA] for A (DOX–DNA) and B (FDOX–DNA).
these values are greater than the maximum collisional quenching constant (1010 M−1 /s), the static quenching is dominant in these drug-DNA complexes [36–38]. 3.4. Docking study and antiproliferative activity on colon carcinoma Our spectroscopic results are accompanied by docking study in which the DOX and FDOX were docked to DNA to determine the preferred binding sites on DNA. The dockings results are shown in Fig. 6 and Table 1. The models show that both DOX and FDOX are intercalated into DNA duplex and surrounded by A-7, C-5, *C19 (H-bonding with doxorubicin NH2 group), G-6, T-8 and T-18 with the free binding energy of −4.99 for DOX and −4.92 kcal/mol Table 1 Deoxyribonucleotides in the vicinity of DOX and FDOX docked with DNA (PDB 6TNA) and the free binding energies of the docked complexes. *Hydrogen bonding involved with this nucleotide. Complex
Deoxyribonucleotides involved in drug interactions
Gbinding (kcal/mol)
DOX–DNA FDOX–DNA
A-7, C-5, *C-19, G-6, T-8, T-18. A-7, C-5, C-19, G-6, T-8, T-18.
−4.99 −4.92
for FDOX–DNA complexes (Fig. 6 and Table 1). The docking results showed the binding sites used are similar for DOX and FDOX with no H-bonding in the FDOX–DNA adduct (Table 1). The binding energy (G) shows more stable complexes formed with DOX than FDOX (Table 1). The extra stability is related to the presence of H-bonding between cytosine-19 base and the NH2 group in DOX–DNA complexes (not present in FDOX–DNA adduct). This further confirms the importance of the free daunosamine amino group in DOX for its optimal interaction with DNA and induction of its biological activity. The results obtained in this study parallel the biological activities of DOX and FDOX obtained on SXC01 human tumour cancer cells using the MTT assay as described earlier [39,40]. The latter colon carcinoma is very sensitive to DOX and thus, was selected as a screen test to assess the possible loss of biological activity of FDOX in comparison to that of DOX. The IC50 for DOX is 73 nM and was found to be twice as active as FDOX showing an IC50 of 156 nM. 4. Discussion Anthracyclines rank among the most effective anticancer drugs ever developed [2]. Doxorubicin is widely used in chemotherapy due to its efficacy in fighting a wide range of cancers. The injury to non-targeted tissues, particularly to the heart, often complicates cancer treatment by limiting doxorubicin dosage and diminishing the patients’ quality of life during and after doxorubicin treatments [1,41]. Furthermore, the development of multiple drug resistance is an additional limitation to the long term use of doxorubicin [1]. Even though the intercalation of doxorubicin into DNA duplex has been studied both experimentally [6] and theoretically [42], much about the importance of role of free NH2 group in drug-DNA
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Fig. 5. Stern–Volmer plots of fluorescence quenching constant (KQ ) for the DNA and its drug complexes at different DNA concentrations (A) DOX–DNA and (B) FDOX–DNA complexes.
interaction has to be investigated. Furthermore, most of the anthracyclines marketed in the world today (doxorubicin, daunorubicin, idarubicin, epirubicin, pirarubicin, zorubicin) contains a free amino functional group [43]. This is no coincidence; it has to play a crucial role for their interaction with DNA. Hence, the aim of our study was to determine the importance of the free daunosamine amino group in DOX–DNA interaction and its biological implication. For this purpose, the amino group of daunosamine was acetylated with S-ethyltrifluoroacetate under standard reaction conditions [8]. This particular derivative was selected in order to change the physicochemical property of the amino group (being a base likely to from H-bonds) into an amide that reduces drastically the basic character of the nitrogen atom. Our infrared results showed no major differences between DOX and FDOX interactions with DNA since both are intercalated into DNA duplex. However, our CD spectroscopic results showed major differences between DOX and FDOX complexes. DOX interaction induced a partial B to A-DNA conformational changes, while FDOX did not alter DNA conformation (Fig. 2). Even docking results showed interaction for both DOX and FDOX, the hydrogen bonding between cytosine-19 and DOX amino group was not present in FDOX–DNA adduct (Table 1). Such H-bonding network brought more stability for DOX–DNA complex than FDOX–DNA adduct. It is proposed that these structural changes are responsible for antitumor activity of doxorubicin and are consistent with the antitumor activities of DOX and FDOX obtained on SXC01 human tumour cancer cells using the MTT assay.
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Fig. 6. Best conformations for drug docked to DNA (PDB entry 6TNA). The drug is shown in green color. (A) shows DNA in stick model with DOX intercalation in sticks and (A ) shows DOX binding sites represented in sticks with the corresponding base residues (B) shows DNA in stick model with FDOX intercalation in sticks and (B ) shows FDOX binding sites represented in sticks.
In conclusion, DOX and FDOX intercalate into DNA duplex causing major DNA structural changes. While DOX intercalation induced a partial B to A-DNA transition, no major structural changes occurred upon FDOX–DNA complexation. The presence of NH2 group on DOX causes major differences between DOX and FDOX interactions with DNA duplex, highlighting the important role of NH2 group in drug-DNA interaction. Hydrophobic and hydrophilic contacts play major role in both DOX and FDOX intercalation. The major DNA conformational changes induced by doxorubicin can be the main reason for its enhanced anticancer activity. Our results substantiate the in vitro biological activity on colon carcinoma showing that DOX is twice as active as FDOX. Acknowledgments This work is supported by grant from Natural Sciences and Engineering Research Council of Canada (NSERC). We wish to thank Dr. C.H.J. Ford for providing the drug in vitro biological activity. References [1] P. Ma, R.J. Mumper, Nano Today 8 (2013) 313–331. [2] C. Carvalho, R.X. Santos, S. Cardoso, S. Correia, P.J. Oliveira, M.S. Santos, P.I. Moreia, Curr. Med. Chem. 16 (2009) 3267–3285. [3] G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, L. Gianni, Pharmacol. Rev. 56 (2004) 185–229. [4] A. Turner, L.C. Li, T. Pilli, L. Qian, E.L. Wiley, S. Setty, K. Christov, L. Ganesh, A.V. Maker, P. Li, P. Kanteti, T.K. D. Gupta, B.S. Prabhakar, PLOS ONE 8 (2013) 1–8, e56817.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Intercalation of antitumor drug doxorubicin and its analogue by DNA duplex: Structural features and biological implications Daniel Agudelo, Philippe Bourassa, Gervais Bérubé, Heidar-Ali Tajmir-Riahi ∗ Department of Chemistry, Biochemistry and Physics, University of Québec at Trois-Rivières, C.P. 500, Trois-Rivières G9A 5H7 Québec, Canada
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 10 February 2014 Accepted 13 February 2014 Available online 20 February 2014 Keywords: Doxorubicin DNA Intercalation Conformation Molecular modeling
a b s t r a c t The intercalation of antitumor drug doxorubicin (DOX) and its analogue N-(trifluoroacetyl) doxorubicin (FDOX) with DNA duplex was investigated, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Both DOX and FDOX were intercalated into DNA duplex with the free binding energy of −4.99 kcal for DOX–DNA and −4.92 kcal for FDOX–DNA adducts and the presence of H-bonding network between doxorubicin NH2 group and cytosine-19. Spectroscopic results showed FDOX forms more stable complexes than DOX with KDOX-DNA = 2.5(±0.5) × 104 M−1 and KFDOX-DNA = 3.4(±0.7) × 104 M−1 . The number of drug molecules bound per DNA (n) was 1.2 for DOX and 0.6 for FDOX. Major alterations of DNA structure were observed by DOX intercalation with a partial B to A-DNA transition, while no DNA conformational changes occurred upon FDOX interaction. This study further confirms the importance of unmodified daunosamine amino group for optimal interactions with DNA. The results of in vitro MTT assay carried out on SKC01 colon carcinoma corroborate the observed DNA interactions. Such DNA structural changes can be related to doxorubicin antitumor activity, which prevents DNA duplication. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Doxorubicin (Scheme 1) remains as one of the most effective chemotherapeutic anticancer drugs of the past 50 years and is crucial to the treatment of a range of neoplasms including acute leukemia, malignant lymphoma, and breast cancer [1,3]. However, like all the other anticancer drugs, the efficacy of DOX is associated with high systemic toxicity to healthy tissue [3]. In particular, the dose-dependent cardiotoxicity induced by DOX is cumulative and life-threatening, making the development of targeted DOX delivery systems of particular importance [1,4]. Furthermore, the development of multiple drug resistance to anthracyclines is also an impediment for its long term use [1]. Others type of secondary effects includes hair loss, gastrointestinal discomfort, anemia, leucopenia, etc. [1,3]. DOX is an anticancer drug that inhibits topoisomerase II, interfering with the topoisomerase IIDNA complex, leading to the formation of double-stranded breaks of DNA or direct intercalation with DNA, which in turn inhibits DNA duplication and transcription to mRNA [5,6]. Production of reactive oxygen species (ROS) is also thought to be a cytotoxic effect of DOX on cancer cells [7]. Despite the fact that much work has
been done, the antitumor antibiotic DOX still remains to this day a drug of choice for the treatment leukemia, Hodgkin’s disease, breast carcinoma, ovarian carcinoma and other cancers and is still a subject of intense research [1]. In this study, we sought to verify the impact of a relatively subtle molecular modification on the daunosamine moiety of doxorubicin towards its interaction with DNA. Hence, using drug analogue FDOX (Scheme 1) with blocked NH2 group helps us to understand the role of NH2 in drug efficacy and drug-DNA complexation. This comprehensive structural analysis of doxorubicin-DNA adducts could have major biological and biomedical implications. We now report the structural analysis of calf-thymus-DNA complexes with DOX and FDOX by FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Structural information regarding the drug intercalation into DNA duplex and the effect of drug on DNA stability and secondary structure are provided. This is the first spectroscopic and structural analysis of DNA interaction with DOX and FDOX at molecular level, which can contribute to further clarifying the mechanism of action of antitumor activity of doxorubicin. 2. Experimental
Abbreviations: DOX, doxorubicin; FDOX, N-(trifluoroacetyl) doxorubicin; FTIR, Fourier transform infrared; CD, Circular dichroism. ∗ Corresponding author. Tel.: +1 819 376 5011x3310; fax: +1 819 376 5084. E-mail address: [email protected] (H.-A. Tajmir-Riahi). http://dx.doi.org/10.1016/j.ijbiomac.2014.02.028 0141-8130/© 2014 Elsevier B.V. All rights reserved.
2.1. Materials Doxorubicin hydrochloride was generously provided by Pharmacia/Farmitalia Carlos Erba, Italy and N-(trifluoroacetyl)
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Scheme 1. Chemical structures of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX).
doxorubicin was synthesized according to the published methods [8,9]. Highly polymerised type I calf-thymus DNA sodium salt (7% Na content) was purchased from Sigma Chemical Co., and was deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. In order to check the protein content of DNA solution, the absorbance at 260 and 280 nm was recorded. The A260 /A280 ratio was 1.85, showing that the DNA was sufficiently free from protein [10]. Other chemicals were of reagent grade and used without further purification. 2.2. Preparation of stock solutions Sodium-DNA was dissolved to 1% (w/w) (10 mg/ml) in Tris–HCl (pH 7.3) at 5 ◦ C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the stock DNA solution was determined spectrophotometrically at 260 nm by using molar extinction coefficient of 6600 cm−1 M−1 (expressed as molarity of phosphate groups) [11,12]. The UV absorbance at 260 nm of a diluted solution (40 M) of calf-thymus DNA used in our experiments was measured to be 0.25 (path length was 1 cm) and the final concentration of the stock DNA solution was calculated to be 25 mM in DNA phosphate. The average length of the DNA molecules, estimated by gel electrophoresis was 9000 base pairs (molecular weight ∼ 6 × 106 Da). The solutions of DOX and FDOX (0.15 M to 1 mM) were prepared in water for DOX and in ethanol/water 25/75% for FDOX and diluted in Tris–HCl (pH 7.4). The drug solution was added drop-wise to DNA solution with constant stirring to ensure the formation of homogeneous solution. 2.3. FTIR spectroscopic measurements Infrared spectra were recorded on a BOMEM DA3-0.02 Fourier transform infrared spectrometer, equipped with a nitrogen cooled HgCdTe detector and a KBr beam splitter. Solution spectra were recorded in solution on AgBr windows with resolution of 2 cm−1 and 100 scans with drug concentrations 0.125, 0.25 and 0.5 mM and a final DNA concentration of 12.5 mM (P). The water subtraction was carried out with 0.1 M NaCl solution used as a reference at pH 7.3 [13]. A good water subtraction was achieved as shown by a flat baseline around 2200 cm−1 where the water combination mode is located. This method is a rough estimate, but removes the water content in a satisfactory way. The difference spectra [(DNA solution + drug) − (DNA solution)] were obtained, using the sharp DNA band at 968 cm−1 as internal reference. This band, which is due to deoxyribose C-C stretching vibrations, exhibits no spectral
changes (shifting or intensity variation) upon drug-DNA complexation, and cancelled out upon spectral subtraction. The spectra are smoothed with Savitzky-Golay procedure [13]. 2.4. CD spectroscopy Spectra of DNA and its drug complexes were recorded at pH 7.4 with a Jasco J-720 spectropolarimeter. For measurements in the Far-UV region (200–320 nm), a quartz cell with a path length of 0.01 cm was used. Three scans were accumulated at a scan speed of 50 nm per minute, with data being collected at every nm from 200 to 320 nm. Sample temperature was maintained at 25 ◦ C using a Neslab RTE-111 circulating water bath connected to the waterjacketed quartz cuvettes. Spectra were corrected for buffer signal and conversion to the Mol CD (ε) was performed with the Jasco Standard Analysis software. The drug concentrations used in our experiment varied from 125 M to 500 M with the final DNA concentration of 2.5 mM. 2.5. Fluorescence spectroscopy Fluorimetric experiments were carried out on a Perkin-Elmer LS55 Spectrometer. Stock solution of drug (30 М) in Tris–HCl (pH 7.4) was also prepared at 24 ± 1 ◦ C. Various solutions of DNA (1 to 200 M) were prepared from the above stock solutions by successive dilutions at 24 ± 1 ◦ C. Samples containing 0.06 ml of the above drug solution and various DNA solutions were mixed to obtain final DNA concentrations ranging from 1 to 100 М with constant drug content (30 М)· The fluorescence spectra were recorded at ex = 480 nm and em from 500 to 750 nm. The intensity of the band at 592 nm from doxorubicin and its analogue [14] was used to calculate the binding constant (K) as reported [15–20]. Results from fluorescence measurements can be used to estimate the binding constant of drug-DNA complex. From eq. (1): log [(F0 − F)] = log KA + n log [Q ]
(1)
The accessible fluorophore fraction (f) can be calculated by modified Stern–Volmer equation: 1 F0 1 = + F0 − F fK [Q ] f
(2)
where, F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting molecule). K is the Stern–Volmer quenching constant, [Q] is the molar concentration of quencher and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional
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Fig. 1. FTIR spectra and difference spectra [(DNA solution + drug solution) − (DNA solution)] in the region of 1800–600 cm−1 for the free calf-thymus DNA and its DOX (A) and FDOX (B) complexes in aqueous solution at pH 7.4 with various drug concentrations and constant DNA content (12.5 mM).
fluorescence contribution of the total emission for an interaction with a hydrophobic quencher [21,22]. The K will be calculated from F0 /F = K[Q] + 1. 2.6. Molecular modeling The docking studies were performed with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, Wa, http://www.arguslab.com). DNA structure were obtained from the PDB (ID: 6TNA) [23] and the three dimensional structures of DOX and FDOX were generated from PM3 semi-empirical calculations using Chem3D Ultra 6.0. The docking runs were performed on the ArgusDock docking engine using high precision with a maximum
of 150 candidate poses. The conformations were ranked using the Ascore scoring function, which estimates the free binding energy. Upon docking of drug to DNA, the current configurations were optimized using a steepest decent algorithm until convergence, within 40 iterations and nucleobase residues within a distance of 3.5 A˚ relative to drug were involved in the complexation. 3. Results 3.1. FTIR spectra of drug-DNA complexes Drug-DNA intercalation causes major alterations of DNA in-plane vibrational frequencies that are presented in Fig. 1.
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(0.125 and 0.25 mM), no major shifting of CD bands was observed, while major intensity changes occurred, which is indicative of no alterations of B-DNA conformation (Fig. 2). However, at high DOX content (0.5 mM), major shifting of the bands at 211 to 214 and 246 to 240 nm was observed and the band at 280 was split to 270 and 258 nm in the spectra of DOX–DNA complexes (Fig. 2A). The spectral shifting was accompanied with major intensity increase of these bands upon DOX intercalation (Fig. 2A). These spectral shifting are due to a partial B to A-DNA transition upon drug interaction, whereas the major intensity changes observed are due to base destacking as DOX intercalation occurs. Similar spectral changes observed when B to A-DNA conformational changes occurred [29,31]. These spectral shifting was not observed here in the CD spectra of FDOX–DNA complexes and therefore DNA remains in B-family structure upon FDOX complexation (Fig. 2B). The reason for the differences can be due to the blockage of the drug NH2 group in DOX by trifluoroacetyl moiety in FDOX. This emphasizes the important role of free NH2 group in drug-DNA interaction. 3.3. Fluorescence spectra and stability of drug-DNA complexes
Fig. 2. Circular dichroism of the free calf-thymus DNA and its DOX (A) and FDOX complexes (B) in aqueous solution with 2.5 mM DNA concentration and 0.125 to 0.5 mM drug concentrations at pH 7.4.
Intercalation of DOX and FDOX into DNA duplex brought major spectral changes for DNA in-plane vibrational frequencies [11,24–28]. Spectral shifting and intensity increase were observed for the guanine band at 1710, thymine band at 1661 and adenine band at 1610 cm−1 , upon drug complexation. The guanine band at 1710 shifted toward a lower frequency at 1707 (DOX) and 1708 (FDOX), while thymine band at 1661 appeared at 1653 in the spectra of both DOX and FDOX–DNA complexes (Fig. 1, complex 0.5 mM)). The shifting was accompanied by a major intensity increase for these vibrations as shown by positive spectral features at 1700–1683 (guanine), 1653–1649 (thymine), 1512–1513 and 1458 cm−1 (cytosine) in the difference spectra of DOX and FDOX–DNA adducts (Fig. 1 diffs, 0.125 and 0.5 mM). Drug intercalation also induced spectral changes for DNA phosphate group as the band at 1225 (PO2 asymmetric) gained intensity, while the band at 1088 cm−1 (PO2 symmetric) shifted to a lower frequency at 1084 cm−1 , in the spectra of drug-DNA complexes (Fig. 1, complex 0.5 mM). These spectral changes are due to drug intercalation into DNA duplex, which alters the nucleobase vibrational frequencies). Similar infrared spectral changes were observed for DNA when well known intercalating agents ethidium bromide, acridine orange and methylene blue were intercalated into DNA duplex [28]. 3.2. CD spectra and DNA conformation The CD spectra of the free calf-thymus DNA and DOX and FDOX complexes with different drug concentrations are shown in Fig. 2. The CD spectrum of the free DNA composed of four major peaks at 211 (negative), 222 (positive), 246 (negative) and 280 nm (positive) (Fig. 2). This is consistent with CD spectra of double helical DNA in B conformation [29–31]. Upon DOX complexation
Since DNA is a weak fluorophore, the titrations of DOX and FDOX were done against various DNA concentrations, using drug excitation at 480 nm and emission at 500–750 nm [12]. When drug interacts with DNA, fluorescence may change depending on the impact of such interaction on the drug conformation, or via direct quenching effect. The decrease of fluorescence intensity of DOX or FDOX has been monitored at 592 nm for drug-DNA systems (Fig. 3A and B). The plot of F0 /(F0 − F) vs. 1/[DNA] is shown in Fig. 3A and B . Assuming that the observed changes in fluorescence come from the interaction between the drug and DNA, the quenching constant can be taken as the binding constant of the complex formation. The K value obtained is the averages of four and sixreplicate run for drug-polymer systems. Each run involves several different concentrations of DNA (Fig. 3A and B). The overall binding constants KDOX-DNA = 2.5 (±0.5) × 104 M−1 and KFDOX-DNA = 3.4 (±0.7) × 104 M−1 (Fig. 3A and B ) showing more stable complexes formed with FDOX than DOX. The f value calculated from Eq. (7) represents the mole fraction of the accessible population of fluorophore to quencher. The f values were from 0.20 to 0.60 for these drug-DNA complexes indicating a large portion of fluorophore was exposed to quencher. The number of drug molecules bound per polymer (n) is calculated from log [(F0 − F)/F] = logKS + nlog[DNA] for the static quenching [32–35]. The n values from the slope of the straight line plot showed 1.2 (DOX) and 0.6 (FDOX) drug molecules that are bound per DNA molecule (Fig. 4). The results indicate some degree of cooperativity for drug–DNA interaction. To calculate the fluorescence quenching coefficient constant (KQ) for drug-DNA complexes we have plotted F0 /F against Q and the results are show in Fig. 5. The plot of F0 /F versus Q is a straight line for drug-DNA adducts indicating that the quenching is mainly static in these drug-biopolymer complexes (Fig. 5). The quenching constant KQ was estimated according to the Stern–Volmer equation: F0 = 1 + kQ t0 [Q ] = 1 + Ksv [Q ] F
(3)
where F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration and Ksv is the Stern–Volmer quenching constant [36,37], which can be written as Ksv = kQ t0 ; where kQ is the drug quenching rate constant and t0 is the lifetime of the fluorophore in the absence of quencher about 1.1 ns for free DOX and FDOX around neutral pH [14]. The quenching constants (KQ ) are 3.4 × 1013 M−1 /s for DOX–DNA and 1.2 × 1012 M−1 /s for FDOX–DNA complexes (Fig. 5). Since
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Fig. 4. The plot of log(F0 − F)/F as a function of log [DNA] for calculation of number of bound drug molecule (n) per DNA (A) DOX–DNA and (B) FDOX–DNA complexes.
Fig. 3. Fluorescence emission spectra of drug-DNA systems in 10 mM Tris-HCl buffer pH 7.4 at 25 ◦ C for (A) DOX–DNA: (a) free DOX (20 M), (b–j) with DOX–DNA complexes at 5, 10, 15, 20, 30, 40, 50, 60 and 70 M; (B) FDOX–DNA: (a) free FDOX (20 M), (b–j) with FDOX–DNA complexes at 5, 10, 20, 30, 40, 60, 80, 90 and 100 M. Inset: F0 /(F0 − F) vs. 1/[DNA] for A (DOX–DNA) and B (FDOX–DNA).
these values are greater than the maximum collisional quenching constant (1010 M−1 /s), the static quenching is dominant in these drug-DNA complexes [36–38]. 3.4. Docking study and antiproliferative activity on colon carcinoma Our spectroscopic results are accompanied by docking study in which the DOX and FDOX were docked to DNA to determine the preferred binding sites on DNA. The dockings results are shown in Fig. 6 and Table 1. The models show that both DOX and FDOX are intercalated into DNA duplex and surrounded by A-7, C-5, *C19 (H-bonding with doxorubicin NH2 group), G-6, T-8 and T-18 with the free binding energy of −4.99 for DOX and −4.92 kcal/mol Table 1 Deoxyribonucleotides in the vicinity of DOX and FDOX docked with DNA (PDB 6TNA) and the free binding energies of the docked complexes. *Hydrogen bonding involved with this nucleotide. Complex
Deoxyribonucleotides involved in drug interactions
Gbinding (kcal/mol)
DOX–DNA FDOX–DNA
A-7, C-5, *C-19, G-6, T-8, T-18. A-7, C-5, C-19, G-6, T-8, T-18.
−4.99 −4.92
for FDOX–DNA complexes (Fig. 6 and Table 1). The docking results showed the binding sites used are similar for DOX and FDOX with no H-bonding in the FDOX–DNA adduct (Table 1). The binding energy (G) shows more stable complexes formed with DOX than FDOX (Table 1). The extra stability is related to the presence of H-bonding between cytosine-19 base and the NH2 group in DOX–DNA complexes (not present in FDOX–DNA adduct). This further confirms the importance of the free daunosamine amino group in DOX for its optimal interaction with DNA and induction of its biological activity. The results obtained in this study parallel the biological activities of DOX and FDOX obtained on SXC01 human tumour cancer cells using the MTT assay as described earlier [39,40]. The latter colon carcinoma is very sensitive to DOX and thus, was selected as a screen test to assess the possible loss of biological activity of FDOX in comparison to that of DOX. The IC50 for DOX is 73 nM and was found to be twice as active as FDOX showing an IC50 of 156 nM. 4. Discussion Anthracyclines rank among the most effective anticancer drugs ever developed [2]. Doxorubicin is widely used in chemotherapy due to its efficacy in fighting a wide range of cancers. The injury to non-targeted tissues, particularly to the heart, often complicates cancer treatment by limiting doxorubicin dosage and diminishing the patients’ quality of life during and after doxorubicin treatments [1,41]. Furthermore, the development of multiple drug resistance is an additional limitation to the long term use of doxorubicin [1]. Even though the intercalation of doxorubicin into DNA duplex has been studied both experimentally [6] and theoretically [42], much about the importance of role of free NH2 group in drug-DNA
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Fig. 5. Stern–Volmer plots of fluorescence quenching constant (KQ ) for the DNA and its drug complexes at different DNA concentrations (A) DOX–DNA and (B) FDOX–DNA complexes.
interaction has to be investigated. Furthermore, most of the anthracyclines marketed in the world today (doxorubicin, daunorubicin, idarubicin, epirubicin, pirarubicin, zorubicin) contains a free amino functional group [43]. This is no coincidence; it has to play a crucial role for their interaction with DNA. Hence, the aim of our study was to determine the importance of the free daunosamine amino group in DOX–DNA interaction and its biological implication. For this purpose, the amino group of daunosamine was acetylated with S-ethyltrifluoroacetate under standard reaction conditions [8]. This particular derivative was selected in order to change the physicochemical property of the amino group (being a base likely to from H-bonds) into an amide that reduces drastically the basic character of the nitrogen atom. Our infrared results showed no major differences between DOX and FDOX interactions with DNA since both are intercalated into DNA duplex. However, our CD spectroscopic results showed major differences between DOX and FDOX complexes. DOX interaction induced a partial B to A-DNA conformational changes, while FDOX did not alter DNA conformation (Fig. 2). Even docking results showed interaction for both DOX and FDOX, the hydrogen bonding between cytosine-19 and DOX amino group was not present in FDOX–DNA adduct (Table 1). Such H-bonding network brought more stability for DOX–DNA complex than FDOX–DNA adduct. It is proposed that these structural changes are responsible for antitumor activity of doxorubicin and are consistent with the antitumor activities of DOX and FDOX obtained on SXC01 human tumour cancer cells using the MTT assay.
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Fig. 6. Best conformations for drug docked to DNA (PDB entry 6TNA). The drug is shown in green color. (A) shows DNA in stick model with DOX intercalation in sticks and (A ) shows DOX binding sites represented in sticks with the corresponding base residues (B) shows DNA in stick model with FDOX intercalation in sticks and (B ) shows FDOX binding sites represented in sticks.
In conclusion, DOX and FDOX intercalate into DNA duplex causing major DNA structural changes. While DOX intercalation induced a partial B to A-DNA transition, no major structural changes occurred upon FDOX–DNA complexation. The presence of NH2 group on DOX causes major differences between DOX and FDOX interactions with DNA duplex, highlighting the important role of NH2 group in drug-DNA interaction. Hydrophobic and hydrophilic contacts play major role in both DOX and FDOX intercalation. The major DNA conformational changes induced by doxorubicin can be the main reason for its enhanced anticancer activity. Our results substantiate the in vitro biological activity on colon carcinoma showing that DOX is twice as active as FDOX. Acknowledgments This work is supported by grant from Natural Sciences and Engineering Research Council of Canada (NSERC). We wish to thank Dr. C.H.J. Ford for providing the drug in vitro biological activity. References [1] P. Ma, R.J. Mumper, Nano Today 8 (2013) 313–331. [2] C. Carvalho, R.X. Santos, S. Cardoso, S. Correia, P.J. Oliveira, M.S. Santos, P.I. Moreia, Curr. Med. Chem. 16 (2009) 3267–3285. [3] G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, L. Gianni, Pharmacol. Rev. 56 (2004) 185–229. [4] A. Turner, L.C. Li, T. Pilli, L. Qian, E.L. Wiley, S. Setty, K. Christov, L. Ganesh, A.V. Maker, P. Li, P. Kanteti, T.K. D. Gupta, B.S. Prabhakar, PLOS ONE 8 (2013) 1–8, e56817.
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