Journal of Photochemistry and Photobiology B: Biology 130 (2014) 281–286
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Spectroscopic analysis of the interaction of lomustine with calf thymus DNA Shweta Agarwal, Deepak Kumar Jangir, Parul Singh, Ranjana Mehrotra ⇑ Quantum Optics and Photon Physics, CSIR-National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India
a r t i c l e
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Article history: Received 22 July 2013 Received in revised form 9 November 2013 Accepted 16 November 2013 Available online 28 November 2013 Keywords: Lomustine Drug–DNA interaction FTIR spectroscopy CD spectroscopy
a b s t r a c t Investigation of drug–DNA interaction is important for understanding the drug action at molecular level and for designing specific DNA targeted drug. Lomustine (CCNU = 1-[2-chloroethyl]-3-cyclohexyl1-nitroso-urea) is an alkylating antineoplastic nitrosourea derivative, used to treat different types of cancer. In the present study, conformational and structural effects of lomustine on DNA are investigated using different spectroscopic approaches. Different drug/DNA molar ratios are analyzed to determine the binding sites and binding mode of lomustine with DNA. Fourier transform infrared spectroscopic (FTIR) results suggest binding of lomustine with nitrogenous bases guanine and cytosine along with weak interaction to the sugar-phosphate backbone of DNA. Circular dichroism (CD) spectroscopic results show perturbation in the local conformation of DNA upon binding of lomustine with DNA helix. These local conformational changes may act as recognition site for alkylating enzymes that further causes alkylation of DNA. Spectroscopic results confirm the formation of an intermediate stage of DNA that occurs during the transition of B-conformation into A-conformation. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] (Fig. 1) is an alkylating antineoplastic drug, which inhibits cell cycle progression at S- and G2-M phase [1,2]. It is an extremely potent member of nitrosourea (class of anticancer agents) widely used in the treatment of brain tumors, resistant or relapsed Hodgkin’s disease, small cell lung cancer, lymphomas, malignant melanoma and various solid tumors [1–4]. Lomustine is used alone or in combination with radiotherapy and surgery for the treatment of brain tumors and brain metastasis [5]. Solubility of lomustine in alcohol specifies its affinity towards lipids at physiological level; hence, it can cross the blood brain barrier and effectively treat brain tumors [1–3,5,6]. Antineoplastic action of lomustine is believed to involve the inhibition of DNA replication, RNA transcription and protein translation by the means of alkylation [7]. Moreover, lomustine also affects a number of cellular events including ribosomal and nucleoplasmic messenger RNA processing, DNA base structure and DNA polymerase activity [2–4,8]. It has been shown that the primary mechanism of action of lomustine at molecular level involves the alkylation of nitrogenous bases in DNA double helix [5,9–12]. However, till now no experimental data is available on interaction between lomustine and DNA in particular. Drug–DNA interaction studies have been shown to be of significant importance
not only to delineate the interaction mechanism of the drug under investigation but also to provide inputs to rational drug designing efforts [13–16]. Thus, establishment of a correlation between the drug’s molecular structure and its cytotoxicity would be instrumental in the designing and synthesis of new drugs possessing reduced secondary cytotoxicity. It allows the identification of structural motifs of the drug that interact with the DNA. Therefore, it becomes important to understand the mechanism of interaction of drug with DNA in vitro. Recently, FTIR spectroscopy is gaining importance for studying the interaction mechanism of ligand with biomolecules. It offers an excellent way to investigate the alterations arise in the biomolecular conformation due to interaction with ligand [13–17]. Alongwith FTIR, circular dichroism (CD) spectroscopy is suitable to investigate the conformational effects in biomolecule after the complex formation with drug/ligand [18–20]. In the present work, interaction mechanism of lomustine with calf thymus DNA at physiological pH is investigated using Attenuated Total Reflectance-FTIR, difference spectroscopy and circular dichroism spectroscopic techniques.
2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel.: +91 11 45608366; fax: +91 11 45609310. E-mail address: [email protected] (R. Mehrotra). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.11.017
Lomustine (M.W-233.695) and highly polymerized type I calf thymus DNA (sodium content 6%) are procured from Sigma–Aldrich
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Fig. 2. FTIR spectrum of lomustine.
Fig. 1. Chemical structure of lomustine.
chemicals, USA. Purity of DNA is determined by taking ratio of the absorbance of DNA at 260 nm (A260) and 280 nm (A280). The calculated ratio of (A260)/(A280) in DNA sample is found to be 1.82 indicating sufficient purity of DNA. Other chemicals and reagents used in the study are of the analytical grade and used without any further purification. Deionized ultra pure water (resistance 18.2 MX) from Scholar-UV Nex UP 1000 system is used for the preparation of buffer solution and lomustine drug solution. 2.2. Preparation of stock solutions Stock solution of DNA is prepared in Tris–HCl buffer (pH-7.4, 10 mM). 15 mg of calf thymus DNA is dissolved per milliliter of buffer and kept at 8 °C for 24 h. The solution is stirred at the regular intervals to make sure the formation of homogeneous DNA solution. Final concentration of the DNA stock solution is measured spectrophotometrically using extinction coefficient of 6600 cm1 M1 [21–24]. The estimated final concentration of DNA stock solution is 42 mM (molarity of phosphate group). Lomustine solution of varying concentrations are prepared by a series of dilutions of stock solution. For the FTIR studies, lomustine solutions of different concentrations so prepared are added dropwise to DNA solutions of constant concentration to get 1/20, 1/40 and 1/60 M ratios. This is followed by continuous vortexing for 15 min and incubation at room temperature for two hours to facilitate the complete complexation of drug–DNA. Circular dichroism spectroscopic measurements are performed using drug solution of various concentrations in the range of 0.08–0.25 mM with constant DNA concentration of 2.5 mM followed by two hours incubation at room temperature. 2.3. FTIR spectroscopic measurements FTIR spectra of free DNA and lomustine–DNA complexes are recorded on Varian-660-IR spectrophotometer equipped with deuterated triglycine sulfate (DTGS) detector and KBr beam splitter. All the spectra are collected in 10 mM Tris–HCl buffer as a solvent (pH 7.4). Sampling is done in ATR mode using PIKE micro horizontal attenuated total internal reflection (HATR) assembly with tightly packed ZnSe crystal. All the spectra are collected in the range of 2400–700 cm1 by averaging two hundred fifty-six scans with a resolution of 4 cm1. Background spectra are collected with ZnSe before each measurement. All the spectra are baseline corrected and normalized for DNA band at 967 cm1 [25–28]. To perform water subtraction, a spectrum of tris buffer is recorded and then subtracted from the spectra of free DNA and drug–DNA complexes. A satisfactory water subtraction is achieved when the intensity of water combination band at about 2200 cm1 became zero in the spectra of free DNA and drug–DNA complexes [29]. Infrared spectrum of free lomustine is also recorded (Fig. 2) and
subtracted from the spectra of drug–DNA complexes. This is done to make sure that the observed changes (shift in peak position and intensity) in DNA are due to interaction with lomustine. 2.4. Circular dichroism spectroscopic measurement CD spectroscopic measurements are performed on Applied Photophysics (Chirascan) spectrophotometer. Quartz cuvette with a pathlength of 1 mm is used for spectral collection in the far UV range (200–320 nm). All the measurements are taken at room temperature. Five scans are collected for each sample with a scan speed of 1 nm/s and then averaged. A spectrum of buffer solution is recorded and subtracted from the spectra of DNA and lomustine–DNA complexes. 3. Results and discussion 3.1. FTIR spectral outcome 3.1.1. Interaction with nitrogenous bases The infrared spectral features observed in the spectrum of calf thymus DNA in the free and complex form due to its interaction with lomustine are shown in Fig. 3. Phosphate (PO2) stretching vibrations (symmetric and asymmetric), deoxyribose sugar stretching of phosphate-sugar backbone and ring vibrations of nitrogenous bases (C@O, C@N stretching) of DNA are confined from 1800 cm1 to 700 cm1 infrared region. The band at 1712 cm1 is due to the in plane vibrations of guanine (G) stretching [30–33]. The band at 1659 cm1 is attributed primarily to thymine (T) stretching vibrations [30–33]. The bands at 1607 cm1 and 1493 cm1 appear due to the ring stretching vibrations of adenine and cytosine respectively [30–33]. The band emerges at 1528 cm1 is assigned to in plane stretching vibrations of guanine and cytosine residues [30–33]. Infrared band observed in the spectrum of free DNA at 1712 cm1 (guanine) shows major downshift of 5 cm1 (1712 cm1 to 1707 cm1) at lomustine/DNA molar ratio of 1/20 and 1/40 while 4 cm1 downshift is observed at lowest drug/DNA molar ratio (1/60). Infrared band observed at 1493 cm1 (cytosine) in the spectrum of free calf thymus DNA also shifts downward to 1490 cm1 at all the drug/DNA molar ratios. Band at 1528 cm1 assigned to stretching vibrations of guanine-cytosine residues shows downshift of 2 cm1 after the formation of drug–DNA complexes. Minor upward shift of 2 cm1is observed for the infrared band around 1659 cm1 (thymine) at the molar ratio of 1/40 while at other drug/DNA molar ratios (1/20 and 1/60) shift of 3 cm1is observed. No shift is observed in the band of adenine at 1607 cm1 in all the drug/DNA complexes at different molar ratios. Shifts in the band positions for the nitrogenous bases of DNA are also accompanied by the changes in intensities at all molar ratios of lomustine– DNA complexes. Difference spectra of lomustine–DNA complexes [(DNA solution + lomustine solution) – DNA solution] (Fig. 4) show
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Fig. 3. Stacked view of FTIR spectra in the region of 1800–700 cm1 for free calf-thymus DNA and lomustine–DNA complexes at different molar ratio.
Fig. 4. Difference spectra of lomustine–DNA complexes in the region of 700–1800 cm1.
the remarkable negative band at 1718 cm1 that specifies major reduction in intensity of guanine absorptions. Positive bands are also observed around 1653 cm1 and 1489 cm1. These positive features can be attributed to the infrared hyperchroism for the stretching vibrations of thymine and cytosine respectively [30– 33]. Increment in intensity is also observed for the stretching vibrations of guanine and cytosine at 1528 cm1; indicated by the positive band in the difference spectra of lomustine–DNA complexes. No change in the intensity of adenine (at 1602 cm1) is observed at all the three drug/DNA molar ratios. These band shift and variations in intensity can be attributed to the direct interaction of lomustine with heterocyclic bases of DNA [15–18,31–33]. Variations in intensity and shift in the bands associated with guanine and cytosine suggest direct interaction of lomustine with the active sites of guanine and cytosine in the minor and major groove of DNA double helix [13]. The plausible explanation of these results
is that the reactive form of lomustine binds with DNA via guanine and transfers its chloroethyl moiety to guanine. Spectral results confirm no interaction of drug with adenine as no shift and intensity variation is observed at 1607 cm1. Spectral features appear in the spectra of DNA after binding with lomustine reveal minor interaction of drug with nitrogenous base thymine, evident by shift and intensity variation of infrared band at 1659 cm1 [30–33]. There are reports that suggest lomustine undergoes oxidation and form geometric monohydroxylated isomers [3]. These monohydroxylated metabolites (trans-40 -CCNU and cis-40 -CCNU, shown in Fig. 5) could either bound to O6 position of guanine (produces O6-chloroethyl guanine) or N7 atom of guanine (produces N7-chloroehtly guanine) [3–5,9]. O6-chloroethyl guanine can stimulate the formation of intramolecular N1-O6-ethano guanine adduct which subsequently should react with N3 of adjacent cytosine (in complementary strand) to produce G-C crosslink (1-(3-cytosinyl)-2-(1-
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Fig. 5. Chemical structure of (A) trans-40 -lomustine and (B) cis-40 -lomustine.
guanosinyl)-ethane) as a secondary product [2–5,9]. Lomustine binding and cross-linking make DNA unable to undergo replication and ultimately stops its normal functioning. The band at about 1578 cm1is attributed to in plane C8@N7 stretching vibrations of purine ring mainly guanine residues [34]. This band shifts downward by 3 cm1, which is an indication of complexation with guanine-N7 residues. Conclusively shift and intensity variations in the band of cytosine (1493 cm1) and guanine-cytosine (1528 cm1) in the infrared spectra of drug/DNA complexes affirm interaction of these nitrogenous bases of DNA with the drug [3–5,34]. 3.1.2. Interaction with phosphate-sugar backbone In the spectrum of free calf thymus DNA (Fig. 3), infrared bands arise at 1220 cm1 and 1084 cm1 are due to the vibrations caused by phosphate asymmetric and symmetric stretching respectively [13–16]. Infrared band attributed to phosphate asymmetric vibrations (1220 cm1) shows minor upward shift of 2 cm1 while the band assigned to phosphate symmetric vibrations (1084 cm1) shifts downward by 1 cm1 on complexation. However, in the difference spectra, (Fig. 4) negative and positive bands are observed at 1220 cm1 and 1099 cm1 respectively. It indicates variations in intensity of phosphate symmetric and asymmetric vibrations. Deoxyribose sugar vibrations due to C@O and CAC stretching is denoted by infrared bands at 1051 cm1 and 967 cm1 respectively in the spectrum of free calf thymus DNA [35]. After addition of lomustine, both sugar bands show downward shift of 1 cm1 alongwith increase in intensity (positive bands at 1044 cm1 and 957 cm1 in difference spectra). It suggests minor external binding of drug with phosphate-sugar backbone of DNA double helix. Infrared band at 837 cm1 is primarily attributed to the vibrations of phosphodiester bonds [13–15,35]. This band shows minor shift (2 cm1 downward) at higher drug/DNA ratios proving slight binding of lomustine with backbone of DNA. Other bands at 1373 cm1, 1295 cm1, 779 cm1 and 727 cm1 that are attributed to sugar conformations, show minor shifts when the lomustine–DNA interaction takes place [35,36]. In the difference spectra of lomustine– DNA complexes, positive features are observed around at 1370, 780 and 734 cm1 due to the increase in intensity of sugar vibrations respectively. Spectral changes observed for the sugar conformations, deoxyribose-phosphodiester chain vibrations and sugar-phosphate backbone stretching vibrations might be attributed to the slight external binding of lomustine with DNA double helix [16–18]. 3.1.3. Changeover in DNA double helix conformation Infrared band at 837 cm1 is considered to be a B-DNA marker band that is attributed to S-C2 endo/anti sugar pucker-phosphodiester mode. Besides this, band at 1220 cm1 (assigned to antisymmetric phosphate stretching) and 893 cm1 (assigned to sugar-phosphate stretch) also regarded as B-DNA marker band [35–37]. Moreover, the band at 1461 cm1 that is assigned to CAN glycosyl bond, is also responsible for the B-conformation of DNA double helix. While A-form of DNA shows band in higher fre-
quency region from 1230 to 1240 cm1 [37] for the antisymmetric phosphate stretching, which is absent in the spectra of lomustine/ DNA complexes at all molar ratio (Fig. 3). Minor shift of 2 cm1 (893–895 cm1 and 837–835 cm1) along with increase in intensity (positive band at 897 cm1) is observed on complexation, which indicates local perturbation in DNA conformation. Band at 1461 cm1 shows 4 cm1 downshift after interaction of DNA with lomustine, indicating presence of some A-type conformation of DNA double helix. Moreover, in all the spectra of drug/DNA complexes a new band originates at 861 cm1 that is a definitive A-form marker band [35]. Generation of new characteristic A-form marker bands is coupled with intensity variation as evident by positive bands at 1457 cm1 and 864 cm1. Besides this, positive bands are also observed at 1244 cm1 (antisymmetric phosphate stretching in A-form of DNA) and 1184 cm1 (sugar-phosphate backbone with sugar moiety in C-30 endo/anti type of puckering, A-form) in the difference spectra of drug/DNA complexes [35–37]. Spectral features observed here are not entirely representative of either A-form or B-form of DNA but incorporation of both conformations. This should be due to the formation of an intermediate stage during the transition of B-form of DNA into A-form [34]. From the results, it has been cleared that drug interaction with DNA does not induce any final transitions in DNA double helix conformation and ultimately DNA remains in an intermediate B-A form with more towards B-conformation. 3.2. Circular dichroism spectral outcome 3.2.1. Conformational analysis in DNA double helix Circular dichroism spectra of free calf thymus DNA and lomustine–DNA complexes at different molar ratios were collected as a function of time to get detailed insights in the alkylation mechanism and conformational changes in DNA (Fig. 6). Generally, DNA possesses B-DNA form, which has a conservative CD spectrum comprised of positive as well as negative bands. CD spectrum of free B-DNA has four major characteristic bands: 214 nm (negative), 224 nm (positive), 245 nm (negative) and 277 nm (positive) [13,14,18,19]. These B-DNA markers bands appear due to helical arrangements of its component and asymmetric sugar-phosphate backbone. Band at 277 nm occurs due to right-handedness of BDNA helix while the band observed at 245 nm is attributed to the stacking interactions between nucleic acid bases. Appearance of the bands at 224 nm depends upon hydrogen bonding interactions between bases of the two helices and band at 214 nm arises due to interaction between the nucleic acid bases and deoxyribose sugar respectively [38,39]. Thus, the alterations in position and in intensity of these spectral bands are used to identify the conformational transitions in double helix due to interaction with any molecule/ligand [18]. CD spectra collected immediately after mixing of lomustine and DNA (at 0 h) shows no significant changes in the B-DNA bands at all the molar ratios except the highest one (ratio 1:20). The band attributed to right handed helicity (at 277 nm) shows increase in intensity alongwith minor 3 nm blue shift at 1:20 M ratio (red line). These changes indicate initiation of alternation in duplex helicity due to lomustine interaction and signify early stages of lomustine binding with DNA, where drug molecule forms reversible non-covalent complex and attempt to make stable contact with nucleic acid [40]. After ½ h of incubation, band assigned to base stacking (at 245 nm) shows decrease in negative ellipticity along with hypsochromic shift at 1:20 and 1:40 M ratios while no change is observed for lowest ratio i.e. 1:60 (blue line). Besides this, the band at 277 nm (ascribed to helicity) shows increase in positive ellipticity accompanied with blue shift at 1:20 and 1:40 M ratios. Minor increase in positive ellipticity is also observed at the band
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Fig. 6. Circular dichroism spectra of the free calf-thymus DNA and lomustine/DNA ratios of 1/60 (blue line), 1/40 (green line) and 1/20 (red line) as a function of incubation time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
224 nm (refer to hydrogen bonding). CD spectra recorded at 1 h represents the formation of an intermediate complex with DNA [40]. At 2 h of incubation of lomustine–DNA complexes, CD spectra show maximum changes in band intensities and positions. Band attributed to base stacking (at 245 nm) shows hypsochromic shift along with the decrease in negative ellipticity in all the three molar ratios of drug–DNA complexes. Highest molar ratio of complex, i.e. 1:20 shows negative minimum of ellipticity (red line) with the blue shift of 5 nm (from 245 nm to 240 nm). Blue shift and decrease in negative intensity observed for this band can be attributed to the changes in local base pair geometry and reduction in stacking interaction between the bases of double helix upon the lomustine–DNA complex formation respectively [38–41]. These observed phenomena might be due to intercalation of drug between the base pair of nucleic acid during the formation of G-C crosslink. No appreciable changes in CD bands at 224 nm and 214 nm are observed. However, slight increase is evident in molar ellipticity with increasing concentration of lomustine. This subtle alteration for these bands should be due to the reduction of water around DNA due to the presence of additional hydrophobic alkyl groups. These alkyl groups not only protrude the water around DNA, but also cause the loss of the hydrogen bonding resulting in the formation of loose conformation [14,19]. Band observed at 277 nm in CD spectrum of free calf thymus DNA shows hypsochromic shift (from 277 nm to 260 nm) alongwith increase in positive ellipticity after DNA complexation. Positive band at 260 nm is the marker band of A-conformation of DNA [41,42]. Although, other characteristic bands in the CD spectrum for A-DNA conformation (negative band at 210 nm and positive band at 195 nm) are not observed. However, blue shift and increase in intensity in CD spectral band at 277 nm is due to local structural distortion in B-DNA. No appreciable alteration (shifts and intensity variations) is observed in CD spectra at 3 and 4 h incubations rather all the molar ratios show nearly uniform shift
and ellipticity changes. These local structural distortions may lead to the formation of an intermediate stage of DNA during the transition from B- to Aconformation [38–43]. These observations are in accordance with our FTIR spectroscopic results, which show formation of an intermediate stage of DNA upon interaction with drug. Localized structural distortion in native B-DNA conformation is suggestive of perturbation of only few base pairs of DNA; where the binding of lomustine takes place. These local variations are of considerable significance for the occurrence of DNA–protein and DNA-ligand recognition processes. It has been reported in literature that DNA adopts A-DNA conformation locally during the processes of replication and transcription, which is necessary for the correctness and fidelity of these phenomena [44]. These local conformational changes can act as recognition patterning for alkylating enzymes alongwith lomustine binding at the site of interaction. This assumption should be acceptable because ‘‘enzymes and other ligands generally recognize the three-dimensional structure of DNA rather its direct read-out’’ [43–45].
4. Conclusion In the present work, we investigated the interaction of antitumor drug lomustine with DNA double helix using various spectroscopic techniques. Our spectroscopic observations indicate the major/minor groove binding with alkylating mode of action. FTIR results suggest that lomustine interaction occurs via guanine (N7, O6) and cytosine (C4) along with slight binding with sugar-phosphate backbone of DNA. The CD spectroscopic observation suggests the formation of an intermediate stage (B-A-form) of DNA double helix after its interaction with lomustine. These findings can contribute to further understanding the action mode of lomustine at molecular level. Finally, we can conclude that interaction studies
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are fundamental to unravel the mystery of molecular recognition in general and DNA binding in particular.
Acknowledgement The authors thank Director, CSIR-National Physical Laboratory for granting the permission for publication of the work. S.A., D.K.J. and P.S. is thankful to Council of Scientific & Industrial Research for financial support.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Spectroscopic analysis of the interaction of lomustine with calf thymus DNA Shweta Agarwal, Deepak Kumar Jangir, Parul Singh, Ranjana Mehrotra ⇑ Quantum Optics and Photon Physics, CSIR-National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India
a r t i c l e
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Article history: Received 22 July 2013 Received in revised form 9 November 2013 Accepted 16 November 2013 Available online 28 November 2013 Keywords: Lomustine Drug–DNA interaction FTIR spectroscopy CD spectroscopy
a b s t r a c t Investigation of drug–DNA interaction is important for understanding the drug action at molecular level and for designing specific DNA targeted drug. Lomustine (CCNU = 1-[2-chloroethyl]-3-cyclohexyl1-nitroso-urea) is an alkylating antineoplastic nitrosourea derivative, used to treat different types of cancer. In the present study, conformational and structural effects of lomustine on DNA are investigated using different spectroscopic approaches. Different drug/DNA molar ratios are analyzed to determine the binding sites and binding mode of lomustine with DNA. Fourier transform infrared spectroscopic (FTIR) results suggest binding of lomustine with nitrogenous bases guanine and cytosine along with weak interaction to the sugar-phosphate backbone of DNA. Circular dichroism (CD) spectroscopic results show perturbation in the local conformation of DNA upon binding of lomustine with DNA helix. These local conformational changes may act as recognition site for alkylating enzymes that further causes alkylation of DNA. Spectroscopic results confirm the formation of an intermediate stage of DNA that occurs during the transition of B-conformation into A-conformation. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] (Fig. 1) is an alkylating antineoplastic drug, which inhibits cell cycle progression at S- and G2-M phase [1,2]. It is an extremely potent member of nitrosourea (class of anticancer agents) widely used in the treatment of brain tumors, resistant or relapsed Hodgkin’s disease, small cell lung cancer, lymphomas, malignant melanoma and various solid tumors [1–4]. Lomustine is used alone or in combination with radiotherapy and surgery for the treatment of brain tumors and brain metastasis [5]. Solubility of lomustine in alcohol specifies its affinity towards lipids at physiological level; hence, it can cross the blood brain barrier and effectively treat brain tumors [1–3,5,6]. Antineoplastic action of lomustine is believed to involve the inhibition of DNA replication, RNA transcription and protein translation by the means of alkylation [7]. Moreover, lomustine also affects a number of cellular events including ribosomal and nucleoplasmic messenger RNA processing, DNA base structure and DNA polymerase activity [2–4,8]. It has been shown that the primary mechanism of action of lomustine at molecular level involves the alkylation of nitrogenous bases in DNA double helix [5,9–12]. However, till now no experimental data is available on interaction between lomustine and DNA in particular. Drug–DNA interaction studies have been shown to be of significant importance
not only to delineate the interaction mechanism of the drug under investigation but also to provide inputs to rational drug designing efforts [13–16]. Thus, establishment of a correlation between the drug’s molecular structure and its cytotoxicity would be instrumental in the designing and synthesis of new drugs possessing reduced secondary cytotoxicity. It allows the identification of structural motifs of the drug that interact with the DNA. Therefore, it becomes important to understand the mechanism of interaction of drug with DNA in vitro. Recently, FTIR spectroscopy is gaining importance for studying the interaction mechanism of ligand with biomolecules. It offers an excellent way to investigate the alterations arise in the biomolecular conformation due to interaction with ligand [13–17]. Alongwith FTIR, circular dichroism (CD) spectroscopy is suitable to investigate the conformational effects in biomolecule after the complex formation with drug/ligand [18–20]. In the present work, interaction mechanism of lomustine with calf thymus DNA at physiological pH is investigated using Attenuated Total Reflectance-FTIR, difference spectroscopy and circular dichroism spectroscopic techniques.
2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel.: +91 11 45608366; fax: +91 11 45609310. E-mail address: [email protected] (R. Mehrotra). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.11.017
Lomustine (M.W-233.695) and highly polymerized type I calf thymus DNA (sodium content 6%) are procured from Sigma–Aldrich
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Fig. 2. FTIR spectrum of lomustine.
Fig. 1. Chemical structure of lomustine.
chemicals, USA. Purity of DNA is determined by taking ratio of the absorbance of DNA at 260 nm (A260) and 280 nm (A280). The calculated ratio of (A260)/(A280) in DNA sample is found to be 1.82 indicating sufficient purity of DNA. Other chemicals and reagents used in the study are of the analytical grade and used without any further purification. Deionized ultra pure water (resistance 18.2 MX) from Scholar-UV Nex UP 1000 system is used for the preparation of buffer solution and lomustine drug solution. 2.2. Preparation of stock solutions Stock solution of DNA is prepared in Tris–HCl buffer (pH-7.4, 10 mM). 15 mg of calf thymus DNA is dissolved per milliliter of buffer and kept at 8 °C for 24 h. The solution is stirred at the regular intervals to make sure the formation of homogeneous DNA solution. Final concentration of the DNA stock solution is measured spectrophotometrically using extinction coefficient of 6600 cm1 M1 [21–24]. The estimated final concentration of DNA stock solution is 42 mM (molarity of phosphate group). Lomustine solution of varying concentrations are prepared by a series of dilutions of stock solution. For the FTIR studies, lomustine solutions of different concentrations so prepared are added dropwise to DNA solutions of constant concentration to get 1/20, 1/40 and 1/60 M ratios. This is followed by continuous vortexing for 15 min and incubation at room temperature for two hours to facilitate the complete complexation of drug–DNA. Circular dichroism spectroscopic measurements are performed using drug solution of various concentrations in the range of 0.08–0.25 mM with constant DNA concentration of 2.5 mM followed by two hours incubation at room temperature. 2.3. FTIR spectroscopic measurements FTIR spectra of free DNA and lomustine–DNA complexes are recorded on Varian-660-IR spectrophotometer equipped with deuterated triglycine sulfate (DTGS) detector and KBr beam splitter. All the spectra are collected in 10 mM Tris–HCl buffer as a solvent (pH 7.4). Sampling is done in ATR mode using PIKE micro horizontal attenuated total internal reflection (HATR) assembly with tightly packed ZnSe crystal. All the spectra are collected in the range of 2400–700 cm1 by averaging two hundred fifty-six scans with a resolution of 4 cm1. Background spectra are collected with ZnSe before each measurement. All the spectra are baseline corrected and normalized for DNA band at 967 cm1 [25–28]. To perform water subtraction, a spectrum of tris buffer is recorded and then subtracted from the spectra of free DNA and drug–DNA complexes. A satisfactory water subtraction is achieved when the intensity of water combination band at about 2200 cm1 became zero in the spectra of free DNA and drug–DNA complexes [29]. Infrared spectrum of free lomustine is also recorded (Fig. 2) and
subtracted from the spectra of drug–DNA complexes. This is done to make sure that the observed changes (shift in peak position and intensity) in DNA are due to interaction with lomustine. 2.4. Circular dichroism spectroscopic measurement CD spectroscopic measurements are performed on Applied Photophysics (Chirascan) spectrophotometer. Quartz cuvette with a pathlength of 1 mm is used for spectral collection in the far UV range (200–320 nm). All the measurements are taken at room temperature. Five scans are collected for each sample with a scan speed of 1 nm/s and then averaged. A spectrum of buffer solution is recorded and subtracted from the spectra of DNA and lomustine–DNA complexes. 3. Results and discussion 3.1. FTIR spectral outcome 3.1.1. Interaction with nitrogenous bases The infrared spectral features observed in the spectrum of calf thymus DNA in the free and complex form due to its interaction with lomustine are shown in Fig. 3. Phosphate (PO2) stretching vibrations (symmetric and asymmetric), deoxyribose sugar stretching of phosphate-sugar backbone and ring vibrations of nitrogenous bases (C@O, C@N stretching) of DNA are confined from 1800 cm1 to 700 cm1 infrared region. The band at 1712 cm1 is due to the in plane vibrations of guanine (G) stretching [30–33]. The band at 1659 cm1 is attributed primarily to thymine (T) stretching vibrations [30–33]. The bands at 1607 cm1 and 1493 cm1 appear due to the ring stretching vibrations of adenine and cytosine respectively [30–33]. The band emerges at 1528 cm1 is assigned to in plane stretching vibrations of guanine and cytosine residues [30–33]. Infrared band observed in the spectrum of free DNA at 1712 cm1 (guanine) shows major downshift of 5 cm1 (1712 cm1 to 1707 cm1) at lomustine/DNA molar ratio of 1/20 and 1/40 while 4 cm1 downshift is observed at lowest drug/DNA molar ratio (1/60). Infrared band observed at 1493 cm1 (cytosine) in the spectrum of free calf thymus DNA also shifts downward to 1490 cm1 at all the drug/DNA molar ratios. Band at 1528 cm1 assigned to stretching vibrations of guanine-cytosine residues shows downshift of 2 cm1 after the formation of drug–DNA complexes. Minor upward shift of 2 cm1is observed for the infrared band around 1659 cm1 (thymine) at the molar ratio of 1/40 while at other drug/DNA molar ratios (1/20 and 1/60) shift of 3 cm1is observed. No shift is observed in the band of adenine at 1607 cm1 in all the drug/DNA complexes at different molar ratios. Shifts in the band positions for the nitrogenous bases of DNA are also accompanied by the changes in intensities at all molar ratios of lomustine– DNA complexes. Difference spectra of lomustine–DNA complexes [(DNA solution + lomustine solution) – DNA solution] (Fig. 4) show
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Fig. 3. Stacked view of FTIR spectra in the region of 1800–700 cm1 for free calf-thymus DNA and lomustine–DNA complexes at different molar ratio.
Fig. 4. Difference spectra of lomustine–DNA complexes in the region of 700–1800 cm1.
the remarkable negative band at 1718 cm1 that specifies major reduction in intensity of guanine absorptions. Positive bands are also observed around 1653 cm1 and 1489 cm1. These positive features can be attributed to the infrared hyperchroism for the stretching vibrations of thymine and cytosine respectively [30– 33]. Increment in intensity is also observed for the stretching vibrations of guanine and cytosine at 1528 cm1; indicated by the positive band in the difference spectra of lomustine–DNA complexes. No change in the intensity of adenine (at 1602 cm1) is observed at all the three drug/DNA molar ratios. These band shift and variations in intensity can be attributed to the direct interaction of lomustine with heterocyclic bases of DNA [15–18,31–33]. Variations in intensity and shift in the bands associated with guanine and cytosine suggest direct interaction of lomustine with the active sites of guanine and cytosine in the minor and major groove of DNA double helix [13]. The plausible explanation of these results
is that the reactive form of lomustine binds with DNA via guanine and transfers its chloroethyl moiety to guanine. Spectral results confirm no interaction of drug with adenine as no shift and intensity variation is observed at 1607 cm1. Spectral features appear in the spectra of DNA after binding with lomustine reveal minor interaction of drug with nitrogenous base thymine, evident by shift and intensity variation of infrared band at 1659 cm1 [30–33]. There are reports that suggest lomustine undergoes oxidation and form geometric monohydroxylated isomers [3]. These monohydroxylated metabolites (trans-40 -CCNU and cis-40 -CCNU, shown in Fig. 5) could either bound to O6 position of guanine (produces O6-chloroethyl guanine) or N7 atom of guanine (produces N7-chloroehtly guanine) [3–5,9]. O6-chloroethyl guanine can stimulate the formation of intramolecular N1-O6-ethano guanine adduct which subsequently should react with N3 of adjacent cytosine (in complementary strand) to produce G-C crosslink (1-(3-cytosinyl)-2-(1-
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Fig. 5. Chemical structure of (A) trans-40 -lomustine and (B) cis-40 -lomustine.
guanosinyl)-ethane) as a secondary product [2–5,9]. Lomustine binding and cross-linking make DNA unable to undergo replication and ultimately stops its normal functioning. The band at about 1578 cm1is attributed to in plane C8@N7 stretching vibrations of purine ring mainly guanine residues [34]. This band shifts downward by 3 cm1, which is an indication of complexation with guanine-N7 residues. Conclusively shift and intensity variations in the band of cytosine (1493 cm1) and guanine-cytosine (1528 cm1) in the infrared spectra of drug/DNA complexes affirm interaction of these nitrogenous bases of DNA with the drug [3–5,34]. 3.1.2. Interaction with phosphate-sugar backbone In the spectrum of free calf thymus DNA (Fig. 3), infrared bands arise at 1220 cm1 and 1084 cm1 are due to the vibrations caused by phosphate asymmetric and symmetric stretching respectively [13–16]. Infrared band attributed to phosphate asymmetric vibrations (1220 cm1) shows minor upward shift of 2 cm1 while the band assigned to phosphate symmetric vibrations (1084 cm1) shifts downward by 1 cm1 on complexation. However, in the difference spectra, (Fig. 4) negative and positive bands are observed at 1220 cm1 and 1099 cm1 respectively. It indicates variations in intensity of phosphate symmetric and asymmetric vibrations. Deoxyribose sugar vibrations due to C@O and CAC stretching is denoted by infrared bands at 1051 cm1 and 967 cm1 respectively in the spectrum of free calf thymus DNA [35]. After addition of lomustine, both sugar bands show downward shift of 1 cm1 alongwith increase in intensity (positive bands at 1044 cm1 and 957 cm1 in difference spectra). It suggests minor external binding of drug with phosphate-sugar backbone of DNA double helix. Infrared band at 837 cm1 is primarily attributed to the vibrations of phosphodiester bonds [13–15,35]. This band shows minor shift (2 cm1 downward) at higher drug/DNA ratios proving slight binding of lomustine with backbone of DNA. Other bands at 1373 cm1, 1295 cm1, 779 cm1 and 727 cm1 that are attributed to sugar conformations, show minor shifts when the lomustine–DNA interaction takes place [35,36]. In the difference spectra of lomustine– DNA complexes, positive features are observed around at 1370, 780 and 734 cm1 due to the increase in intensity of sugar vibrations respectively. Spectral changes observed for the sugar conformations, deoxyribose-phosphodiester chain vibrations and sugar-phosphate backbone stretching vibrations might be attributed to the slight external binding of lomustine with DNA double helix [16–18]. 3.1.3. Changeover in DNA double helix conformation Infrared band at 837 cm1 is considered to be a B-DNA marker band that is attributed to S-C2 endo/anti sugar pucker-phosphodiester mode. Besides this, band at 1220 cm1 (assigned to antisymmetric phosphate stretching) and 893 cm1 (assigned to sugar-phosphate stretch) also regarded as B-DNA marker band [35–37]. Moreover, the band at 1461 cm1 that is assigned to CAN glycosyl bond, is also responsible for the B-conformation of DNA double helix. While A-form of DNA shows band in higher fre-
quency region from 1230 to 1240 cm1 [37] for the antisymmetric phosphate stretching, which is absent in the spectra of lomustine/ DNA complexes at all molar ratio (Fig. 3). Minor shift of 2 cm1 (893–895 cm1 and 837–835 cm1) along with increase in intensity (positive band at 897 cm1) is observed on complexation, which indicates local perturbation in DNA conformation. Band at 1461 cm1 shows 4 cm1 downshift after interaction of DNA with lomustine, indicating presence of some A-type conformation of DNA double helix. Moreover, in all the spectra of drug/DNA complexes a new band originates at 861 cm1 that is a definitive A-form marker band [35]. Generation of new characteristic A-form marker bands is coupled with intensity variation as evident by positive bands at 1457 cm1 and 864 cm1. Besides this, positive bands are also observed at 1244 cm1 (antisymmetric phosphate stretching in A-form of DNA) and 1184 cm1 (sugar-phosphate backbone with sugar moiety in C-30 endo/anti type of puckering, A-form) in the difference spectra of drug/DNA complexes [35–37]. Spectral features observed here are not entirely representative of either A-form or B-form of DNA but incorporation of both conformations. This should be due to the formation of an intermediate stage during the transition of B-form of DNA into A-form [34]. From the results, it has been cleared that drug interaction with DNA does not induce any final transitions in DNA double helix conformation and ultimately DNA remains in an intermediate B-A form with more towards B-conformation. 3.2. Circular dichroism spectral outcome 3.2.1. Conformational analysis in DNA double helix Circular dichroism spectra of free calf thymus DNA and lomustine–DNA complexes at different molar ratios were collected as a function of time to get detailed insights in the alkylation mechanism and conformational changes in DNA (Fig. 6). Generally, DNA possesses B-DNA form, which has a conservative CD spectrum comprised of positive as well as negative bands. CD spectrum of free B-DNA has four major characteristic bands: 214 nm (negative), 224 nm (positive), 245 nm (negative) and 277 nm (positive) [13,14,18,19]. These B-DNA markers bands appear due to helical arrangements of its component and asymmetric sugar-phosphate backbone. Band at 277 nm occurs due to right-handedness of BDNA helix while the band observed at 245 nm is attributed to the stacking interactions between nucleic acid bases. Appearance of the bands at 224 nm depends upon hydrogen bonding interactions between bases of the two helices and band at 214 nm arises due to interaction between the nucleic acid bases and deoxyribose sugar respectively [38,39]. Thus, the alterations in position and in intensity of these spectral bands are used to identify the conformational transitions in double helix due to interaction with any molecule/ligand [18]. CD spectra collected immediately after mixing of lomustine and DNA (at 0 h) shows no significant changes in the B-DNA bands at all the molar ratios except the highest one (ratio 1:20). The band attributed to right handed helicity (at 277 nm) shows increase in intensity alongwith minor 3 nm blue shift at 1:20 M ratio (red line). These changes indicate initiation of alternation in duplex helicity due to lomustine interaction and signify early stages of lomustine binding with DNA, where drug molecule forms reversible non-covalent complex and attempt to make stable contact with nucleic acid [40]. After ½ h of incubation, band assigned to base stacking (at 245 nm) shows decrease in negative ellipticity along with hypsochromic shift at 1:20 and 1:40 M ratios while no change is observed for lowest ratio i.e. 1:60 (blue line). Besides this, the band at 277 nm (ascribed to helicity) shows increase in positive ellipticity accompanied with blue shift at 1:20 and 1:40 M ratios. Minor increase in positive ellipticity is also observed at the band
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Fig. 6. Circular dichroism spectra of the free calf-thymus DNA and lomustine/DNA ratios of 1/60 (blue line), 1/40 (green line) and 1/20 (red line) as a function of incubation time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
224 nm (refer to hydrogen bonding). CD spectra recorded at 1 h represents the formation of an intermediate complex with DNA [40]. At 2 h of incubation of lomustine–DNA complexes, CD spectra show maximum changes in band intensities and positions. Band attributed to base stacking (at 245 nm) shows hypsochromic shift along with the decrease in negative ellipticity in all the three molar ratios of drug–DNA complexes. Highest molar ratio of complex, i.e. 1:20 shows negative minimum of ellipticity (red line) with the blue shift of 5 nm (from 245 nm to 240 nm). Blue shift and decrease in negative intensity observed for this band can be attributed to the changes in local base pair geometry and reduction in stacking interaction between the bases of double helix upon the lomustine–DNA complex formation respectively [38–41]. These observed phenomena might be due to intercalation of drug between the base pair of nucleic acid during the formation of G-C crosslink. No appreciable changes in CD bands at 224 nm and 214 nm are observed. However, slight increase is evident in molar ellipticity with increasing concentration of lomustine. This subtle alteration for these bands should be due to the reduction of water around DNA due to the presence of additional hydrophobic alkyl groups. These alkyl groups not only protrude the water around DNA, but also cause the loss of the hydrogen bonding resulting in the formation of loose conformation [14,19]. Band observed at 277 nm in CD spectrum of free calf thymus DNA shows hypsochromic shift (from 277 nm to 260 nm) alongwith increase in positive ellipticity after DNA complexation. Positive band at 260 nm is the marker band of A-conformation of DNA [41,42]. Although, other characteristic bands in the CD spectrum for A-DNA conformation (negative band at 210 nm and positive band at 195 nm) are not observed. However, blue shift and increase in intensity in CD spectral band at 277 nm is due to local structural distortion in B-DNA. No appreciable alteration (shifts and intensity variations) is observed in CD spectra at 3 and 4 h incubations rather all the molar ratios show nearly uniform shift
and ellipticity changes. These local structural distortions may lead to the formation of an intermediate stage of DNA during the transition from B- to Aconformation [38–43]. These observations are in accordance with our FTIR spectroscopic results, which show formation of an intermediate stage of DNA upon interaction with drug. Localized structural distortion in native B-DNA conformation is suggestive of perturbation of only few base pairs of DNA; where the binding of lomustine takes place. These local variations are of considerable significance for the occurrence of DNA–protein and DNA-ligand recognition processes. It has been reported in literature that DNA adopts A-DNA conformation locally during the processes of replication and transcription, which is necessary for the correctness and fidelity of these phenomena [44]. These local conformational changes can act as recognition patterning for alkylating enzymes alongwith lomustine binding at the site of interaction. This assumption should be acceptable because ‘‘enzymes and other ligands generally recognize the three-dimensional structure of DNA rather its direct read-out’’ [43–45].
4. Conclusion In the present work, we investigated the interaction of antitumor drug lomustine with DNA double helix using various spectroscopic techniques. Our spectroscopic observations indicate the major/minor groove binding with alkylating mode of action. FTIR results suggest that lomustine interaction occurs via guanine (N7, O6) and cytosine (C4) along with slight binding with sugar-phosphate backbone of DNA. The CD spectroscopic observation suggests the formation of an intermediate stage (B-A-form) of DNA double helix after its interaction with lomustine. These findings can contribute to further understanding the action mode of lomustine at molecular level. Finally, we can conclude that interaction studies
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are fundamental to unravel the mystery of molecular recognition in general and DNA binding in particular.
Acknowledgement The authors thank Director, CSIR-National Physical Laboratory for granting the permission for publication of the work. S.A., D.K.J. and P.S. is thankful to Council of Scientific & Industrial Research for financial support.
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