Curcumin-DNA interaction studied by Fourier Transform Infrared spectroscopy.
Cancer results from a multistage carcinogenesis process that involves three distinguishable but closely connected stages initiation, promotion and progression . The multistage cancer may be due to the initial uptake of a carcinogen and subsequent stable genotoxic damage caused by its metabolic activation . Other causes of cancer initiation include oxidative stress, chronic inflammation and hormonal imbalance [3,4]. The role of inflammation in cancer can be understood by the frequent upregulation of inflammation mediators like NF-KappaB. NF-KappaB is an excellent target for anticancer therapy . The activated NF-KappaB signaling pathway plays a major role in tumorigenesis. Experimental evidence has suggested that NF-KappaB has an important role in not just cancer initiation but cancer promotion and progression. The key protein NF-KappaB binds to DNA and results in transcription of genes that contribute to tumorigenesis, such as inflammatory, antiapoptotic, and positive regulators of cell proliferation and angiogenesis [6,7]. Curcumin has been found to increase expression of conjugation enzymes and has been shown to be one of the most potent inhibitors of NF-KappaB, thereby exerting anti-inflammatory effects . When unmodified, carcinogens can form a covalent adduct with DNA, resulting in DNA damage. Irreparable damage leads to mutations in critical genes involved in growth, proliferation, and apoptosis, resulting in initiation and subsequent development of cancer. By modulating cytochrome P450 function, curcumin reduces the aflatoxin B1-DNA adduct formation, thereby showing its potential to inhibit chemical carcinogenesis . Hence binding studies of curcumin with DNA are important for the understanding of the reaction mechanism and for the application of the new and more efficient drugs targeted to DNA.The interaction of curcumin with DNA is of major biochemical importance. There are two main modes of non-covalent interaction of these small molecules with DNA, intercalation and groove-binding. Intercalation is usually independent of the DNA sequence context (a slight GCspecificity has been observed), while the groove-binding molecules are commonly specific to adenine-thymine(AT) -rich sequences.
In the present study FT-IR spectroscopic analysis of curcumin interactions with DNA in aqueous solution at physiological conditions are reported. The drug binding sites, and the effects of curcumin complexation on the stability and conformation of DNA are discussed.
Highly polymerized calf thymus DNA Sodium salt (7% sodium content) was purchased from Genei Chemical Co., Bangalore and was deproteinated by the addition of chloroform and isoamyl alcohol in NaCl solution. Curcumin (1,7-bis[4hydroxy-3-methosyphenyl]-1,6-heptadiene-3,5-dione, catalog no.C-7727) was purchased from Sigma Chemical Co.(St.Louis,Mo).
Preparation of stock solutions
Sodium-DNA(8mg/ml) was dissolved in distilled water(pH7) and kept at 4[degrees]C for 24h with occasional stirring to ensure the formation of a homogeneous solution. A solution of calf thymus DNA in aqueous solution gave a ratio of UV absorbance at 260-280nm Ca 1.85 indicating that the DNA was sufficiently free of protein. The final concentration of the calf thymus DNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient [epsilon.sub.260]=6600[cm.sup.-1] [M.sup.-1] (expressed as molarity of phosphate groups) [11, 12].
The UV absorbance at 260nm of a diluted solution (1/200) of calf thymus was 0.81 (path length was lcm), and the final concentration of the original DNA solution was 25mM in DNA phosphate. Curcumin was dissolved in 95% ethanol at a concentration of 25mM and added drop-wise to DNA solution with constant stirring in order to attain the desired curcumin/DNA(P) molar ratios(r) of 1/50,1/20,1/10 and 1/5.
FT- IR spectra
Infrared spectra were recorded on a Thermo Nicolet NEXUS model 670 FTIR Spectrometer with DTGS dectector and KBr beam splitter with samples prepared as KBr pellets. Each set of infrared spectra was taken twice. Spectra were collected and treated using the SPECTRA MANAGER software. For each spectrum 128 scans were collected at a resolution of 4[cm.sup.-1]. The difference spectra [(DNA solution + ligand)(DNA solution)] were obtained using a sharp DNA band at 968[cm.sup.-1] as internal reference. This band, which is due to sugar C-C and C-0 stretching vibrations, exhibits no spectral (shifting or intensity) variation upon curcumin-DNA complexation, and cancelled out upon spectral subtraction.
The intensity ratios of bands due to several DNA in-plane vibrations related to AT, G-C base pairs and the P02 stretching vibrations were measured with respect to the reference band at 968[cm.sup.-1] (DNA) as a function of curcumin concentrations with an error of 3%. Similar intensity variations have been used to determine the ligand binding to DNA bases and backbone phosphate groups[19,14].
The plots of the relative intensity [ R;) of several peaks of DNA in-plane vibrations related to A-T and G-C base pairs and the P02- stretching vibrations, such as 1710(guanine), 1661(thymine), 1610(adenine), 1493(cytosine) and 1229[cm.sup.-1](PO2 groups), versus the curcumin concentrations was obtained after peak normalization using [R.sub.i] =[I.sub.i]/I96a where [I.sub.i] is the intensity of absorption peak for pure DNA in the complex with i as ligand concentration, and 1968 is the intensity of the 968[cm.sup.-1] peak (internal reference).
Results and Discussion:
FT-IR spectra of curcumin-DNA complexes:
FT-IR spectra curcumin-DNA complexes between 1800 and 600[cm.sup.-1] are presented in Fig. 1. The spectral changes (intensity and shifting) of several prominent DNA inplane vibrations at 1710(sh)(G,T; mainly G), 1649(T,G,A,C; mainly T), 1610(A), 1492(C,G; mainly C), 1229([PO.sub.]2 asymmetric), 1088 [sm.sup-1] ([PO.sub.2]-symmetric) (15-21) were monitored at different curcumin-DNA molar ratios. The calculated intensity ratios of several DNA in-plane vibrations (related to A,T,G,C bases and the backbone [P.sub.O2]- groups) as a function of curcumin concentrations are presented in Fig. 2.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
At r=1/50, decrease of intensity was observed for the guanine band at 1710(20%), thymine at 1649(37%), adenine at 1610(35%), cytosine at 1492(14%), [PO.sub.2]- asymmetric at 1229(17%) and P02 -symmetric at 1088[cm.sup.-1] (19%) upon addition of curcumin. Earlier studies indicate that cation binding to DNA bases and the phosphate groups causes major increase of intensity of these vibrations, while the loss of intensity has been attributed to DNA helix stabilization[18-23). Thus, the decrease in intensity can be suggested due to hypochromic effect associated with partial helix stabilization aggregation in the presence of curcumin. The intensity variations were associated with the shift of the band at 1710(G) to 1703cm 1 upon curcumin-DNA complexation(Fig. 2). However, no shiftin was observed for the thymine(1661[cm.sup-1]), adenine(1610[cm.sup.-1]) and cytosine(1492[cm.sup.-1] bands. The observed spectral changes are indicative of curcumin interaction(indirectly through H-bonding) with the guanine N7 atoms [24-26).
At r=1/20, increase in intensity of DNA in-plane vibrations was observed as a result of curcumin's interaction with duplex and helix destabilization(fig. 2). Evidence for this comes from the spectral changes observed for the DNA in-plane vibrations at 1710(G), 1661(T), 1610(A) and 1492(C)[cm.sup.-1]. The intensity of the bands at 1710 (29%), 1661(26%),1610(20%) and 1492(29%) increased relative to free DNA upon curcumin complexation. The intensity variations were associated with the shift of the bands at 1710(G) to 1703[cm.sup.-1] and 1661(T) to 1659[cm.sup.-1](fig. 2). No shifting was observed for the adenine band at 1610[cm.sup.-1] and cytosine band at 1492[cm.sup.-1]. The observed spectral changes are attributed to curcumin interaction (indirectly through H-bonding) with the guanine N-7 atoms and to a lesser extent with thymine O-2 atoms that are not normally involved in Watson-Crick hydrogen bonding network. Kanakis et al have observed similar spectral changes due to an indirect interaction of flavonoids with guanine N-7 atom. Such interaction does not bring about helix destabilization. Thus curcumin shows a preferential binding affinity for DNA bases and the order could be visualized as G>T>C>A.
At (r>1/10), the infrared in-plane vibrations in the free DNA spectrum at 1710[cm.sup-1](G), 1661(T), 1610(A), 1530(C) and 1493(C) were shifted to lower frequencies at 1703, 1658, 1602, 1527 and 1487[cm.sup-1] respectively(Fig. 1). The observed spectral changes can be due to the interaction of curcumin (via C=O, C-C, O-[CH.sub.3] and OH groups) with G-C and A-T rich regions of DNA duplex (adenine and guanine N7 atoms as well as thymine O-2 atom) or intercalation of curcumin between G-C and AT base pairs and partial conformational change of DNA duplex upon curcumin interaction. The observed shiftings were accompanied by a major decrease in intensity of these vibrations which reached a minimum at r=1/5(Fig .1). The intensity of the guanine band at 1710[cm.sup.-1] decreased by 47%, the thymine band at 1661[cm.sup.-1] decreased by 55%, the adenine band at 1610[cm.sup.-1] decreased by 37% and the cytosine bands at 1530 and 1493[cm.sup.-1] decreased respectively by 33% and 43% (relative to free DNA) upon curcumin-DNA complexation. Additionally the intensity of asymmetric phosphate vibration decreased by 44%, which can be related to curcumin P02 interaction(Fig .2). The observed decrease in intensities can be related to curcumin weak interaction with or intercalation into DNA duplex and helix stabilization of DNA duplex at higher concentration(r=1/10). Nafisi et al. have observed similar spectral changes (intensity loss and low frequency shift) in FT-IR spectra of Apigenin-DNA complex at low concentration(r=1/40) [14,29].
Additional evidence for drug-DNA interaction was obtained from the spectral shifting of curcumin vibrartional frequencies upon DNA binding. The major shifting of infrared bands of in-plane stretching vibrations of curcumin at 1733(C=0 stretch) to 1703[cm.sup.-1], 1513(in-plane C-C str) to 1493[cm.sup.-1],1463(in-plane C-C str) to 1423 [cm.sup.-1], 1131(C-0 and C-C stretch) to 1092 [cm.sup.-1] and 671(out of plane ring C-C stretch) to 661[cm.sup.-1] [30,31] upon DNA complexation are evidence for a major curcumin-DNA interaction through curcumin C=O, C-C,C-0 groups.
The drug complexation did not alter DNA conformation. It is known that in a Bto-A transition, the B-DNA marker band at 835 (phosphodiester mode) shifts toward a lower frequency at -800 [cm.sup.-1] and a new band appear at -870-860[cm.sup.1]. Similarly, the band at 1710-1717(G) appears at 1700 [cm.sup.-1], where as the band 1222-1229(PO2asymmetric) shifts toward a higher frequency at 1240 [cm.sup.-1] . In a B-to-Z conformational change, the sugar-phosphate band at 835 appears at 800, and the band at 1710-1717 displaces to 1690[cm.sup.-1], where as the phosphate band at 1222-1229 shifts to 1216[cm.sup.-1] . Since, the sugar-phosphate bands at 836 and 889[cm.sup.-1] exhibited no major shifting in the spectra of the curcumin-DNA adducts, the DNA remains in the B-family structure. However, minor spectral changes (shifting and intensity changes) in the region of 1060-600 [cm.sup.-1] of curcumin-DNA complexes can be attributed to the alterations of the sugar-phosphate geometry while DNA remains in the B-family structures (Fig. 1 and 2)[24,32-35].
In conclusion, the vibrational spectroscopic results presented here, show that at low concentration(r=1/50) curcumin binding is mainly through the guanine N-7 atom (indirectly through H-bonding). When the concentration increases (r=1/20), curcumin binding extends to thymine O-2 atoms. This type of complexation does not cause helix perturbation. However, at higher curcumin concentration (r>1/10), drug binding to G,T,A and C bases occurs (indirectly) with a partial helix stabilization as a result weak intercalation into DNA duplex.
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K. Senthil (1) and R. Sarojini (2)
(1) Research scholar, R&D Department of Physics, KASC Coimbatore-29, TamilNadu, India E-mail: email@example.com
(2) Reader, R&D Department of Physics, KASC Coimbatore-29, Tamil Nadu, India E-mail: firstname.lastname@example.org
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|Title Annotation:||deoxyribonucleic acid|
|Author:||Senthil, K.; Sarojini, R.|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Sep 1, 2009|
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