DNA as a Possible Target for Antitumor Ruthenium(III) Complexes

Archives of Biochemistry and Biophysics Vol. 376, No. 1, April 1, pp. 156 –162, 2000 doi:10.1006/abbi.1999.1654, available online at http://www.idealibrary.com on

DNA as a Possible Target for Antitumor Ruthenium(III) Complexes A Spectroscopic and Molecular Biology Study of the Interactions of Two Representative Antineoplastic Ruthenium(III) Complexes with DNA Enzo Gallori,† Cristina Vettori,† Enzo Alessio,‡ Francisco Gonzalez Vilchez,§ Rosario Vilaplana,§ Pierluigi Orioli,* ,1 Angela Casini,* and Luigi Messori* *Department of Chemistry, University of Florence, 50121 Florence, Italy; †Department of Genetics, University of Florence, 50121 Florence, Italy; ‡Department of Chemistry, University of Trieste, 34127 Trieste, Italy; and §Department of Chemistry, University of Sevilla, 41071 Sevilla, Spain

Received September 28, 1999, and in revised form November 18, 1999

The interaction of two experimental ruthenium(III)containing antitumor complexes—Na[trans-RuCl 4(DMSO)(Im)] (NAMI) and dichloro(1,2-propylendiaminetetraacetate)ruthenium(III) (RAP)—with DNA was investigated through a number of spectroscopic and molecular biology techniques, including spectrophotometry, circular dichroism, gel shift analysis, and restriction enzyme inhibition. It was found that both complexes slightly alter DNA conformation, modify its electrophoretic mobility, and inhibit DNA recognition and cleavage by some restriction enzymes, though they were less effective than cisplatin in producing such effects. Notably, the effects produced by NAMI on DNA were much larger than those induced by RAP. Implications of these results for the mechanism of action of ruthenium(III) antitumor complexes are discussed. © 2000 Academic Press

Key Words: ruthenium complexes; cancer; DNA; circular dichroism; restriction enzymes; electrophoresis.

INTRODUCTION

There is growing interest in the design, synthesis, and pharmacological evaluation of ruthenium(III) complexes as potential antineoplastic agents (1–3). Indeed, in recent years, ruthenium(III) complexes, designed on the model of cisplatin, have demonstrated favorable 1 To whom correspondence should be addressed. Fax: ⫹⫹390552757555. E-mail: [email protected].

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antitumor properties toward a number of in vitro and in vivo tumor models while showing lower systemic toxicity than platinum(II) compounds (4). In spite of a few structural similarities, the spectrum of the antitumor action of these complexes differs significantly from that of cisplatin; notably, an important antimetastatic activity of ruthenium compounds has been described (5, 6). It is commonly believed that the main target for ruthenium(III) drugs and other antitumor metal complexes is DNA; as shown for platinum(II) drugs, the antitumor action of ruthenium(III) complexes would be the consequence of direct DNA binding and damage. A recent paper by Clarke et al. (7) showed that, upon incubation of tumor cells in vitro with a well-known antitumor ruthenium(III) complex—the bis(indazole)tetrachlororuthenate anion—a significant percentage of added ruthenium ions associates to nuclear DNA; on average, cytotoxicity develops when ruthenium concentration exceeds the value of 1 ruthenium equivalent per 6000 base pairs (7). Novakova et al. previously reported that the complex mer-[Ru(III)(terpy)Cl 3], exhibiting relevant cytotoxic properties, is able to bind DNA firmly and to modify importantly its conformation (8); remarkably, this study showed that mer-[Ru(III)(terpy)Cl 3] unwinds DNA and coordinates preferentially to isolated guanine bases. The effects produced by mer-[Ru(III)(terpy)Cl 3], on DNA are large and comprise formation of a certain number of interstrand crosslinks. Extensive DNA damage correlates with a high cytotoxicity. Footprinting experiments carried out by Coluccia et al. also showed a clear preference of 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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tained after digestion of ruthenium-treated pHV14 plasmid DNA with restriction enzymes. For comparison purposes, parallel experiments were carried out with cisplatin under the same experimental conditions. MATERIALS AND METHODS

SCHEME I.

Schematic drawings of NAMI and RAP

NAMI 2 for guanine bases (6). Barca et al. recently reported that a number of ruthenium(III) complexes, including NAMI, are able to bind DNA but induce very few interstrand crosslinks (9). Some years ago, Esposito et al., through NMR and molecular modeling studies, showed that the octahedral ruthenium(II) complex trans-RuCl 2(DMSO) 4 reacts with d(GpG), giving rise to a product in which the ruthenium(II) center is coordinated to the N7 of two adjacent guanines, to two dimethyl sulfoxides, and to one chloride; this suggests that ruthenium, in its reduced form, exhibits some preference for adduction to GG sites (10). In contrast to the view that DNA is the main target for ruthenium drugs, other authors claim that DNAindependent mechanisms such as inhibition of metalloproteinases, interference with adhesion processes, and scavenging of nitric oxide are responsible for the antitumor and antimetastatic activity of these compounds (11–13). To better establish the nature of the damage that ruthenium(III) drugs infer on DNA and to determine if DNA may be a reasonable target for this family of drugs, we investigated in vitro the reactions of two representative and well-established antitumor ruthenium(III) complexes, NAMI and RAP (Scheme I), with calf thymus and plasmidic DNA. NAMI, Na[transRuCl 4(DMSO)(Im)], strictly related to ICR (4), was developed in Trieste (Italy) by the group of Mestroni and Alessio (14) and is presently undergoing the first clinical trials; RAP, dichloro(1,2-propylendiaminetetraacetate)ruthenium(III), a ternary complex of ruthenium(III) with two chlorides and a polyaminocarboxylate PDTA anion, was designed, prepared, and characterized in Sevilla (Spain) by the group of Vilchez (15). Reactions of both NAMI and RAP with DNA were monitored through absorption and CD spectroscopies; in addition, modifications of DNA conformation subsequent to ruthenium binding were analyzed, directly, through gel shift assays and, indirectly, through identification and quantitation of the DNA fragments ob2

Abbreviations used: NAMI, Na[trans-RuCl 4(DMSO)(Im); RAP, dichloro(1,2-propylendiaminetetraacetate)ruthenium(III).

Synthesis and characterization of ruthenium(III) complexes. NAMI and RAP were prepared according to the standard procedures (14, 15). The purity of both compounds was checked through elemental analysis and 1H NMR spectra. All other reagents used in this study were of analytical grade. The 1H NMR spectra were obtained on a Bruker 200 MSL spectrometer. DNA. Calf thymus DNA was purchased from Sigma Chemical Co. (St. Louis, MO) and dialyzed extensively against buffer before use. Plasmid pHV14, 7269 bp (16), was prepared and purified with Qiagen Tip 500 (Qiagen Inc., Chatsworth, CA); the preparation contained an average of 90% supercoiled form. The physical map of the plasmid with all cleavage sites for the restriction enzymes used in this work is reported in Scheme II. Preparation of ruthenium adducts with DNA for electronic and CD spectra. The reactions between cisplatin, NAMI, or RAP and calf thymus DNA were carried out by adding the required volume of a freshly prepared solution of cisplatin, NAMI, or RAP, dissolved in 100 mM sodium phosphate and 4 mM sodium chloride buffer (pH 7.4), to calf thymus DNA solutions (40 ␮g/ml) and incubating at 25°C for 24 h. Samples were prepared in such a way to have final ruthenium/DNA base pair ratios (r) of either 0.5 or 0.1. Electronic and CD spectra were recorded at increasing times after mixing for 24 h. Electronic spectra were recorded on a Lambda-Bio 20 Perkin Elmer spectrophotometer operating at room temperature. The circular dichroism spectra were recorded on a Jasco J715 dichrograph interfaced with a PC and analyzed through the standard Jasco software package. Preparation of ruthenium adducts with DNA for electrophoretic mobility assays. Adducts with pHV14 plasmid DNA were prepared by adding the required volume of a freshly prepared solution of cisplatin, NAMI, or RAP obtained using 50 mM sodium phosphate and 4 mM sodium chloride (pH 7.4) buffer. The concentration of pHV14 DNA in the reaction mixture was 75 ng/␮l, while the concentration of either cisplatin or ruthenium complexes was varied to give different metal-to-base pair stoichiometries (0.5, 0.1, 0.05, and 0.025). The mobility of the cisplatin-, NAMI-, and RAP-treated pHV14 samples was analyzed by gel electrophoresis on 0.8% (w/v) agarose gel (Boehringer-Mannheim, Mannheim, Germany) at 10 V/cm at 4°C for 8 h in Tris-acetate/EDTA buffer, and then the gel was stained for 1 h in 0.5 ␮g/ml (w/v) ethidium bromide (17). The bands were photographed and analyzed with a UVP gel scanner (GDS2000; Ultra Violet Products Ltd., Cambridge, UK). Digestion of plasmid DNA by restriction enzymes. Enzyme digestions were carried out by incubating the untreated and cisplatin-, NAMI-, or RAP-treated pHV14 samples (at r ⫽ 0.5) with AluI, BamHI, ClaI, DraI, EcoRI, HindIII, or SalI (Boehringer-Mannheim), respectively. Eight hundred nanograms of each sample was incubated with the restriction enzyme at 37°C for 2 h in the appropriate buffer recommended by the manufacturer. Each sample was filtrated using specific DNA cutoffs (Microcon Microconcentrators, Amicon, Beverly, MA) to remove excess metal complex not bound to DNA. Then DNA restriction fragments were run on 0.8% (w/v) agarose gel (Boehringer-Mannheim) or on 1% (w/v) Separide gel (GIBCO BRL, Grand Island, NY) containing 0.5 ␮g/ml (w/v) ethidium bromide at 10 V/cm at 4°C for 4 h in Tris-acetate/EDTA buffer, and the bands were photographed and analyzed as above.

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SCHEME II. Physical map of plasmid pHV14 showing restriction sites for AluI, BamHI, ClaI, DraI, EcoRI, HindIII, and SalI.

RESULTS AND DISCUSSION

Electronic and Circular Dichroism Spectra Hydrolysis reactions of both NAMI and RAP in a physiological buffer were previously described in detail (18, 19). We have investigated here hydrolysis of RAP and NAMI in the presence of saturating concentrations of calf thymus DNA (r ⫽ 0.1) by spectrophotometry. The hydrolysis process was followed for several hours at room temperature. Remarkably, NAMI and RAP hydrolysis profiles in the presence of DNA do not differ significantly from those obtained in the pure buffer (data not shown), implying that the interactions of both complexes with DNA are weak. Also, it can be ruled out that addition of DNA causes fast reduction of ruthenium(III) to ruthenium(II); indeed in the case of NAMI, reduction of ruthenium(III) results into a drastic decrease of the intense visible bands of NAMI (13), a feature that is not observed in our case. More detailed information on the interactions of both ruthenium complexes with calf thymus DNA is obtained by circular dichroism spectroscopy. CD spectra of calf thymus DNA, dissolved in the buffer, after ad-

dition of either RAP or NAMI at r ⫽ 0.1 and r ⫽ 0.5 are shown in Fig. 1. It is evident that both compounds, at r ⫽ 0.1, do not produce appreciable alterations of the characteristic CD bands of B-type DNA at 275 and 240 nm (Figs. 1A and 1C); modest spectral changes are detected, for both complexes, at the higher stoichiometry (r ⫽ 0.5) (Figs. 1B and 1D). The observed spectral changes take several hours to develop—in agreement with the view that both ruthenium(III) complexes bind DNA only after chloride hydrolysis—and may be interpreted in terms of some minor loss of DNA helicity; in any case, DNA remains in the B-type conformation. At variance with the present ruthenium(III) complexes, cisplatin induces much larger distortions of calf thymus DNA conformation that are clearly revealed by the CD spectra (Figs. 1E and 1F). As previously reported by Brabec et al. (20), addition of cisplatin at r ⫽ 0.1 causes a net increase of the positive band at 275 nm whereas addition of larger amounts of cisplatin induces a significant decrease of the same band. These results suggest a different mode of interaction with DNA between cisplatin and ruthenium(III) complexes.

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FIG. 1. CD spectra in the UV of calf thymus DNA following addition of RAP (A, B), NAMI (C, D), or cisplatin (E, F) for a final metal-to-base pair ratio of 0.1 (A, C, E) and 0.5 (B, D, F). In all cases, spectra were recorded before mixing (a) and after 24 h (b). The molar ellipticity [␪] is expressed in units of deg M ⫺1 cm ⫺1.

Electrophoretic Mobility Studies Platinum(II) complexes are known to bind plasmid DNA and modify the electrophoretic mobility of its various forms (21–23). Classically, binding of cisplatin to plasmid DNA results in a decrease in mobility of the supercoiled form (CCC (covalently closed circular)

DNA) and an increase in mobility of the open circular (OC) form; formation of multimeric species is detected as well. Following the pioneering work on gel mobility shifts caused by platinum compounds, changes in DNA mobility are usually taken as evidence of the occurrence of direct metal–DNA interactions leading to DNA

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CD on calf thymus DNA are nearly identical. On the other hand, treatment of pHV14 DNA with either NAMI or RAP, at r ⫽ 0.05 and r ⫽ 0.025, does not affect significantly the electrophoretic mobility of the supercoiled form. Inhibition of Restriction Enzymes

FIG. 2. Electrophoresis of pHV14 DNA incubated with cisplatin, NAMI, or RAP at different stoichiometries. Lanes 1 and 16, molecular mass markers: ␭ DNA digested with HindIII (GIBCO BRL); lanes 2 and 15, plasmid pHV14; lanes 3– 6, cisplatin-treated pHV14 at 0.5, 0.1, 0.05, and 0.025 stoichiometry, respectively; lanes 7–10, NAMI-treated pHV14 at 0.5, 0.1, 0.05, and 0.025 stoichiometry, respectively; lanes 11–14, RAP-treated pHV14 at 0.5, 0.1, 0.05, and 0.025 stoichiometry, respectively.

unwinding. Generally, the larger the retardation of supercoiled DNA, the larger the DNA unwinding produced by the metal complex (24). We used the agarose gel shift assay to monitor the changes in mobility of pHV14 samples caused by the addition of increasing amounts of either RAP or NAMI; for comparison purposes, parallel experiments were carried out on pHV14 alone and on cisplatin-treated pHV14 samples. The obtained gels are shown in Fig. 2. Plasmid pHV14 alone gives a single major electrophoretic band corresponding to the supercoiled form and a weaker band corresponding to oligomeric DNA (25). Cisplatin causes large mobility shifts, in agreement with literature data (23); the fast-running supercoiled form is greatly retarded as the amount of added cisplatin increases. Treatment with ruthenium(III) complexes results in significant decreases in mobility of the supercoiled form for both intermediate (r ⫽ 0.1) and high (r ⫽ 0.5) NAMI concentrations and in a slight decrease for the high RAP concentration (r ⫽ 0.5); notably, NAMI turns out to be more effective than RAP in unwinding pHV14 DNA, although the conformational effects observed via

To better describe the damage that ruthenium binding causes on DNA, we measured inhibition of some selected restriction enzymes brought about by treatment with ruthenium(III) complexes. This approach was previously applied with success to illustrate the effects and the sequence selectivity of platinum binding to DNA (26, 27). In practice, the action of various restriction enzymes on treated and control plasmid DNA samples was determined through identification and quantitation of the fragments resulting from enzymatic digestion of DNA. The decrease in concentration of the various restriction fragments directly correlates with the degree of inhibition of the corresponding restriction enzymes. We chose seven representative restriction enzymes, which differ in their target sequence (namely, AluI, BamHI, ClaI, DraI, EcoRI, HindIII, and SalI), and tested their ability to cleave ruthenated pHV14 samples. This analysis was performed on the high-concentration ruthenium(III)–pHV14 samples (r ⫽ 0.5) that showed the largest effects in the electrophoretic studies. For comparison purposes, the ability of cisplatin to inhibit the same restriction enzymes, under the same experimental conditions, was checked. A representative gel of enzymatic digestion— with BamHI— of pHV14 alone and of NAMI– and RAP–pHV14 samples is shown in Fig. 3. Remarkably, binding of NAMI to the pHV14 plasmid results in important inhibition of BamHI activity; a slight inhibition of BamHI is also observed in the case of RAP. In any case, inhibition produced by NAMI is smaller than that caused by cisplatin. Parallel experiments were carried out with various other restriction endonucleases; results are shown in Table I. These results indicate that cisplatin is able to inhibit pHV14 cleavage by all tested enzymes, with the exception of DraI. NAMI inhibits BamHI and SalI to a large extent and ClaI, EcoRI, and HindIII only slightly. No effect is observed with AluI and DraI. On the other hand, treatment with RAP inhibits, slightly, only BamHI. Notably, DraI, a restriction enzyme that recognizes a target sequence characterized by the absence of G (see Table I), is able to cleave all the treated pHV14 samples. To rule out that the observed inhibition is a consequence of a direct interaction of the ruthenium(III) complexes with restriction enzymes, additional experiments were carried out in which ruthenated pHV14 samples, previously precipitated by ethanol, were resuspended in the buffer. This treatment did not affect enzyme inhibition,

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the SalI site with cisplatin presumably arises from a 1,3 interstrand d(G),d(G) crosslink. Notably, NAMI has been shown to protect to a large extent BamHI and SalI sites and partially ClaI, EcoRI, and HindIII sites; no protection is observed for AluI and DraI sites. Thus, NAMI reproduces somehow the pattern of cisplatin, with the only exception of AluI sites. The latter sites are fully protected by cisplatin but completely cleaved in the presence of NAMI. This result seems to suggest that NAMI does not form 1,2 intrastrand d(ApG) crosslinks. On the contrary, RAP exhibits very weak effects, producing only partial inhibition of BamHI. In any case, both NAMI and RAP protect somehow BamHI sites with a GGATCC target sequence, suggesting formation of 1,2 intrastrand d(GpG) crosslinks. Overall, the results obtained with restriction enzymes suggest some preference of ruthenium complexes for guanine; however, the presence of a single guanine per se is not sufficient to afford protection. CONCLUSIONS FIG. 3. Electrophoresis of untreated and cisplatin-, NAMI-, and RAP-treated pHV14 samples after digestion with BamHI. Lanes 1 and 10, molecular mass markers: ␭ DNA digested with HindIII (GIBCO BRL); lane 2, BamHI-treated pHV14; lane 3, pHV14; lane 4, BamHI-treated pHV14 – cisplatin; lane 5, pHV14 – cisplatin; lane 6, BamHI-treated pHV14 –NAMI; lane 7, pHV14 –NAMI; lane 8, BamHI-treated pHV14 –RAP; lane 9, pHV14 –RAP.

supporting the view that ruthenated DNA—and not the free ruthenium complex—is indeed responsible for enzyme inhibition. The results obtained with restriction enzymes deserve some further comments. Cisplatin gives rise preferentially to 1,2 intrastrand d(GpG) or d(ApG) crosslinks (28); this property nicely explains full protection of AluI and BamHI restriction sites and partial protection of the EcoRI and HindIII sites. Protection of

The present results demonstrate that both NAMI and RAP, in vitro, are able to alter DNA conformation and inhibit, at least partially, DNA recognition and cutting by restriction enzymes. Remarkably, NAMI is far more effective than RAP in producing these effects although less effective than cisplatin. DNA damage produced by these ruthenium(III) complexes is detected only at relatively high concentrations—significantly larger than those at which cisplatin produces similar effects—in good agreement with the lower cytotoxicity and lower systemic toxicity of these ruthenium compounds. Notably, the cytotoxic effects of NAMI and RAP are much lower than those induced by mer-[Ru(III)(terpy)Cl 3], whose ability to drastically modify DNA conformation was previously well documented. No evidence of ruthenium(III) reduction has been found. These findings lead us to conclude that DNA may represent a feasible target for the action of

TABLE I

Cleavage of pHV14 Plasmid by Various Restriction Enzymes Following Treatment with Cisplatin, NAMI, or RAP Restriction enzyme

Sample

AluI 2 AG CT TC GA 1

BamHI 2 G GATCC CCTAG G 1

ClaI 2 AT CGAT TAGC TA 1

DraI 2 TTT AAA AAA TTT 1

EcoRI 2 GA ATTC CTTA AG 1

HindIII 2 A AGCTT TTCGA A 1

SalI 2 G TCGAC CAGCT G 1

pHV14 pHV14–cisPt pHV14–NAMI pHV14–RAP

⫹ ⫺ ⫹ ⫹

⫹ ⫺ ⫾a ⫾c

⫹ ⫾a ⫾c ⫹

⫹ ⫹ ⫹ ⫹

⫹ ⫾b ⫾c ⫹

⫹ ⫾b ⫾c ⫹

⫹ ⫺ ⫾a ⫹

⫹, total enzymatic digestion; ⫾, partial inhibition of enzymatic digestion ( a 90,

b

50, c 10%); ⫺, total inhibition of enzymatic digestion.

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these ruthenium(III) complexes provided that substantial amounts of ruthenium are able to enter cells and to reach nuclear DNA. In any case, other reactions involving direct binding of ruthenium(III) complexes to different biomolecular targets (e.g., proteins and enzymes) might play as well a crucial role in the mechanism of action of ruthenium metallodrugs. ACKNOWLEDGMENTS MURST is acknowledged for financial support in the frame of the project Pharmacological and Diagnostic Aspects of Metal Complexes. The Cassa di Risparmio di Firenze is gratefully acknowledged for a generous grant. L.M. gratefully acknowledges stimulating discussions with Dr. Viktor Brabec.

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