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Cytotoxic activity, cell imaging and photocleavage of DNA induced by a Pt( II )

cyclophane bearing 1,2 diamino ethane as a terminal ligand Niraj Kumari, a Brajesh Kumar Maurya, b Raj Kumar Koiri, b Surendra Kumar Trigun, b Srikrishna Saripella, c

Michael P. Coogan d and Lallan Mishra *a

Received 21st June 2011, Accepted 17th September 2011DOI: 10.1039/c1md00159k

A Pt II complex [{Pt(en)L} 2 ]$4PF 6 (Ptcyp ) (LH 2 N ,N 0-bis(salicylidene)- p-phenylenediamine, en 1,2-diamino ethane) shows high cytotoxicity against HeLa cells (IC 50 11.5 mM) and against Daltonslymphoma (DL) cells (IC 50 0.65 nM); UV-vis titration of Ptcyp with calf thymus DNA (CT-DNA)demonstrated its DNA binding, which could be further quantied by competitive uorescence titrationof DNA, Ptcyp and ethidium bromide. Circular dichroism studies suggest that Ptcyp interacts with CT-DNA by intercalation in an aqueous medium containing a minimum amount of DMSO. Agarose gelelectrophoresis showed that Ptcyp is able to convert a supercoiled pBR322 plasmid DNA into a nickedcircular DNA in DMSO, but to a much lower extent in an aqueous medium. However, with UVirradiation, Ptcyp is able to cause concentration-dependent nicking of supercoiled DNA in an aqueousmedium. These ndings indicate the DNA binding and UV exposure-dependent DNA cleavageproperties of Ptcyp . Cell imaging studies using the HeLa cell line carried out in the presence of Ptcyprepresent one of the rst examples of Pt complexes applied as uorophores in cell imaging and stronglysupport its interaction with DNA.

Introduction

Nucleic acids are important cellular targets for many anticancerand antiviral drugs and the identication of new agents that caninteract with DNA molecules in cellular environments isconsidered important for the design and development of newchemotherapeutic agents. 1 In this context, transition metalcomplexes which can cleave DNA under physiological condi-tions are of current interest. Moreover, DNA is considered to bean unselective target at the cellular level, and thus, identicationof compounds that can interact with DNA and can be activatedfor its nuclease activity by a specic stimulus e.g. light would bea relevant approach to ensure drug action at a specic site in thebody. Additionally, photoactivation-promoted enhancedactivity of pro-drug/modestly active compounds is an emerging

concept in the area of cancer therapy,2

and some platinumcomplexes have also been reported with such properties. 35 Thus,

platinum complexes which can bind with and cleave DNA underphysiological conditions are attractive therapeutic targets.

Recently, it has been reported that inclusion of amine ligandssuch as methylamine and isopropylamine can signicantlyimprove the cytotoxicity of Pt II anticancer complexes. 6 There-fore, our aim was to incorporate amine ligands such as ethylenediamine as terminal ligands conjugated in a Pt II species to a bio-logically active Schiffs base. 7 The present article describesstudies aimed at evaluating the cytotoxicity of a recentlysynthesized and characterized complex of Pt II 8 with a focus on itsinteraction with and photocleavage of DNA. Recently an elegantapplication of a neutral cyclometallated Pt II complex basedaround the 1,3-bispyridylbenzene skeleton was applied in uo-rescence cell imaging, taking advantage of both two-photon andtime-resolved techniques to provide high resolution images. 9

These species were found to be of low cytotoxicity but showedgood uptake, with an interesting pattern of localization, mainlywithin the nucleus, and concentrated in nucleoli. In contrast tothese species, complex Ptcyp is isolated as a tetracation (althoughthe phenolic hydroxyls may ionize under physiological condi-tions), so it should assist in cellular uptake due to the membranepotential. Once inside the cell, the high charge and lipophilicitymay be expected to lead to accumulation of the complex inorganelles such as mitochondria. As is discussed below, ina ground breaking application of such Ptamine complexes incell imaging, Ptcyp showed very little accumulation in the cyto-plasm, with only weak Pt-based luminescence being observed,

a Department of Chemistry, Faculty of Science, Banaras Hindu University,Varanasi, 221 005, Indiab Biochemistry & Molecular Biology Section, Centre of Advanced Studies inZoology, Banaras Hindu University, Varanasi, 221 005, Indiac Department of Biochemistry, Faculty of Science, Banaras HinduUniversity, Varanasi, 221 005, Indiad Department of Chemistry, Cardiff University, CF103AT, UK Electronic supplementary information (ESI) available: Synthetic andcharacterization data, further gures and an optimized structure of complex Ptcyp are available. See DOI: 10.1039/c1md00159k

1208 | Med. Chem. Commun. , 2011, 2 , 12081216 This journal is The Royal Society of Chemistry 2011

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and no discernible pattern of localization indicating uptake byany cytoplasmic organelles. However, there was an obviousconcentration of luminescence in the nuclear region in the centreof the cells.

Result and discussion

In this study the interaction between the Pt( II ) complex, [Pt(en)

LH 2 ]2 $4PF 6 (S1) (Ptcyp ; LH 2 N ,N 0

-bis(salicylidene)- p-phe-nylenediamine; Fig. 1) and DNA has been investigated by meansof absorption, emission, circular dichroism spectroscopy, gelelectrophoresis and cell imaging studies.

In order to check the stability of the complex in aq. medium, its1 H NMR and UV-vis spectra were recorded in the presence of D 2 O and water respectively. 1 H-NMR data show the existence of the intact complex in aq. medium. However, the absorption bandundergoes a blue shift of about 10 nm negative solvatochromicshifts (S2) (from DMSO to water) indicating that the groundstate is more polar than the excited state as has been previouslyreported for related PtSchiff base derivatives. 1015 To evaluatethe effect of anionson Ptcyp in solution, titrations of the complex

with anions like tetrabutyl ammonium chloride (TBAC) andtetrabutyl ammonium phosphate (TBAP) in DMSO were carriedout. No signicant changes were observed between the absorp-tion spectra of the platinum( II) complex Ptcyp in the absence andpresence of anions (S3 and S4). This indicated that anions donot compromise the stability of the platinum( II ) complex insolution.

In vitro cytotoxicity evaluation

The HeLa cell line is one of those which are most extensively usedto evaluate the cytotoxicity of the novel complexes. Daltonslymphoma (DL) is another tumor cell line which has been

successfully used to evaluate the anticancer potential of newlysynthesized complexes. 1618 In order to have a comparativemeasure of Ptcyp cytotoxicity on different cell lines, the cyto-toxicity assay was performed against both these cell lines. TheMTT test, which measures mitochondrial dehydrogenase activityas a measure of cell viability, 19,20 was carried out to calculate IC 50values of the complex against these cells.

The HeLa cells were incubated with an increasing concentra-tion of Ptcyp and, for comparison, with cisplatin in a separateexperiment for 24 h. The result, shown in Fig. 2, showsa concentration-dependent decline in the number of viable HeLa

cells with the IC 50 values of 11.5 and 22.2 mM for Ptcyp andcisplatin respectively.

The value with cisplatin corroborated the range of IC 50 valuesreported against many human cell lines. 2123 Thus, as comparedto cisplatin, the IC 50 value of Ptcyp against HeLa cells suggestsa stronger cytotoxic action of the complex than that of cisplatin.Additionally, the IC 50 value of Ptcyp against DL cells wascalculated to be 0.65 0.11 mM, which, in comparison to theIC 50 of cisplatin (6.96 0.85 mM) for DL cells, obtained in ourlaboratory as well as with still higher values reported from othergroups 24 suggests stronger cytotoxicity of Ptcyp against DL cellsalso. Other platinum complexes bearing similar ligands haveshown much lower IC 50 than those of the cisplatin. 22,25 Forexample, incorporation of a p acceptor pyridine ligand in placeof an ammine to a Pt IV complex caused $ 13 to 80 fold decline inIC 50 and $ 15 times more cytotoxicity than cisplatin towardsa cisplatin resistant human ovarian cell line. 25 Also, as comparedto a 3-picoline ligated Pt II complex, 2-picoline containingcomplex showed 2-fold greater cytotoxicity than the cisplatintowards the HeLa cells. 22 Thus, in the present context, it may beargued that as compared to the cisplatin, ligation of 1,2-diaminoethane as terminal ligand to a Pt II complex ( Ptcyp ) couldsignicantly enhance cytotoxicity towards HeLa cells, a widelyused drug discovery model for human cancers and towards theDL cells of mouse origin.

UV-vis absorption studies for the binding of complex Ptcyp toCT-DNA

The mechanism of action of many platinum-based chemothera-peutic agents relies upon DNA damage. 26,27 Therefore, the DNA-binding properties of Ptcyp have been evaluated in order to shedlight upon its cytotoxicity against a cancerous cell line. The UV-vis absorption spectrum of Ptcyp recorded in DMSO showedpeaks at l max 265, 340, 384 and 439 sh nm assigned to intra-ligandand MLCT transitions respectively.

However, in phosphate buffer solution these peaks shiftedsignicantly and the corresponding peaks appear at l max 253, 328Fig. 1 Structural formula of Ptcyp .

Fig. 2 Cytotoxicity of Ptcyp (P ) and cisplatin ( : ) against the HeLacells. After 24 h incubation with different concentration of thecompounds, the cell viability was assayed using the MTT dye reductionmethod. The data at each point in the logarithmic doseresponse plotrepresent mean SD ( n 3; 5 well data repeated 3 times) as percent of surviving cells taking the value of the untreated control set as 100%.

This journal is The Royal Society of Chemistry 2011 Med. Chem. Commun. , 2011, 2 , 12081216 | 1209

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and 390 sh nm. The addition of CT-DNA to a solution of Ptcypinduced remarkable shifts in l max as well as in absorptionintensity. The absorption band at 254 nm showed a hyper-chromic shift by 30% and a bathochromic shift of $ 3 nm. Incontrast, the absorption band at 328 nm showed hypochromismof 31% with no signicant change in the peak position. Suchspectral changes suggest that Ptcyp binds DNA. Hypochromismin DNA-based bands may result from the contraction of DNA in

the helical axis and also from the conformational change inDNA. In contrast, hyperchromism may result from the damageto the DNA double-helix structure. 28 It was observed thataddition of CT-DNA to a solution of Ptcyp induces remarkablehyperchromic as well as hypochromic shifts. These changes arelikely to result from Ptcyp intercalating with CT-DNA: thebathochromic shift could be derived from lowering of the energyof the ligand p * orbital upon intercalation by interaction with porbitals of the base pairs, and thus reducing the energy of thep p * transition. 29 According to the literature, substantialhypochromism, extensive broadening, and red shift of theabsorption band are characteristic of an intercalative interac-tion. 30 In order to calculate the binding strength of Ptcyp with

CT DNA, the intrinsic binding constant K b is calculated bymonitoring the changes in absorbance of Ptcyp in the presenceof increasing concentration of DNA (Fig. 3). K b is obtainedfrom the ratio of slope to the intercept from the plots of [DNA]/( 3a 3f ) vs. [DNA]. The K b value is 4.5 105 M 1 forPtcyp . Cisplatin shows a hyperchromic shift after subsequentaddition of CT DNA. The intrinsic binding constant, K b forcisplatin is found to be 3.20 104 M 1 .31

Photophysical studies

Ptcyp was found to exhibit intense photoluminescence withemission at $ 500 nm (a useful low energy wavelength for cellimaging applications) following excitation at a variety of wave-lengths, and in view of the interest in applying such species in cellimaging a fuller study of its photophysical properties wasundertaken. Examination of the excitation and emission spectraof Ptcyp (S8 and S9) showed a number of broad bands in the

excitation spectrum with maxima around 300, 350 and 380 nm.Signicantly, while the overall prole matched the electronicabsorption spectra described above, the relative intensities arevery different with the 380 nm band being the strongest in theexcitation spectrum, indicating that this is directly excitingMLCT emission, whereas higher energy bands observed to havestronger absorption in the UV-vis spectrum are less efcient atpopulating MLCT states indicating inefcient energy transfer

between states. Importantly, while the maximum of the lowestenergy band was 380 nm, as is typical of 1 MLCT excitation bandsthis peak is so broad that very efcient excitation is possible atwavelengths >400 nm, which is signicant for tissue penetrationand tissue damage considerations in cell imaging experiments.The emission spectrum shows a weak band centred at 475 anda much stronger one at 550 nm, assigned to ligand-centred and3 MLCT bands respectively. The 550 nm band becomes moredominant at longer wavelength excitation, supporting the3 MLCT assignment. The broad bands and solvatochromism inthe UV-vis spectrum indicate that these are charge transferbands, and the large Stokes shift (120 nm) indicates a tripletorigin for the emission. It is likely that there is signicant mixing

of the ligand centred and 3 MLCT states and although the lowerenergy band is the most intense regardless of excitation wave-length, interestingly, the relative intensity is excitation dependentindicating true dual emission. The luminescence lifetime ( s ) of theemission of Ptcyp was measured following excitation at ( l ex ) 340nm and tted a mono-exponential decay indicating a lifetime of 4.73 ns (Fig. 4). While this is short for 3 MLCT the presence of a large number of non-coordinated heteroatoms in the ligandstructure is likely to lead to self-quenching giving a short lifetime.Although this lifetime in itself could be thought characteristic of a singlet process, the large Stokes shift makes the alternativeexplanation of a quenched triplet state more appealing.

A reproducible 15% decrease in the steady-state emission

intensity is observed on progressing from an N 2 -saturated to anO 2 -saturated solution (S5), again indicating likely contributionsfrom triplet states. In any event, the large Stokes shift renders thiscomplex appealing for cell imaging applications, regardless of theelectronic nature of the excited state.

Fluorescence titrations

A binding assay of Ptcyp with CT-DNA was performed bymonitoring the changes in the emission spectral pattern of Ptcyp

Fig. 3 UV-vis absorption spectra of Pt II -complex Ptcyp in the presenceof increasing concentration of DNA. [DNA] 06 mM, [Ptcyp ] 10 mM.

Fig. 4 Time-resolved uorescence decay spectrum of Ptcyp measuredwith the excitation wavelength l ex 304 nm in DMSO.


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(0.5 mM, excited at 328 nm) in the presence of increasingconcentration of CT-DNA (05 mM) in an aqueous buffer (Na phosphate buffer; pH 7.4). Even though no appreciable change inthe position of the charge transfer band of the complex wasobserved upon addition of DNA, the uorescence intensity of thecomplex increases progressively with increasing concentration of DNA (Fig. 5), suggesting that Ptcyp -based emission is enhancedwhen it is bound to DNA. The concentration of the free complex

C F was determined by eqn (1): 32

C F C T [(I /I o ) P ]/(1 P ) (1)

where C T is the total concentration of the free and bound formsof the platinum( II ) complex, I and I o are the emission intensity inthe presence and absence of DNA, respectively, and P is the ratioof the observed emission intensity of the bound complex relativeto that of the free complex. The I o and P values are obtained byplotting I /I o versus 1/[DNA]. The concentration of the boundcomplex C B is equal to C T C F . A plot of r/C F versus r , where r C B /[DNA], is constructed according to eqn (2):

r/C F

K (1

nr){(1

nr)/[1

(n

1)r]}n 1

(2)

The binding constant K was 4.62 105 mol 1 dm 3 , which issimilar to that of 4.5 105 mol 1 dm 3 obtained by absorptiontitration.

It has been suggested that DNA bound complexes have lessaccess to water and therefore, they are likely to show increaseduorescence in aqueous solutions. 33,34 One of the primary modesof quenching of the excited states of luminescent transition metalcharge transfer species is the collisional quenching with watermolecules, or interactions with dissolved dioxygen. It is generallythought that the intercalation protects the planar ligand fromsuch interactions with the environment and thus leads to the

enhanced emission intensity. While traditionally the interactionwith water has been thought to be the most signicant, the tripletground state of dioxygen makes it a particularly efcientquencher for triplet excited states of certain transition metalcomplexes. It is also likely that the increased rigidity of thesystem leads to the reduced vibronic energy losses, and all thesefactors may have some role in explaining the characteristicincrease in intensity observed upon interaction with DNA.

Competitive binding with ethidium bromide

The ability of a complex to affect the uorescence intensity of ethidium bromide (EB) in the EBDNA adduct is a reliable toolto measure afnity of the complex for DNA because unbound(displaced) EB is known to rapidly lose its emission intensity dueto surrounding water. 35 Thus a measure of the relative propen-sities of EB and Ptcyp was assessed by uorescence-based

competitive binding experiments. The emission spectra of theEBDNA adduct in the presence and absence of Ptcyp is shownin Fig. 6. The emission spectrum of Ptcyp recorded in the pres-ence of EB only (S10), discards any probable interaction of Ptcyp with EB. Addition of increasing concentrations of Ptcyp toCT-DNA solution pretreated with EB showed a linear decline inthe emission intensity from the EBDNA adduct, indicatingdisplacement of EB by Ptcyp . It is also evident that in a solutionof 10 mM of CT-DNA pretreated with EB, 0.75 mM of thecomplex caused $ 90% EB displacement. These results aresuggestive of efcient DNA binding by Ptcyp . This surmise iswell supported from the quenching plot of I o /I vs. [Ptcyp ]/[DNA](the inset of Fig. 6). It was found in good agreement with the

linear SternVolmer equation with SternVolmer quenchingconstant ( K sv ) 1.08 M 1 .In order to check, whether Ptcyp displaces ethidium bromide

(EB) in the EBDNA adduct, a UV-vis titration experiment isperformed. Free ethidium bromide shows absorbance at 490 nm.After addition of CT-DNA the intensity of the absorbancedecreases. Addition of increasing concentrations of Ptcyp to theEB-DNA adduct showed a linear increase in the absorptionintensity, indicating increasing concentration of free EB. It rea-ches saturation after addition of $ 0.75 mM of the complex (S6).

Circular dichroism

Circular dichroism spectroscopy is useful in diagnosing changesin DNA morphology during drugDNA interactions. The banddue to base stacking ( $ 275 nm) and that due to right-handedhelicity ( $ 245 nm) are quite sensitive to the mode of DNAinteractions with small molecules. 36 Therefore, changes in the

Fig. 5 Fluorescence spectra of Pt II -complex Ptcyp in the presence of increasing concentration of DNA. [DNA] 05 mM, [Ptcyp ] 0.5 mM.

Fig. 6 Fluorescence quenching pattern of ethidium bromide bound toDNA by the Pt complex. [DNA] 10 mM, [ Ptcyp ] 00.75 mM. {Inset:quenching plot of I o /I vs. [Ptcyp ]/[DNA]}.


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CD spectrum of DNA upon interaction with extraneous speciesare often assigned as being related to corresponding changes inthe DNA structure. 37 Simple groove binding and electrostaticinteractions of small molecules induce little or no perturbation inthe base-stacking and helicity bands. In the case of the classicalDNA intercalator methylene blue, 38 the intercalation enhancesthe intensities of both the bands related to right-handed Bconformation of CT DNA. The CD spectral variations of

CT-DNA (100.0 mM, in 0.1 mM Naphosphate buffer (pH 7.4)) were recorded in the presence of increasing amounts of Ptcyp up to [1]/[DNA] molar ratios of approximately 0.8 (Fig. 7).The observed CD spectrum showed a positive band at 278 nmdue to base stacking and a negative band at 242 nm due to hel-icity which is characteristic of B-DNA (Fig. 7). It is interesting toobserve that the CD spectrum of native DNA (solid line in Fig. 7)is drastically modied by the addition of increasing amounts of Ptcyp . The spectral patterns showed an increase in the intensityof both the positive and the negative bands. Similar changes inCD spectra have previously been shown to correlate with inter-calation with the DNA. 39 The results observed in Fig. 6 thereforesuggest that Ptcyp binds with DNA by intercalation. Moreover,

the negative DNA band was blue shifted by 15 nm, which may becorrelated with the resultant change in the conformation of CT-DNA due to the binding of Pycyp . This observation is furthersupported from the gel electrophoresis result showing conversionof a circular supercoiled plasmid DNA into a nicked openconformation (Fig. 8 and 9).

DNA cleavage study

Monitoring changes in the supercoiled structure of pBR322DNA is a reliable tool to ascertain the DNA cleavage potential of a compound. The native circular plasmid DNA assumesa supercoiled structure (SC) which shows faster migration on

agarose gel electrophoresis than its relaxed (non-supercoiled)form. In general, the relaxed form of plasmid DNA is generateddue to the cleavage of one of the DNA strands, known as nickingof DNA, and the resultant opened circular DNA is known as thenicked circular (NC) form which migrates more slowly in theagarose gel. If both strands are cleaved, a linear L form isgenerated which migrates between SC and NC forms. 40 In order

to study whether Ptcyp enables DNA cleavage, differentconcentrations (50 and 500 mM) of Ptcyp were incubated with 0.5mg pBR322 DNA for 24 and 48 h separately in 25 mL of the twoseparate reaction mixtures (a) aqueous: TrisCl buffer (pH 7.4)containing 50 mM NaCl and (b) organic: 0.02 and 0.2% DMSO.

Fig. 7 Circular dichroism spectra of CT-DNA in the presence of increasing concentration of Pt II complex Ptcyp in 0.1 mM Naphosphatebuffer. [DNA] 100 mM, [Ptcyp ] 2080 mM.

Fig. 8 Agarose gel electrophoretic pattern of pBR322 plasmid DNA inthe presence of Ptcyp . The plasmid DNA was incubated at indicatedconcentrations of Ptcyp in aqueous buffer and DMSO separately for 24 h(A) and 48 h (B) followed by separation of DNA bands by 1% agarose gelelectrophoresis. DNA bands were stained with ethidium bromide. The gelphotomicrograph shown is a representative out of three such experi-mental repeats. SC, supercoiled DNA; NC, nicked circular DNA.

Fig. 9 UV light induced cleavage of supercoiled pBR322 plasmid DNAin the presence of increasing concentration of complex Ptcyp in aqueousbuffered solution. The plasmid DNA (0.5 mg) + complex mix. wasincubated for 10 min, followed by UV exposure at 312 nm (64 W) for 10min. The samples were then subjected to 1% agarose gel electrophoresisand bands were developed by ethidium bromide staining. (A) Repre-sentative photomicrograph from the three gel repeats. (B) Mean densi-tometric values of SC and NC DNA bands form the three gel repeats. SC,supercoiled DNA; NC, nicked circular DNA.


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in contrast to the neutral cyclometallated species, Ptcyp does not

appear to localize in the nucleoli, suggesting its preferentialafnity for DNA than RNA. As all the cells appeared to main-tain a healthy morphology throughout the experiment, Ptcypcould be useful for live cell imaging in cell biology research.

Experimental section

Materials and measurements

Ethidium bromide (EB) was purchased from Fluka Co. whereascalf thymus (CT) DNA and the supercoiled (SC) plasmid DNA,pBR322 (4361 bp) were purchased from Bangalore Genei, India.The UV-visible and luminescence measurements were recorded

in the range of 200800 nm on a Shimadzu UV-1601 and a PerkinElmer LS-45 luminescence spectrophotometer respectively.

Synthesis of Pt II complex Ptcyp

Complex Ptcyp [C44 H 48 O 4 N 8 P 4 F 24 Pt 2 ] was prepared and char-acterized as described previously 8 by us. The structure of thecomplex is shown in Fig. 1 and it is also optimized (S7). 48

In vitro cytotoxicity assay

The HeLa cells obtained from the NCCS, Pune, India, werecultured in DMEM supplemented with 10% fetal bovine serumand antibiotics (penicillin 100 unit per mL andstreptomycin 50mg mL 1 ) and an antifungal agent (amphotericin B, 2.5 mg mL 1 ).Cells were maintained in a CO 2 incubator with 5% CO 2 at 37 C.Approximately 2 103 exponentially growing cells were seededin a 96 well plate and after 24 h incubation, they were incubatedwith different concentrations (10 4 109 M) of Ptcyp andcisplatin separately (ve wells for each concentration) for further24 h. The DL cells were collected from the mouse ascite asdescribed previously. 12 The experiments using DL cells, collectedfrom the DL bearing mice, were performed in compliance withthe institutional animal ethical committee guidelines. The viableDL cells, determined by the Trypan blue exclusion test, wereseeded on to a 96 well plate in 100 mL of RPMI-1640 medium.DL cells were also maintained in a CO 2 incubator and treatedwith Ptcyp and cisplatin as described for the HeLa cells. Cellviability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on theability of viable cells to reduce a soluble yellow tetrazolium saltinto a blue formazan crystal. 19,20 Briey, after 24 h of the treat-ment, the MTT dye (10 mL/100 mL of medium), prepared inphosphate buffered saline (PBS), was added to all the wells. The

plates were then incubated for 4 h at 37 C, the medium wasdiscarded and 100 mL of DMSO was added to each well. Opticaldensity was measured at 570 nm. The percentage of viable cellswas determined by taking the cell counts in the untreated sets as100%. By the help of semi-logarithmic doseresponse plots,constructed using Graph Pad Prism5 software, the IC 50 valueswere determined as concentration of the compound that resultedin DL cell death by 50%.

UV-vis absorption spectroscopy

The absorption spectral titrations were performed using a xedconcentration of complex (1 105 M) with incremental addi-tion of the solution of the CT-DNA in [Pt( II ) complex] : [DNA]ratios ranging from 0.1 to 0.6. ComplexDNA solutions wereallowed to stand for 5 min before recording their absorptionspectra. Absorbance was corrected for the minor dilution effectsby monitoring the absorbance of CT-DNA in the reference setalso. All absorptions scans were saved as ACSII les and furtherprocessed in Origin Pro 6.1 to produce all graphs shown.

Photophysical studies

Time-resolved uorescence intensity of the dye was measured byemploying a CW passively mode-locked frequency-doubled Nd-YAG laser (Vanguard, Spectra Physics, USA) driven rhodamine

Fig. 11 Projections of confocal sections showing specic localization of Ptcyp in HeLa cells (DE). DNA is stained with Hoechst (blue, B) andthe merge image of DIC and Hoechst is shown in panel C. The orthog-onal view (XX, XY) of the merge image is shown in panel F. Green circlesin A, C, D and E represent the nuclear boundary while white ovalsrepresent cell boundaries. White arrows in panel D and E point to the low

concentration of Ptcyp while green arrows show intense localization of Ptcyp inside the nucleus. Scale bar represents 20 mm.

Fig. 12 Confocal images showing specic localization of Ptcyp in HeLacells (B). Blank experiment without Ptcyp was carriedout in HeLa cells tosee whether any auto uorescence emanates from unstained cells. Notethat there is no detectable auto uorescence from HeLa cells (E) whilePtcyp positive cells show strong intracellular localization (red in B).Merge images of A and B, D and E are shown in C and F, respectively.


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6G dye tunable laser, which generates pulses of width $ 1 ps(repletion rate 4 MHz). The sample was excited by using thesecond harmonic output (304 nm) of an angle-tuned KDPcrystal. Fluorescence decay curves were obtained by using a time-correlated, single-photon counting setup, coupled to a micro-channel plate photomultiplier (model 2809u; HamamatsuCorp.). The instrument response function (IRF) was obtained at304 nm using a dilute colloidal suspension of dried non-dairy

coffee whitener. The half-width of the instrument responsefunction was $ 40 ps, and the time per channel was 40 ps. Thecut-off lter was used to prevent scattering of the excitation beamfrom the samples. The number of counts in the peak channel wasat least 10 000. In uorescence lifetime measurements, theemission was monitored with the polarizer at the magic angle(54.7 ) to eliminate any contribution from the decay of anisot-ropy. Time-resolved uorescence decay data were t to a func-tion that is a sum of discrete exponentials:

I (t) P a iexp( t/s i)

where S a i 1, by the iterative deconvolution method. The

correction factors for a i and s i were determined by the Mar-quardt method in non-linear least-squares analysis. Numericalcalculation of the convolution integrals for intensity and partialderivatives was done with the GrinvaldSteinberg recursionequations. The mean lifetime, s m S a i s i, correlates with theaverage uorescence yield of the system.

Luminescence titration

DNA binding experiments of the platinum complex Ptcyp arecarried out in Naphosphate buffer (aqueous solution). Theabsorption ratio at 260 nm and 280 nm of CT DNA was found tobe 1.9 : 1, showing that the DNA is sufciently free from protein.The concentration of DNA is determined by UV-vis absorbanceusing the molar absorption coefcient of 6600 M 1 cm 1 at 260nm. The complex Ptcyp exhibits uorescence $ l em 500 nm at theexcitation wavelength ( l ex ) 340 nm. Therefore, binding of theplatinum complex to CT DNA was also studied by uorescencespectroscopy. The binding of the platinum complex to CT DNAis performed by monitoring the changes in the emission spectralpattern of platinum complex Ptcyp (0.5 mM) in Naphosphatebuffer (pH 7.4) in the presence of increasing concentrations of CT DNA (05 mM). After addition of DNA to the platinumcomplex, the resulting solution was allowed to equilibrate for 5min at 25 C, excited at 340 nm followed by recording thespectral changes.

Competitive DNA binding studies of the complex and ethidiumbromide

Competitive DNA binding studies of Ptcyp with that of theethidium bromide were carried out using recording of ethidiumbromide uorescence quenching after successive addition of 00.75 mM of complex Ptcyp to 10 mM DNA solutions con-taining 10 mM ethidium bromide in Naphosphate buffer (pH7.4). The extent of ethidium bromide uorescence quenching dueto addition of Ptcyp was taken as a measure of strength of intercalation of Ptcyp with the DNA. For this experiment,samples were excited at l ex 510 nm and emission spectra were

recorded between 550 and 700 nm. SternVolmer quenchingconstants were calculated using the equation I o /I 1 + K sv r,where I o and I are the uorescence intensities in the absence andpresence of Ptcyp respectively, K sv is a linear SternVolmerquenching constant and r is the ratio of the total concentration of the complex to that of DNA. The value of K sv was given by theratio of slope to intercept in a plot of I o /I versus [Ptcyp ]/[DNA].

DNA cleavage studies

Monitoring the changes in a supercoiled structure of pBR322DNA is a reliable tool to ascertain the DNA cleavage potential of a compound. In order to study whether the Ptcyp was able tocleave DNA, different concentrations (50 and 500 mM) of Ptcypwere incubated with 0.5 mg pBR322 DNA for 24 and 48 hseparately in 25 mL of the two separate reaction mixtures: (a)aqueous: TrisCl buffer (pH 7.4) containing 50 mM NaCl and(b) organic: 0.02 and 0.2% DMSO. After incubation was over,samples were analyzed on native agarose gel electrophoresisfollowed by ethidium bromide staining of the DNA bands. Inorder to study the photo-cleavage property of the platinum

complex Ptcyp in aqueous solution, different concentrations of the compound were incubated with 0.5 mg pBR322 DNA in 5mM TrisCl buffer (pH 7.4) containing 50 mM NaCl. Thereaction mixtures are exposed to UV light (312 nm, 64 W) for 10min. The samples are analyzed on 1% agarose gel electrophoresisin TAE buffer for 3 h at 50 V. The ethidium bromide stainedDNA bands are photographed and quantied by AlphaImager2200 software.

Circular dichroism studies

CD spectra were recorded on a JASCO J-810 spectropolarimeterat increasing complex/DNA ratio. Each sample solution wasscanned in the range of 220320 nm. The CD spectrum thusgenerated represents an average of three scans from which thebuffer background had been subtracted. The concentration of DNA solution was 1.0 104 M.

Cell imaging

HeLa cells, obtained from NCCS (National Centre for CellScience, Pune, India), were grown in a culture ask containingDMEM (Delbeco Modied Eagles Medium) supplemented with10% FBS (Fetal Bovine Serum) and 5% CO 2 at 37 C. Cells weretrypsinized with 0.05% trypsinEDTA (GIBCO-25300) for 2minutes followed by addition of 1 mL of fresh DMEM mediumcontaining serum to inhibit trypsin activity. Cells were spun at

1200 rpm for 10 minutes and the sediment was suspended in 1 PBS (pH 7.4), placed on polylysine coated slides and incubatedfor one hour at room temperature for adherence. Cells wereincubated with Ptcyp (20 mM) for 30 min, briey washed twicewith 1 PBS and then incubated with Hoechst 34580 (1 mg mL 1 )for 5 min followed by washing with PBS. Live cells were imagedby a ZEISS LSM-510 Meta confocal microscope. The excitationwavelength for Ptcyp is 488 nm (Argon 488 Laser line) andemission for Ptcyp was collected at 520 nm. The excitationwavelength for Hoechst was 405 nm (Diode 405) and emissionwas recorded at 440 nm. Fluorescence intensities were measuredby Zeiss LSM software.


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Conclusion

The recently synthesized Pt II complex Ptcyp constructed on theskeleton of a simple Schiff base N ,N 0-bis(salicylidene)- p-phenyl-enediamine shows strong cytotoxicity against cancerous celllines. It also binds with and cleaves DNA readily in aqueousmedium containing 0.02 and 0.2% of DMSO. The UV irradia-tion-induced nicking of supercoiled DNA in a buffer solution by

this complex suggests that Ptcyp also shows photo-inducednuclease action in aqueous medium. The complex also offersa new DNA stain for uorescence microscopic studies in vivo,one of the rst Pt complexes demonstrated to be applicable insuch studies, with the advantages of a transition metal uo-rophore in terms of photophysical attributes.

Acknowledgements

Authors thank UGC, New Delhi, India, for nancial assistance;DST-FIST & UGC CAS programme to the Department of Zoology for infrastructural facilities. The authors BK Maurya,RK Koiri and NK thank CSIR, New Delhi, for the award of

JRF, SRF and RA respectively. One of the authors (SS)acknowledges Dr Geeta Rai, Molecular and Human Genetics,BHU, Varanasi, India for her help in the cell imaging study. Wealso thank Professor J.C. Dabrowiak, University of Syracruse,USA and Dr Vinod Bhakuni CDRI, Lucknow, India, for readingthe manuscript and help in recording the CD spectrarespectively.

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