direct binding of cu(ii)-complexes of oxicam nsaids with dna backbone

JOURNAL OF

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Journal of Inorganic Biochemistry 100 (2006) 1320–1331

InorganicBiochemistry

Direct binding of Cu(II)-complexes of oxicam NSAIDswith DNA backbone

Sujata Roy, Rona Banerjee, Munna Sarkar *

Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Calcutta 700 064, India

Received 17 September 2005; received in revised form 17 March 2006; accepted 21 March 2006Available online 25 April 2006

Abstract

Drugs belonging to the non-steroidal anti-inflammatory drug group (NSAID) are not only used as anti-inflammatory and analgesicagents, but also exhibit chemopreventive and chemosuppressive effects on various cancer cell lines. They exert their anticancer effects byinhibiting both at the protein level and/or at the transcription level. Cu(II) complexes of these NSAIDs show better anti-cancer effectsthan the bare drugs. Considering the above aspects, it is of interest to see if Cu(II) complexes of these drugs can exert their effects directlyat the DNA level. In this study, we have used UV–Vis spectroscopy to characterize the complexation between Cu(II) and two NSAIDsbelonging to the oxicam group viz. piroxicam and meloxicam, both of which exhibit anticancer properties. For the first time, this studyshows that, Cu(II)–NSAID complexes can directly bind with the DNA backbone, and the binding constants and the stoichiometry or thebinding site sizes have been determined. Thermodynamic parameters from van’t Hoff plots showed that the interaction of these Cu(II)–NSAID complexes with ctDNA is an entropically driven phenomenon. Circular dichroism (CD) spectroscopy showed that the bindingof these Cu(II)–NSAIDs with ctDNA result in DNA backbone distortions which is similar for both Cu(II)–piroxicam and Cu(II)–meloxicam complexes. Competitive binding with a standard intercalator like ethidium bromide (EtBr) followed by CD as well asfluorescence measurements indicate that the Cu(II)–NSAID complexes could intercalate in the DNA.� 2006 Elsevier Inc. All rights reserved.

Keywords: Cu(II)–NSAID complex; Drug–DNA interaction; Thermodynamics; UV–Vis absorption; Fluorescence; Circular dichroism

1. Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are awell-known class of drugs that are antipyretic, analgesicand anti-inflammatory agents. They are used to reducepain in different arthritis and other post-operative condi-tions. Besides these functions, they show different typesof other activities also. Many of them exhibit chemopre-ventive and chemosuppressive effects in different cancerssuch as colon cancer, lung cancer, breast cancer, etc. [1–4]. For our work, we have chosen a prototype group ofNSAID viz. oxicams, because the drugs belonging to thisgroup show more or less all the functions that are shownby NSAIDs in general. We have chosen two NSAIDs from

0162-0134/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2006.03.006

* Corresponding author. Tel.: +91 33 23375345; fax: +91 33 23374637.E-mail address: [email protected] (M. Sarkar).

oxicam group viz. piroxicam and meloxicam (Fig. 1),which show chemopreventive and chemosuppressive effectsin different cancer cell lines and animal models [5–7]. Eventhough the molecular mechanism behind their principalfunction, i.e. as analgesic and anti-inflammatory agents isquite well understood, it is not clear exactly how they exerttheir anticancer effects. Anticancer effects of these drugshave been implicated to occur both by the cyclooxygenase(COX)-dependent and COX-independent pathways [8,9].In an effort to explain their anti-cancer effects, researchershave pointed out that inhibition could be both at the levelof protein and/or transcription [8,10]. Keeping in mind thecontroversy behind the molecular mechanism of NSAIDsfor chemoprevention and chemosuppression, their interac-tion directly at the DNA level is of great interest. MostNSAIDs are anionic at physiological pH which obliteratetheir approach to the poly anionic DNA backbone and

mailto:[email protected]

O

N

SO O

O

SO O

S

N

N

OH

NH

N

OH

NH

(a)

(b)

3CH

CH3

CH3

Fig. 1. Structure of piroxicam (a) and meloxicam (b).

S. Roy et al. / Journal of Inorganic Biochemistry 100 (2006) 1320–1331 1321

hence, only a few reports exist about the fact that NSAIDscan interact with DNA [11].

It is well established that metal ions play a wide range ofimportant roles in biological systems [12,13]. The presenceof drugs that can compete with other biological moleculesfor the metal ions, changes the distribution of these ions inblood plasma and other fluids. On the other hand, presenceof these metal ions can affect the bio-availability of thesedrugs. Knowledge of the species formed by combining ametal ion with a drug provides useful information toapproach the mechanisms of action of the drug for a dis-ease under treatment and ultimately this can also diminishcollateral effects and enhance the efficacy of the parentdrug. Cu(II)–NSAID complexes are reported to haveenhanced anti-inflammatory activity and reduced gastrointestinal (GI) toxicity compared to the bare drugs [14].A variety of recent observations indicate that copper whenco-administered with anti-inflammatory drugs exhibit syn-ergistic activity [15]. It is also reported that there is anincreased demand for Cu(II) during inflammatory condi-tions, which is compensated for by enhanced intestinalabsorption and/or decreased intestinal excretion of Cu(II)[16]. It has long been suggested that the mode of actionof many anti-inflammatory drugs may involve the chela-tion with some bioactive metals [17] such as Cu(II), Zn(II),Cd(II) and it facilitates the transfer of the metal to andfrom a site of inflammation or pain. The Cu(II)–NSAIDcomplexes have also been implicated to have better anticancer effect [14]. Till date, there exists no report that metalcomplexes of NSAIDs can directly interact with the DNA.However, both piroxicam and meloxicam exist as anionnear physiological pH that makes their approach to thepoly-anionic backbone prohibitory. Considering the con-troversy, whether the anti-cancer effects of NSAIDs isexerted at the protein level or at the transcription leveland the fact that the Cu(II)–NSAID complex enhancesanticancer efficacy, it is our interest to see if Cu(II)-complexes of piroxicam and meloxicam can directly inter-act at the DNA level. It has been shown that the structureof the Cu(II)–piroxicam complex is a bis complex havingoctahedral geometry and the crystal of the complex fromDMF has monoclinic crystal structure [18]. For these twooxicam NSAIDs viz. piroxicam and meloxicam, Cu(II)complexes are reported in non-aqueous solvents or in

solid state [19,20] but we are interested to monitor themin aqueous conditions because that is more biologicallyrelevant.

In this study, we have first shown that both piroxicamand meloxicam can form complexes with Cu(II) in aque-ous buffer near physiological pH. UV–Vis spectroscopyhas been used to characterize these metal complexes. Bind-ing constants for formation as well as stoichiometry ofthese complexes were determined. We have then studiedthe interaction of these Cu(II)–NSAID complexes viz.Cu(II)–piroxicam and Cu(II)–meloxicam complexes withthe ctDNA. Our results show that both Cu(II)–piroxicamand Cu(II)–meloxicam complexes can bind with ctDNA.The binding constants and the stoichiometries or bindingsite sizes have been determined. The thermodynamic para-meters for binding of these metal complexes with ctDNAare also determined using van’t Hoff equation. The differ-ence in the values of DH and DS highlights the physicalbasis behind the differential binding of the two Cu(II)complexes with ctDNA. Circular dichroism (CD) spectros-copy was used to determine the distortion introduced inthe DNA backbone upon binding with these Cu(II)–NSAID complexes. Competitive binding with a typicalintercalator like ethidium bromide (EtBr) along with thesedrug–metal complexes was studied using CD-spectroscopyand fluorescence spectroscopy. Combining the results ofthermodynamics, fluorescence and CD data allows us tospeculate on the possible mode of binding of these Cu(II)complexes with the DNA backbone.

2. Materials and methods

Piroxicam was purchased from Sigma Chemicals andMeloxicam from LKT laboratories (MN, USA) and wereused without further purification. Since the drugs, piroxi-cam and meloxicam are sparingly soluble in aqueoussolvent, 0.5 mM stock solutions were prepared in spectro-scopic grade ethanol (Merck, Germany) and were dilutedto the desired concentration by 10 mM sodium cacodylatebuffer. Sodium cacodylate was purchased from Merck, Ger-many. The buffer was prepared in triple distilled water andpH of the buffer was adjusted to 6.7 by gradually addingHCl solution. The working buffer is 10 mM sodium cacodyl-ate pH 6.7 is same for all experiments until mentioned other-wise. CuCl2 was purchased from SRL (Sisco ResearchLaboratory, India) and the stock salt solution was preparedin 10 mM sodium cacodylate buffer. Highly polymerizedctDNA was purchased from SRL and after dissolving thefibers in buffer the purity of it was checked from the absor-bance ratio A260/A280. For all the solutions the absorptionratio was in the range 1.8 < A260/A280 < 1.9. Therefore nofurther deproteinization of the DNA solutions was needed.Concentration of DNA in terms of nucleotide was deter-mined taking e260 = 6600 M�1 cm�1 per bases for ctDNA.In all the subsequent experiments the DNA concentrationis expressed in terms of bases. EtBr was purchased fromMerck, Germany.

1322 S. Roy et al. / Journal of Inorganic Biochemistry 100 (2006) 1320–1331

Absorption spectra were recorded using Thermo Spec-tronic Spectrophotometer model UNICAM UV 500.Before taking absorption data, baseline correction wasdone using corresponding solvent, i.e. sodium cacodylatebuffer. A pair of 10 · 10 mm path length quartz cuvettewas used for absorption experiments. Fluorescence mea-surements were performed using Hitachi spectrofluorime-ter model 4010. All emission spectra were corrected forinstrument response at each wavelength. The excitationwas made at the kmax of EtBr, i.e. at 480 nm. A 2 ·10-mm path length quartz cell was used for all fluorescencemeasurements to avoid any blue edge distortion of thespectrum due to inner filter effect [21]. CD spectra wererecorded on a Jasco J-720 spectropolarimeter using cylin-drical quartz cuvette of path length 5 mm. Each spectrumrepresents the average of five successive scans performedat a scan speed 20 nm/min and appropriate baseline sub-tractions were performed. All measurements were done at25 �C.

2.1. UV melting studies

DNA melting curves were determined in the same spec-trophotometer as mentioned above equipped with athermo electronic single cell peltier. DNA sample was incu-bated with saturating concentration of the metal complexfor one hour to ensure saturation of the DNA moleculewith the ligand. During melting experiments the sampleswere heated slowly at the rate of 1 �C/min and the absor-bance values were monitored at 260 nm. To compare thechanges in melting temperatures same concentration ofDNA was used for melting experiment, which was repeatedunder identical conditions.

2.2. Determination of the stoichiometry of the

Cu(II)–NSAID complexes

Determination of stoichiometry of the drug–metal com-plex was done using Job’s plot or mole-ratio method. Inthis method, metal cation and ligand solutions with identi-cal concentrations were mixed in different amounts suchthat the total number of moles of reactants in each mixturewas kept constant. The mole ratio of reactants was variedacross the set of mixtures. Absorbance of each solution wasthen measured at 363 nm (absorbance maxima of thedrugs) and plotted vs. the mole fraction of one of the reac-tants, which was that of the drug in our case. The break-point in the plot corresponds to the mole fraction of thedrug in the drug–metal complex and thereby gave the bind-ing stoichiometry.

2.3. Determination of binding constant of the drug–metal

complex

The overall association constant (K) for the followingtype of reaction:

nDrugþ Cu(II)¡Cu(II)–ðDrugÞnis given by

K ¼ ½Cu(II)� ðDrugÞn�½Drug�n � ½Cu(II)�

If the initial concentration of drug (a) and the concentra-tion of Cu(II) at 50% of the total change of the monitoringparameter as determined from the binding isotherm (b) areknown, the association constant for this reaction can bedetermined from the following equation [22]:

K ¼ ða=2Þ½ða=2Þn � b� ð1Þ

2.4. Determination of binding parameters for Cu(II)–

NSAIDs complexes binding with ctDNA

Separate aliquots were made containing constant con-centration of Cu(II)–NSAID complex and different con-centrations of ctDNA. Absorption spectrum of eachaliquot was recorded and the change in the absorbance val-ues at 363 nm (where DNA has no absorbance) was used toconstruct the binding isotherms. The binding isothermswere analyzed using non-linear curve fitting method. Todo so, the following ligand–DNA equilibrium was consid-ered [23,24]:

LþD¡L–D

Kd ¼½L� � ½D�½L–D� ¼

f½L0� � ½L–D�g � f½D0� � ½L–D�g½L–D�

If C0 be the initial concentration of drug–metal complex ta-ken and CD be the concentration of DNA added to any ali-quot then,

Kd ¼½C0 � ðDA=DAmaxÞ � C0� � ½CD � ðDA=DAmaxÞ � C0�

½ðDA=DAmaxÞ � C0�ð2Þ

C0 � ðDA=DAmaxÞ2 � ðC0 þ CD þ KdÞ� ðDA=DAmaxÞ þ CD ¼ 0 ð3Þ

where DA is the increase in absorbance (at 363 nm) of the li-gand upon each addition of ctDNA. DAmax is the sameparameter when the ligand is totally bound to the oligomer,i.e. here DA/DAmax denotes the fraction of ligand bound toDNA, C0 is the initial concentration of the drug–metal com-plex added and CD is the ctDNA concentration in any aliquot.

All experimental points for binding isotherms were fittedto the above equation by least square analysis and ulti-mately KD was determined. For determination of DAmax,a double reciprocal plot of 1/DA against 1/(CD � C0) wasused according to the following equation:

1

DA¼ 1

DAmax

þ 1

Kapp � DAmax

� 1

½CD � C0�ð4Þ

This approach is based on the assumption that absorbanceis linearly proportional to the concentration of ligand.


Here, the concentration of the ligand, i.e. the drug–metalcomplex was 15–40 lM and for construction of this plotDNA concentration was kept around 8- to 10-fold greaterthan that of the Cu(II)–NSAID complex [25,26].

The binding stoichiometry or the binding site size wasdetermined from the point of intersection of two straightlines obtained from the least square fit plot of normalizedincrease of absorbance against the ratio of input concentra-tion of DNA in bases and Cu(II)–NSAID complex. Thetwo straight lines were drawn by considering points belowsaturation and points after saturation, respectively.

2.5. Determination of thermodynamic parameters

The thermodynamic parameters, DH and DS were eval-uated from the following equation:

ln Kapp ¼ �DHRTþ DS

Rð5Þ

where R and T are the universal gas constant and absolutetemperature, respectively.

The Kapp values were determined at four different tem-peratures and a van’t Hoff plot of lnKapp vs. 1/T gave astraight line. The DH and DS values were obtained fromthe slope (�DH/R) and intercept (DS/R) of the above van’tHoff plot. Knowing these two values DG was calculatedfrom the following standard equation [27]:

DG ¼ DH � TDS ð6ÞFor all cases DG values were calculated at 298 K. It shouldbe mentioned that we do not have an access to an Isother-mal Titration Calorimeter (ITC), which prevented us fromdetermining thermodynamic parameters that are modelindependent.

3. Results and discussions

3.1. Complexation of piroxicam and meloxicam with Cu(II)

Complexation of piroxicam and meloxicam with Cu(II)have been studied using absorption spectroscopy. Fig. 2a(i)shows the absorption spectra obtained on titration of30 lM piroxicam with CuCl2. The absorption maximumof free piroxicam anion is at 363 nm in buffer. It is clearfrom Fig. 2a(i) that, with increase in concentration ofCu(II) the absorbance of the peak at 363 nm decreasesand shifts to 370 nm with a concomitant increase of theabsorbance �410 nm resulting in a broad shoulder around410 nm. However, beyond the concentration of CuCl2 �18.75 lM, only small change in the absorption spectrumoccurred. Presence of a clear isosbestic point near 385 nmclearly indicates that there exists an equilibrium betweenonly two types of species in solution, viz. the free anionicform of piroxicam and its complex with Cu(II). This alsoindicates that there exists only one type of Cu(II)–piroxi-cam complex. In case of meloxicam, the titration has beencarried out in a similar way with 30 lM meloxicam.

Fig. 2b(i) shows that increasing concentration of Cu(II)results in a decrease of 363 nm peak with no peak shift,as observed in case of piroxicam. A concomitant increaseof absorbance occurs at 435 nm. Here too, a clear isosbesticpoint is observed at 391 nm indicating that only one type ofCu(II)–meloxicam complex is produced.

The stoichiometry of the Cu(II)–drug complexes weredetermined using the mole-ratio method or Job’s plot.Figs. 2a(ii) and b(ii) show the Job’s plot of Cu(II)–piroxi-cam and Cu(II)–meloxicam, respectively. For both casesabsorbance at 363 nm was monitored. A clear breakpointis seen for the mole ratio of the drug at 0.68 for piroxicamand at 0.63 for meloxicam, respectively, indicating the stoi-chiometry is drug:metal = 2:1 for both the cases which isin well agreement with the formerly reported value of thestoichiometry from the crystal structure of this type ofcomplexes [18]. Knowing the stoichiometry (n), the associ-ation constant is determined from Eq. (1) using the bindingisotherms of Fig. 2a(iii) and b(iii) for Cu(II)–piroxicamand Cu(II)–meloxicam respectively. For Cu(II)–piroxicamK = 9.8 · 109 M�2 and for Cu(II)–meloxicam K = 3.2 ·109 M�2. It is clear from the values of the binding constantsthat the drugs form very strong complexes with Cu(II).

3.2. Interaction of metal–NSAID complexes with

calf-thymus DNA

Before studying the interaction of Cu(II)–NSAID withctDNA, some important control studies were done toensure that the results obtained are not due to the interac-tion of Cu(II) with ctDNA or that with the bare NSAIDswith ctDNA. Figs. 3a–c show the representative results ofthe control studies. In Fig. 3a, the DNA concentrationwas kept constant but Cu(II) concentration was variedover the range used in subsequent studies. As is evidentno changes occur in the DNA absorption peak and alsoin the 300–450 nm region where the Cu(II)–NSAID com-plexes contribute. Fig. 3b shows the change in the absorp-tion spectra when the concentration of bare meloxicam waskept constant but that of the ctDNA varied. As expected,the 260 nm peak of the ctDNA increased with increasingDNA concentration but the absorption peak of the drugthat appears between 300 and 450 nm remains unaltered.Similar results were obtained in case of piroxicam (datanot shown). Fig. 3c shows the CD spectra of DNA withincreasing concentration of uncomplexed meloxicam. Asexpected, no change is seen in the CD profile of theDNA indicating that the bare drug do not bind with theDNA backbone. Similar results were obtained when Cu(II)was added at concentrations used in all the experiments viz.18.7 lM and 35 lM (data not shown). This also shows thatCu(II) at concentrations used in our experiments do notaffect the DNA backbone. Identical results were alsoobtained in case of the other drug piroxicam. Our controlexperiments indicate that the bare NSAIDs as well asCu(II) do not bind with the DNA backbone. The resultsof the controls are expected since the bare drugs are in their

300 400 500 600

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Increasing [Cu(II)]

abso

rban

ce

wavelength (nm)300 400 500 600

0.0

0.1

0.2

0.3

0.4

0.5

0.6

increasing [Cu(II)]

abso

rban

ce

wavelength (nm)

0.2 0.4 0.6 0.8 1.00.0

0.1

0.2

0.3

0.4

0.5

X=0.68

abso

rban

ce a

t 363

nm

[drug]/{[drug]+[metal]}0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.1

0.2

0.3

0.4

0.5

X=0.63

abso

rban

ce a

t 363

nm

[drug]/{[drug]+[metal]}

0 10 20 30 40 50

0.0

0.2

0.4

0.6

0.8

1.0

ΔA/Δ

Am

ax

[Cu(II)] in μM [Cu(II)] in μM

b(i)

b(ii)

b(iii)a(iii)

a(ii)

a(i)

0 20 40 60 80 100 120 140 160

0.0

0.2

0.4

0.6

0.8

1.0

ΔA/Δ

Am

ax

Fig. 2. Absorption spectra of piroxicam [a(i)] and meloxicam [b(i)] with increasing concentration of CuCl2 [for piroxicam, CuCl2 concentration was variedfrom 0 lM to 18.75 lM and for meloxicam, the range was 0–35.0 lM]. Job’s plots of Cu(II)–piroxicam complex [a(ii)] and Cu(II)–meloxicam complex[b(ii)] monitored at the absorption maxima of the drugs at 363 nm. Binding isotherms of Cu(II)–piroxicam [a(iii)] and Cu(II)–meloxicam complex [b(iii)].


anionic forms in the pH used for the experiments prohibit-ing their approach to the polyanionic DNA backbone.Also for Cu(II), a much higher Cu(II):DNA(P) is requiredto affect the DNA backbone [28]. Keeping in mind theresults of the controls we have monitored the interactionof Cu(II)–NSAID complexes with DNA in the region of300–450 nm where the absorption of the NSAID andCu(II)–NSAID complexes occur.

Increasing the DNA concentration, keeping drug–metalcomplex concentration at constant value results in anincrease in the absorbance value at 363 nm for both

Cu(II)–piroxicam [Fig. 4a(i)] and Cu(II)–meloxicam[Fig. 4b(i)] complexes. It should be mentioned that DNAitself has no absorbance at 363 nm. The increase in absor-bance value for both cases are therefore attributed to thebinding of Cu(II)–piroxicam complex [Fig. 4a(i)] andCu(II)–meloxicam complex [Fig. 4b(i)] with ctDNA.

UV-melting study shows that DNA binding of Cu(II)–NSAID complexes results in an increase in the thermal sta-bility of the DNA duplex. For Cu(II)–piroxicam the Tm

increases from 68.0 �C to 71.8 �C [Fig. 4a(ii)] and forCu(II)–meloxicam the Tm increases from 68.0 �C to

250 300 350 400 450-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

No Cu(II) + 200 mM DNA 18.75 μM Cu(II) + 200 mM DNA 35.0 μM Cu(II) + 200 mM DNA

abso

rban

ce

wavelength (nm)(a)

250 300 350 400 450 500 550 600-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6 30μM meloxicam 30μM meloxicam + 100μM DNA 30μM meloxicam + 200μM DNA

abso

rban

ce

wavelength(nm)(b)

220 240 260 280 300 320 340

-10000

-5000

0

5000

10000

150μM DNA+no meloxicam 150μM DNA+20μM meloxicam 150μM DNA+30μM meloxicamm

olar

elli

ptic

ity (

deg.

cm2 .d

mol

-1)

wavelength (nm)(c)

Fig. 3. (a) Absorption spectra of 200 lM ctDNA with varying Cu(II)concentration. (b) Absorption spectra with increasing ctDNA concentra-tion in presence of 30 lM meloxicam. (c) CD spectra of ctDNA withvarying concentration of meloxicam.


71.3 �C [Fig. 2b(ii)]. The above data clearly show that theCu(II)–NSAID complexes bind with ctDNA leading tothermal stabilization of the duplex.

Fig. 5a(i) and b(i) shows the binding isotherms ofCu(II)–piroxicam and ctDNA as well as Cu(II)–meloxicamand ctDNA, respectively. The binding constants are calcu-lated using Eqs. (2) and (3) as described in Section 2. Thebinding isotherm of Cu(II)–piroxicam and ctDNA

[Fig. 5a(i)] is steeper indicating a stronger binding as com-pared to Cu(II)–meloxicam and ctDNA [Fig. 5b(i)]. Theapparent binding constants obtained are KCu(II)–piroxicam =2.7 · 104 M�1 and KCu(II)–meloxicam = 5.5 · 103 M�1. Theinset of Fig. 5a(i) and b(i) shows the plot of normalizedincrease of absorbance as a function of mole-ratio ofDNA to drug–metal complex. The breakpoints of thetwo straight lines drawn on points below and above satura-tion gives the stoichiometry or the binding site-size (n). ForCu(II)–piroxicam and Cu(II)–meloxicam complexes the ‘n’values are 5.25 and 7.77 bases, respectively. Knowing ‘n’,the binding constant K, i.e. (Kapp · n) is given as 14.4 ·104 M�1 and 4.3 · 104 M�1 for Cu(II)–piroxicam andCu(II)–meloxicam, respectively. Fig. 5a(ii) and b(ii) showsthe corresponding binding isotherms with increasing tem-peratures. The binding constants obtained at different tem-perature by non-linear least square fitting using Eqs. (2)and (3) were used in the van’t Hoff’s plots for Cu(II)–pirox-icam–ctDNA [Fig. 5a(iii)] and Cu(II)–meloxicam–ctDNA[Fig. 5b(iii)].

The changes in enthalpy (DH) and entropy (DS) weredetermined from the slope (�DH/R) and intercept (DS/R)of the corresponding van’t Hoff’s plots [Fig. 5a(iii) andb(iii)]. Table 1 shows the thermodynamic parameters ofthese Cu(II)–NSAID complexes with DNA. In both thecases, negative DG values were obtained which show thatthe binding is favored. However, for the association, thebinding enthalpy is +0.5 kcal/mol for Cu(II)–piroxicam–DNA and +5.7 kcal/mol for Cu(II)–meloxicam–DNAcomplex. TDS for Cu(II)–piroxicam–DNA is +10.8 kcal/mol and for Cu(II)–meloxicam–DNA is +6.5 kcal/mol.This clearly shows that the binding reaction is entropicallydriven for both the cases.

For entropically driven drug–DNA interactions, thesolvent molecules and the counter ions are expelled fromthe bound surface of both the interacting partners whenthey bind to each other leading to entropic gain. Thiseffect is counterbalanced by the enthalpically unfavorableeffects due to breakage of hydrogen bonds and non-cova-lent interactions of the solvent molecules and counterionswith the uncomplexed partners. This is reflected in thepositive value of enthalpy. In such entropically drivendrug–DNA binding reaction, hydrophobic interactionsare known to play a major role [29,30]. For neutralDNA intercalators like actinomycin D and echinomycin,intercalation occurs via entropically driven interaction[31]. Considering the fact that the oxicam NSAIDs aresparingly soluble in water, it is not unlikely that interac-tion of metal-complexes of these drugs with DNA shouldbe stabilized by hydrophobic interactions. Absence of netcharge in the Cu(II)–NSAID complexes compares theircase favorably with that of actinomycin D and echinomy-cin [32]. The fact that the binding reaction is stabilized byhydrophobic interaction is also reflected in the differencesof the DH and DS values of the two types of complexes.The lipophilicity of meloxicam, as reflected in the logP

values (n-octanol/water) is always higher (at pH 7.4,

350 400 450 500 550 6000.000

0.125

0.250

0.375

0.500

[DNA]

abso

rban

ce

300 375 450 525 600

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

[DNA]

abso

rban

ce

wavelength (nm)wavelength (nm)

30 40 50 60 70 80 90 1000.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

norm

aliz

ed a

bsor

banc

e at

260

nm

Temperature (˚C) Temperature (˚C)30 40 50 60 70 80 90 100 110

0.70

0.75

0.80

0.85

0.90

0.95

1.00

norm

aliz

ed a

bsor

banc

e at

260

nm

b(i)a(i)

a(ii) b(ii)

Fig. 4. [a(i)] Changes in the absorption spectra when Cu(II)–piroxicam complex (kept constant at 18.75 lM) was titrated with increasing ctDNAconcentration (0–250 lM). [b(i)] Changes in the absorption spectra when Cu(II)–meloxicam complex (kept constant at 35.0 lM) was titrated withincreasing ctDNA concentration (0–600 lM). [a(ii)] UV-melting profile of 150 lM ctDNA (s) and that of the same concentration of DNA with 18.75 lMof Cu(II)–piroxicam complex (j) under identical conditions. [b(ii)] UV-melting profile of 250 lM ctDNA (s) and that of the same concentration of DNAwith 35.0 lM of Cu(II)–meloxicam complex (j) under identical conditions.


logP = 0.07) than piroxicam (at pH 7.4, logP = �0.14)[33]. Accordingly the DS values for Cu(II)–meloxicam-complex interacting with ctDNA is larger than that ofCu(II)–piroxicam complex. It should be mentioned thatthe enthalpic contribution is more disfavored in case ofCu(II)–meloxicam complex which is expected if theCu(II)–meloxicam complex gets deeper into the hydropho-bic DNA-interior.

3.3. Circular dichroism studies

CD spectrum is a sensitive reporter of any alteration inthe DNA-backbone. We have therefore used CD spectros-copy to identify the backbone distortions in the ctDNAobtained upon binding of Cu(II)–NSAID complex.Fig. 6a(i) shows the CD spectrum of 35 lM DNA withtwo different concentration of Cu(II)–piroxicam complexwith ctDNA. As the concentration of the drug–metalcomplex is increased, there is a very distinct change inthe CD-spectrum of the B-form DNA. Similar changes

were also obtained with increasing concentration ofCu(II)–meloxicam complex added to a constant concentra-tion of 35 lM DNA [Fig. 6b(i)]. It should be mentionedthat the Cu(II)–NSAID complexes did not show anyinduced CD at wavelengths above 300 nm where the firstabsorption band of the Cu(II)–NSAID complex occur.However, it is important to rule out the fact that thechanges observed in the CD spectrum upon addition ofCu(II)–NSAID complex to the DNA in the region between200 and 300 nm is not due to induced CD of the Cu(II)–NSAID in the region of its second absorption band. Todo so, the CD spectra were recorded with increasingDNA concentration keeping the Cu(II)–piroxicam com-plex concentration constant at 18.75 lM. Fig. 6a(ii) showsthose CD spectra expressed in terms of molar ellipticity ofthe DNA. The changes in the CD spectrum clearly indi-cates that binding of Cu(II)–piroxicam complex withctDNA creates backbone distortion. Similar results areobtained when the same experiment was done withCu(II)–meloxicam complex keeping drug–metal complex

-50 0 50 100 150 200 250 300 350

0.0

0.2

0.4

0.6

0.8

1.0

A/

Am

ax

0.0

0.2

0.4

0.6

0.8

A/

Am

ax

0 2 4 6 8 10 12 14 16 18-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

site size(n)=5.25A

/A

max

[DNA]/[Cu(II)-Piro]0 2 4 6 8 10 12 14

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

site size(n)=7.77

A/

Am

ax

[DNA]/[Cu(II)-Melo]

-50 0 50 100 150 200 250 300 350

0 100 200 300 400 500 600 700

0.0

0.2

0.4

0.6

0.8

1.0

1.2

25OC

35OC

45OC

55OC

A/

Am

ax

[DNA] in M [DNA] in M0 100 200 300 400 500 600 700

0.0

0.2

0.4

0.6

0.8

1.0

23oC

35oC

45oC

55oC

A/

Am

ax

a(i)

a(ii)

a(iii) b(iii)

b(ii)

b(i)

3.1x10-3 3.2x10-3 3.3x10-3 3.4x10-3

10.2

10.3

10.4

10.5

10.6

10.7

lnK

app

1/T (K-1)

3.0x10-3 3.1x10-3 3.2x10-3 3.3x10-3 3.4x10-38.4

8.6

8.8

9.0

9.2

9.4

9.6

lnk ap

p

1/T (K-1)

Fig. 5. Binding isotherm of Cu(II)–piroxicam and ctDNA [a(i)] and Cu(II)–meloxicam and ctDNA [b(i)] and the corresponding non-linear fit. Insets showthe plots of normalized increase of absorbance as a function of mole-ratio of ctDNA to corresponding drug–metal complex. [a(ii)] The binding isotherm ofCu(II)–piroxicam complex with ctDNA at different temperatures. [b(ii)] The binding isotherm of Cu(II)–meloxicam complex with ctDNA at differenttemperatures. van’t Hoff plots of binding of Cu(II)–piroxicam complex with ctDNA [a(iii)] and that of Cu(II)–meloxicam complex with ctDNA [b(iii)].

Table 1Thermodynamic parameters for the interaction between Cu(II)–piroxicamand Cu(II)–meloxicam with ctDNA

Sample DG (kcal/molat 298 K)

DH

(kcal/mol)DS (eu)

Cu(II)–piroxicam andDNA complex

�6.0 +0.5 +21.8

Cu(II)–meloxicam andDNA complex

�5.1 +5.7 +36.3

The values are in 10 mM sodium cacodylate buffer (pH 6.7). The methodsto evaluate them are described in the text.


concentration fixed at 35 lM. Fig. 6b(ii) shows the corre-sponding CD spectra expressed as molar ellipticity in termsof DNA concentration. It should be mentioned that themaximum changes are obtained in both cases at low tointermediate DNA concentration. This is because at thisDNA concentration the maximum population is that ofthe bound DNA. On increasing the concentration ofDNA the CD-contribution coming from free or unboundDNA masks the changes obtained in the CD spectra onmetal complex binding to ctDNA.

200 220 240 260 280 300 320 340-2

-1

0

1

2

3

4el

liptic

ity (

mde

g)

wavelength (nm) wavelength (nm)

wavelength (nm)

wavelength (nm)

wavelength (nm)

200 220 240 260 280 300 320 340-2

-1

0

1

2

3

4

ellip

ticity

(m

deg)

220 240 260 280 300 320 340

-10000

-5000

0

5000

10000

mol

ar e

llipt

icity

(de

g.cm

2 .dm

ol-1)

220 240 260 280 300 320 340

-10000

-5000

0

5000

10000

mol

ar e

llipt

icity

(de

g.cm

2 .dm

ol-1)

200 225 250 275 300 325 350-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

ellip

ticity

(m

dg)

(c)

b(ii)a(ii)

a(i) b(i)

Fig. 6. [a(i)] CD spectrum of 35.0 lM ctDNA (——) and that of same concentration of ctDNA with 9.0 lM (� � � � � �) and 18.75 lM (-- - - -) of Cu(II)–piroxicam complex. [b(i)] CD spectrum of 35.0 lM ctDNA (——) and that of same concentration of ctDNA with 15.0 lM (� � � � � �) and 35.0 lM (-- - - -) ofCu(II)–meloxicam complex. [a(ii)] Molar ellipticity plot of ctDNA (——) and 18.75 lM Cu(II)–piroxicam complex containing different concentration ofDNA, 35.0 lM (� � � � � �), 70lM (-- - - -), 140 lM (–ÆÆ–ÆÆ–). [b(ii)] Molar ellipticity plot of ctDNA (——) and 35.0 lM Cu(II)–meloxicam complex containingdifferent concentration of DNA, 35.0 lM (� � � � � �), 70lM (-- - - -), 140 lM (–ÆÆ–ÆÆ–). (c) Difference CD spectra of Cu(ii)-piroxicam (——) and Cu(II)–meloxicam (� � � � � �) bound to 35.0 lM ctDNA. The difference spectra are obtained by subtracting the spectrum of same concentration of ctDNA (35.0 lM)recorded under identical conditions.


Fig. 6(c) shows the CD difference spectrum of Cu(II)–piroxicam bound to DNA and of Cu(II)–meloxicam boundto DNA. For both the cases, the difference spectra wereobtained by subtracting the spectrum of the correspondingDNA. The CD difference spectra for both the Cu(II)–NSAID complexes exhibit a similar profile which is indica-

tive of similar changes in the DNA backbone caused by thebinding of Cu(II)–NSAID complexes to it. The thermody-namic parameters indicate that the DNA binding process isan entropically driven phenomena stabilized by hydropho-bic interactions which is consistent with the thermody-namic parameters obtained in case of other uncharged


intercalators such as echinomycin and actinomycine-D.The above results point to a similar binding mode for thetwo metal complexes with DNA which could beintercalation.

3.4. Competitive binding of Cu(II)–NSAID complexes andEtBr with ctDNA

An effort was made to see if the Cu(II)–NSAID complexcan compete with a typical intercalator like ethidium bro-mide (EtBr). EtBr is an intercalator that approaches theDNA backbone via the minor groove [34]. However, weare aware that EtBr would better seek the polyanionicDNA backbone due to its positive charge, an advantagewhich is absent in the Cu(II)–NSAID complexes. Fig. 7(a)shows three CD spectra. The first spectrum is that of

Fig. 7. (a) CD spectra of 70lM DNA with saturating EtBr (——); 70 lMfollowed by addition of saturating EtBr (� � � � � �); 70 lM DNA with saturatingpiroxicam complex (- - - - -). (b) CD spectra of 70 lM DNA with saturating EtBfor one hour followed by addition of saturating EtBr (- - - - -); 70 lM DNA withCu(II)–meloxicam complex (� � � � � �). (c) Fluorescence spectra of 70 lM DNAcomplex incubated for one hour followed by addition of saturating EtBr (‘b’addition of 35.0 lM Cu(II)–piroxicam complex (‘c’). (d) Fluorescence spectra ofmeloxicam complex incubated for one hour followed by addition of saturatifollowed by addition of 35.0 lM Cu(II)–meloxicam complex (‘c’).

70 lM DNA with saturating EtBr. The second spectrumis that of 70 lM DNA to which Cu(II)–piroxicam complexis added. After incubation for one hour, the same concen-tration of EtBr is added as in the case of the first spectrum.The third spectrum is that of 70 lM DNA to which identi-cal concentration of EtBr is added as the second spectrumfollowed by addition of Cu(II)–piroxicam complex. As isevident, there is little change in the CD spectrum whetherthe Cu(II)–piroxicam complex is added before or afterEtBr and are similar to that of DNA–EtBr complex. Sim-ilar results are obtained when the same samples were mon-itored by the fluorescence of EtBr (Fig. 7c) where onlysmall reductions in EtBr fluorescence were observed whenthe Cu(II)–piroxicam complex was added to the EtBrDNA solutions. This shows that EtBr can be removed bythe Cu(II)–piroxicam complex only to a small extent which

DNA with 18.75 lM Cu(II)–piroxicam complex incubated for one hourEtBr incubated for one hour followed by addition of 18.75 lM Cu(II)–

r (——); 70 lM DNA with 35.0 lM Cu(II)–meloxicam complex incubatedsaturating EtBr incubated for one hour followed by addition of 35.0 lM

with saturating EtBr (‘a’); 70 lM DNA with 35.0 lM Cu(II)–piroxicam); 70 lM DNA with saturating EtBr incubated for one hour followed by70lM DNA with saturating EtBr (‘a’); 70 lM DNA with 35.0 lM Cu(II)–

ng EtBr (‘b’); 70 lM DNA with saturating EtBr incubated for one hour

caat


indicates that Cu(II)–piroxicam complex cannot stronglycompete with EtBr for binding with the DNA backbone.Exactly similar experiments were done for the Cu(II)–meloxicam complex. Interestingly, in this case the CD spec-trum changes significantly when the Cu(II)–meloxicamcomplex is added to DNA either before or after additionof EtBr (Fig. 6b). Consistent with Fig. 7b large reductionin the fluorescence intensity of EtBr was seen (Fig. 7d)when Cu(II)–meloxicam complex was added either beforeor after EtBr in the DNA solution. Taken together, theCD and fluorescence results show that Cu(II)–meloxicamcan better compete with EtBr for binding with the DNAbackbone than Cu(II)–piroxicam. As has been mentionedabove, the EtBr has an advantage over the two metal com-plexes in having a positive charge, which gives it a favor-able electrostatic effect for binding to the poly-anionicDNA backbone. Cu(II)–piroxicam complex cannot signif-icantly overcome this electrostatic advantage of the EtBrmolecule. However, meloxicam being more hydrophobicthan piroxicam as has already been shown by their logPvalues, can overcome the electrostatic advantage of EtBrto a certain extent and compete with it to bind with theDNA backbone.

Putting together the results obtained from UV absorp-tion study fluorescence studies [35] and that of the CDanalysis, one can predict a possible intercalative mode ofbinding of the Cu(II)–NSAID complexes with the DNAbackbone. Further studies will be carried out to find theexact binding mode, which will be the subject of a separatemanuscript.

4. Concluding remarks

Our study shows that Cu(II) can form complexes inaqueous buffer near physiological pH with piroxicam andmeloxicam, the two drugs of the oxicam NSAID groupthat show anticancer effects. We also show for the first timethat these Cu(II)–NSAID complexes can bind directly withthe DNA-backbone. Several important aspects of thisDNA-binding have been highlighted in this study suchas: (a) the binding constants and the binding site sizes havebeen determined which show that these Cu(II)–NSAIDcomplexes bind strongly with the DNA-backbone; (b) thebinding of these neutral Cu(II)–NSAID complexes isentropically driven, stabilized by hydrophobic interactionswhich itself is not a very common phenomena; and (c) anal-ysis of the thermodynamic parameters and competitivenessof binding with EtBr followed by fluorescence studies andCD data points to a similar binding mode for bothCu(II)–piroxicam and Cu(II)–meloxicam complexes withDNA which could be intercalation.

5. Abbreviations

NSAID non-steroidal anti-inflammatory drug
yi
COX c
clooxygenase CD c rcular dichroism
ctDNA
lf-thymus DNA GI g stro intestinal EtBr e hidium bromide ITC I othermal Titration Calorimeter s
Acknowledgements

We are extremely grateful to Prof. Dipak Dasgupta ofBiophysics Division, Saha Institute of Nuclear Physicsfor stimulating discussions and help with the analysis ofthe binding data. We are also thankful to Hirak Chakr-aborty for his help and cooperation and Asima Chakr-aborty for her help with the spectropolarimeter.

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direct binding of cu(ii)-complexes of oxicam nsaids with dna backbone

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