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Comparison of the mode of action of a dinuclear platinum complex containing a pyridine derivative with its monomeric analog
Chemico-Biological Interactions 116 (1998) 19 – 29
Comparison of the mode of action of a dinuclear platinum complex containing a pyridine derivative with its monomeric analog Guanghua Zhao a, Huakoan Lin a,*, Ping Yu b, Hongwei Sun a, Shourong Zhu a, Yunti Chen a b
a Department of Chemistry, Nankai Uni6ersity, Tianjin 300071, PR China Institute of Basic Medical Sciences, Academy of Military Medicine Sciences, Beijing 100850, PR China
Received 4 March 1998; received in revised form 8 July 1998; accepted 23 July 1998
Abstract The DNA binding and interstrand cross-linking properties of the dinuclear platinum complex [{cis-Pt(NH3)2Cl}2bpsu](NO3)2 (bpsu is 4,4%-dipyridyl sulfide) (II) and the mononuclear complex [cis-Pt(NH3)2Cl(4-methylpyridine)]NO3 (I) were compared with those of [{cis-Pt(NH3)2Cl}2H2N(CH2)4NH2](NO3)2 (III) in order to understand the mode of action of complexes I and II. Both compound I and compound II caused significantly different changes of conformation in poly(dG-dC) · poly(dG-dC) than compound III did. Studies of DNA binding, interstrand cross-linking and fluorescence assay suggest that compound I monofunctionally binds to DNA and compound II bifunctionally binds to DNA, that the dinuclear platinum complex II more efficiently interacts with DNA compared to its monomeric analog, and that platinum I and II complexes both interact with DNA in a non-intercalative mode. All the results indicate that the mode of action of the dinuclear complex II is different from that of the mononuclear complex I. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Conformational changes; DNA – metal complexes; Mononuclear or dinuclear complexes; Platinum complexes
* Corresponding author. 0009-2797/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(98)00074-X
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1. Introduction While cis-diamminedichloroplatinum(II) (cisplatin) is efficient against testicular and ovarian cancers and neck and head tumors [1,2], a continuing effort is being made in analog development programs to broaden the spectrum of activity and to improve the therapeutic properties of platinum-based agents. Direct structural analogs of cisplatin have not, however, shown a greatly altered spectrum of clinical efficacy in comparison to the parent drug [3,4]. The explanation for this finding is that all the cisplatin analogs produce a similar array of adducts on DNA. Thus, their biological consequences are also expected to be similar [3,5]. One approach to this problem is to identify new classes of active platinum complexes having structural features that differ from those of the existing cisplatin analogs. In general, the majority of the cisplatin analogs that display antitumor activity are neutral platinum(II) complexes of the form cis-[PtA2X2], in which A is an amine ligand with at least one NH group and X is a moderately strong leaving group such as chloride [6]. The activity of platinum compounds in vivo and in vitro stems from their interaction with DNA. These complexes produce bifunctional lesions on the DNA of the tumor cell that are capable of inhibiting DNA replication and transcription [7,8], whereas the interaction of the platinum compounds with protein is generally believed to be the most likely origin of the several toxic side-effects [9]. However, recently a new class of compounds have been synthesized with the general formula cis-[Pt(NH3)2Cl(N-het)] + (N-het is a heterocyclic amine like
Fig. 1. Structures of the platinum complexes used in this study.
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pyridine), which violates two of the classical rules by being cationic and monofunctional (Fig. 1). These agents show promising antitumor activities [10]. We have also recently synthesized four dinuclear platinum complexes with the general formula [{cis-Pt(NH3)Cl}2L](NO3)2 (L is 4,4%-dipyridyl sulfide or selenide, 4,4%-bis(3-methylpyridyl) sulfide or selenide) [11]. These dinuclear platinum complexes are of special interest because they do not contain a cis-[PtA2X2] unit yet show good antitumor activities in vitro [11] and in vivo (G. Zhao, unpublished results). The structure of the dinuclear platinum complexes has some similarity to that of the above mononuclear complexes. However, the dinuclear platinum complexes are not simple adducts of two monomeric analogs. Some studies indicate that sulfur-containing compounds such as glutathione can antagonize the toxic effects of the drugs [12,13] and an important application of selenium compounds may be their use as chemoprotectors against the toxic side-effects of drugs [14,15]. It is more important that alteration of the structure of dinuclear platinum complexes in comparison to their monomeric analogs may result in alteration of mode of DNA binding, which may result in an altered spectrum of antitumor activity and/or toxicity to non-target cells [3,5,16,17]. Therefore, in the present study, it is of interest to compare binding properties and conformational changes in DNA of the complex [{cisPt(NH3)2Cl}2bpsu](NO3)2 (bpsu is 4,4%-dipyridyl sulfide) (II) with those of its mononuclear analog [cis-Pt(NH3)2Cl(4-methylpyridine)]NO3 (I) which is one of the most active complexes [10].
2. Materials and methods
2.1. Compounds The synthesis of the dinuclear platinum complexes II and III and of the mononuclear platinum complex I has already been described [10,11,18]. Poly(dG-dC) · poly(dG-dC) and calf thymus DNA (CT DNA) were purchased from Pharmacia. Ethidium bromide (EtdBr) was purchased from Sigma; its molar absorbance coefficient in water was 5450 M − 1 cm − 1 at 480 nm. Other chemicals used were of analytical grade or high purity grade.
2.2. Physicaf methods Ultraviolet absorbance were run on a Shimadzu UV-265 UV–Vis recording spectrophotometer. Circular dichroism (CD) spectra were performed on a Jasco J-715 spectropolarimeter. Fluorescence measurements were performed on a Shimadzu RF-540 spectrophotometer. In the experiments, the excitation wavelength was set at 540 nm and the emission at 590 nm. Flameless atomic absorption spectroscopy (FAAS) measurements were carried out on a Perkin-Elmer 560 instrument with a graphite furnace.
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2.3. Determination of relati6e binding (rb) Relative binding, rb, is defined as a number of the compound molecules bound per base pair (bp) of DNA: rb = Cb/CDNA(bp). The procedure is as follows. Poly(dG-dC) · poly(dG-dC) (0.016 mg/ml) was incubated with varying concentrations (10 – 200 mM) of platinum complexes in TEN buffer (10 mM Tris-HCl, 0.1 mM EDTA, 10 mM NaCl, pH 7.2) for 24 h at 37°C, followed by addition of 200 mM NaCl to terminate the reaction. Unbound platinum complexes were removed by extensive dialysis against TEN buffer at 4°C. The CDNA(bp) of the samples was determined by DNA absorption at 260 nm. Cb, the Pt content of the samples, was determined by FAAS by suspending the solutions in 2% nitric acid and heating at 50°C for 24 h to hydrolyze the DNA.
2.4. Measurement of interstrand cross-links Interstrand cross-links formed in CT DNA upon binding of complex I, complex II and [{cis-Pt(NH3)2Cl}2(NH2(CH2)4NH2)](NO3)2 (III), which may be considered as a positive control [18], were studied using an EtdBr fluorescence assay based on that described by Lown and Kharatishvili [19,20]. Ir, the ratio of fluorescence of bound EtdBr of heated samples (Ibd) to that of unheated samples (Ibn), represents the fraction of DNA which has renatured after the ‘temperature jump’.
2.5. Fluorescence spectroscopic measurements EtdBr binding to DNA and DNA–metal complexes (10 mg/ml) was performed using fluorospectrophotometry in 0.3 M KNO3 in order to avoid the second non-intercalative binding site of EtdBr [21]. The results were expressed by the ratio I1/I0, where I1 is the fluorescence intensity of metal–DNA–EtdBr complex minus fluorescence intensity of pure EtdBr and I0 is the fluorescence intensity of DNA– EtdBr complex minus fluorescence intensity of pure EtdBr.
2.6. Kinetics of DNA– metal interaction Two parallel reactions were run at 37°C in the dark and under stirring in 0.3 M KNO3 and 0.3 M NaCl, respectively. At zero time, freshly dissolved compounds in 0.3 M KNO3 or 0.3 M NaCl were added to DNA (25 mg/ml). At various times after initiation, aliquots (1 ml) were added to 1.5 ml EtdBr at a final concentration of 40 mg/ml, corresponding to the saturation of all the intercalation sites of DNA. Fluorescence measurements were performed in the same conditions as described in Section 2.5.
2.7. CD spectroscopy Each Pt – poly(dG-dC) · poly(dG-dC) sample, which was prepared according to the procedure described in Section 2.3, was allowed to warm up to room tempera-
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Fig. 2. Relative binding rb of the mononuclear complex I ( ), the dinuclear complex II () and the dinuclear complex III () with poly(dG-dC) · poly(dG-dC) as a function of dose.
ture (18 91°C) prior to placing a 1-ml sample into the cell for analysis. Each sample was scanned in the range 220–350 nm. A CD spectrum was generated which represented the mean of three scans from which the buffer background had been electronically subtracted.
3. Results The structures of complexes used in this study are shown in Fig. 1. Fig. 2 shows rb values for compound I, compound II and compound III with poly(dG-dC) · poly(dG-dC) plotted versus the initial concentration of the compounds (10 – 200 mM). Both of the dinuclear platinum complexes displayed a plateauing effect at concentrations of approximately 150 mM and this effect was not observed for the mononuclear complex. Under the conditions employed, the two dinuclear complexes showed a similar ability to bind, which was much stronger than that of the mononuclear complex I. Interstrand cross-linking was assayed by the fluorescence methods of Lown and Khavatishvli [19,20]. This assay is useful for the preliminary assessment of crosslinking abilities. Fig. 3 shows the fraction of CT DNA renatured after the temperature jump plotted against the initial dose. Increasing the concentration of the dinuclear compounds significantly increased this fraction. The dinuclear platinum complex III showed the strongest ability to cause cross-links in CT DNA with concentrations as low as 5×10 − 9 M causing a 50% increase in fluorescence. The interstrand cross-linking of another dinuclear complex II containing 4,4%-dipyridyl sulfide, 10 − 8 M, which caused only a 27% increase of fluorescence, was significantly weaker in comparison to the dinuclear complex III bridged by 1,4-butanediamine. For the mononuclear complex I, increasing the concentration induced very little increase in fluorescence.
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Fig. 3. Renaturation of CT DNA complexed with complex I ( ), complex II () amd complex III () upon boiling and fast cooling at pH 11.8 as determined by EtdBr fluorescence. Ir is the ratio of EtdBr fluorescence in heated and cooled samples of DNA treated by compounds against the samples kept at room temperature.
The complexation of a metal to DNA can lead to perturbations of helix DNA which have been revealed by different physicochemical techniques [22,23]. Among these techniques, the use of a fluorescence intercalating agent, EtdBr, offers a useful means of studying the reaction of a compound with DNA. The perturbations induced by complexation of platinum compounds to DNA inhibit the intercalation of EtdBr and lead to a decrease of the fluorescence of the DNA–Pt–EtdBr complex [24,25]. In Fig. 4, the percentage of fluorescence decrease I1/I0 for DNA–cis[Pt(NH3)2Cl(4-methylpyridine)]NO3 and DNA–[{cis-Pt(NH3)Cl}2(bpsu)](NO3)2 complexes was plotted against the concentration. While the dinuclear platinum
Fig. 4. Relationship between the fluorescence decrease (I1/I0) and the concentration of complex I ( ) or complex II. I1 represents the increasing DNA – metal – EtdBr fluorescence above background EtdBr fluorescence and I0 the increasing DNA – EtdBr fluorescence above background EtdBr fluorescence. Final DNA–metal complexes and EtdBr concentrations were 10 and 40 mg/ml, respectively.
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Fig. 5. Kinetics of the interaction between DNA and complex I. The reactions were run at 37°C in 0.3 M NaCl ( ) and 0.3 M KNO3 ( ), respectively. (b) Kinetics of the interaction between DNA and complex II. The reactions were also run at 37°C in 0.3 M NaCl () and 0.3 M KNO3 (), respectively. At the starting point, metal solutions (final concentration 50 mM) were added to the DNA (25 mg/ml). Aliquots (1 ml) were taken at different times and mixed with 1.5 ml of EtdBr (66 mg/ml) in 0.3 M NaCl and 0.3 M KNO3, respectively, and metal – DNA complexes determined.
compound II induced a fluorescence decrease, its monomeric analog I caused very little decrease. Fig. 5a,b show profiles of the kinetics of the interaction between DNA and the metal compounds. With the increase of reaction time, both mononuclear complex I and dinuclear complex II did not induce a decrease in fluorescence when the reactions were run in 0.3 M NaCl. This indicates that the two platinum complexes do not react with DNA in 0.3M NaCl. In contrast, with an increase in reaction time, the dinuclear complex II caused a significant decrease in fluorescence and had a t1/2 of approximately 7.0 (t1/2 is defined as a fluorescence decrease of 50%), and the mononuclear complex I did not cause a significant decrease in fluorescence, when the reactions were run in 0.3 M KNO3.
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Fig. 6a,b represent CD spectra of poly(dG-dC) · poly(dG-dC) which was treated with different concentrations of the two complexes containing an aromatic ligand. Previous studies had indicated that the Z form of poly(dG-dC) · poly(dG-dC) was induced very efficiently upon binding of the dinuclear complex III [18] and mononuclear platinum complex [Pt(dien)Cl] + but not by cis-DDP [26,27] The generality of this feature was examined with the two complexes studied here. Unlike the dinuclear complex III, the dinuclear complex II [{cis-Pt(NH3)2 Cl}2(bpsu)](NO3)2 did not induce a BZ transition as observed earlier for cisplatin. This suggests that the adducts formed by the two dinuclear platinum complexes are structurally distinct. However, the mononuclear platinum complex I shows a significantly different conformation of poly(dG-dC) · poly(dG-dC) in comparison to its dimeric analog. Compound I caused a B Z transition, but it did not induce a complete inversion from B form to Z form even at a concentration of 100 mM.
4. Discussion The DNA binding of the metal complexes studied here is not reversible even upon prolonged dialysis of Pt – DNA samples, indicating that their binding to DNA likely involves covalent interaction. The irreversible binding of metal complex to DNA is strictly true for kinetically inert platinum(II)–DNA complex. However, the binding of complexes I and II to DNA can be completely prevented when the reactions are run in 0 3 M NaCl. Similarly, cisplatin (in its dichloro form) in the
Fig. 6. CD spectra of poly(dG-dC) · poly(dG-dC) modified by complex II (a) and complex I (b). The corresponding rb values are found in Fig. 2.
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presence of at least 10 − 1 M Cl − (the leaving group) does not react with DNA [28]. This indicates that hydrolysis is the first step when the two metal complexes interact with DNA as suggested earlier for the interaction of cisplatin with DNA. At the same time, it suggests that the two metal complexes may bind to DNA by non-intercalation, although both of them contain planar pyridine ring(s). Our previous studies with fluorescence Scatchard assay also suggest that the dinuclear complex II interacts in a non-intercalative mode with DNA [11]. The results of bacterial survival and mutagenesis experiments with Escherichia coli strains show that such an intercalation in the DNA by the pyridine ring moiety of the mononuclear complex I is also unlikely [6]. The mononuclear complex I shows promising antitumor activities [10]. It is expected to behave monofunctionally in reactions with nucleophiles, since the three amine ligands are poor leaving groups. Other related monofunctional compounds such as [Pt(dien)Cl] + and [Pt(NH3)3Cl] + are known to be antitumor inactive [26,27,29]. A possible explanation for the antitumor activity could be the in vivo generation of a bifunctional platinum compound cis-[Pt(NH3)(4-methylpyridine)]2 + , which could then be the active species [10]. In the present work, measurement of interstrand cross-links indicated that complex I shows much weaker ability to cause cross-links in CT DNA in comparison to the reported bifunctional complex cis-[Pt(NH3)(quinoline)Cl2] in which a concentration of 5×10 − 7 M caused a 60% increase in fluorescence [20], and the dinuclear complex III, bifunctionally binding to DNA [18], showed an almost two times stronger ability for DNA binding than that of the monomeric complex I. These results do not support the formation of the bifunctionally active species cis-[Pt(NH3)(4-methylpyridine)]2 + . In addition, like the monofunctional complex [Pt(dien)Cl] + [30], complex I also had no apparent effect on EtdBr binding, further indicating that it monofunctionally binds to DNA. However, the mode of action of complex I with DNA is different from that of [Pt(dien)Cl] + with DNA based on the different changes in conformation in poly(dG-dC) · poly(dG-dC) induced by the two mononuclear complexes. Complex [Pt(dien)Cl] + caused a complete inversion from B form to Z form [26,27] but not complex I. The pyridine ring moiety of complex I may hinder the inversion and, at the same time, it may play an important role in its antitumor activities. This suggests that the conformational changes in DNA induced by platinum complexes are closely related to their antitumor activities. As compared to its monomeric analog, the dinuclear complex II shows a stronger ability for DNA binding and interstrand cross-links, and a more significant decrease in fluorescence with increases in concentration or time. This indicates that it may bifunctionally bind to DNA. From Fig. 2, it is observed that the DNA binding ability of compound II is very similar to that of the bifunctional compound III. This result further supports the conclusion that complex II also bifunctionally interacts with DNA. However, interestingly, complex II has a lower interstrand cross-linking efficiency than compound llI. This may be a reason why complex III induces the BZ transition in poly(dA-dC) · poly(dG-dC) [18] but not complex II which contains the planar pyridine ring ligand, since some studies indicate that interstrand cross-links may be very useful in ‘locking’ the Z conformation [31]. On
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the other hand, due to steric hindrance, complex II possibly causes more intrastrand cross-links than complex III, as suggested for cisplatin [3]. The intrastrand cross-linking interaction of cisplatin with poly(dG-dC) · poly(dG-dC) prevents the rotation of the guanine bases about their glycosyl bonds and prohibits the B Z transition in poly(dG-dC) · poly(dG-dC) [27]. The results presented here suggest that the mononuclear complex I produces monofunctional adducts on DNA. However, the mode of action of complex I with DNA is different than that of [Pt(dien)Cl] + with DNA. The different mode of action of the two mononuclear platinum complexes may be related to their difference in antitumor activities. Compared to its monomeric analog, the dinuclear complex II may produce bifunctional adducts on DNA. The DNA binding of complex II, which is different from that of the dinuclear complex III, is characterized by relatively large numbers of intrastrand cross-links and small numbers of interstrand cross-links. It would be of considerable clinical interest if the novel DNA adducts led to a broader spectrum of antitumor activity.
Acknowledgements This project 29671020 was supported by the National Natural Science Foundation of P.R. China, the Nature Science Foundation of Tianjin and the National Key Laboratory of Coordination Chemistry, Nanjing University.
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[11] G. Zhao, H. Lin, S. Zhu, H. Sun, Y. Chen, Synthesis and biological activity of binuclear platinum complexes containing two monofunctional cis-[Pt(NH3)2Cl] + units bridged by 4,4%-dipyridyl selenides or sulfides, Anti-Cancer Drug Design (in press). [12] S. Ray, F.R. Smith, J.N. Bridson, Q. Hong, V.J. Richardson, S.K. Mandal, Thiopyridyl triazine derivatives and their platinum complexes: a new class of potential antitumor agents, Inorg. Chim. Acta 227 (1994) 175–179. [13] G. Zhao, H. Lin, S. Zhu, H. Sun, Y. Chen, Dinuclear palladium(II) complexes containing two monofunctional [Pd(en)(pyridine)Cl] + units bridged by Se or S. Synthesis, characterization, cytotoxicity and kinetic studies of DNA-binding, J. Inorg. Biochem. 70 (1998) 219 – 226. [14] I. Andreadou, W.P.B. Menge, J.M. Commandeur, E.A. Worthington, N.E. Vermeulen, Synthesis of novel Se-substituted selenocyteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: evaluation of kinetics of b-elimination reactions in rat renal cytosol, J. Med. Chem. 39 (1996) 2040–2046. [15] G.S. Baldew, C.A. van den Hamer, G. Los, N.E. Vermeulen, J.M. de Goeij, J.G. Mcvie, Selenium-induced protection against cis-diamminedichloroplatinum(II) nephrotoxicity in mice and rats, Cancer Res. 49 (1989) 3020–3023. [16] A.L. Kraker, J.D. Hoeschele, W.L. Elliott, H.D. Hollis Showalter, A.D. Sercel, Anticancer activity in murine and human tumor cell lines of bis(platinum) complexes incorporating straight-chain aliphatic diamine linker groups, J. Med. Chem. 35 (1992) 4526 – 4532. [17] N. Farrell, Y. Qu, M.P. Hacker, Cytotoxicity and antitumor activity of bis(platinum) complexes. A novel class of platinum complexes active in cell lines resistant to both cisplatin and 1,2-diaminocyclohexane complexes, J. Med. Chem. 33 (1990) 2179 – 2184. [18] N. Farrell, T.G. Appleton, Y. Qu, J.D. Roberts, A.P. Soares Fontes, K.A. Skov, P. Wu, Y. Zou, Effect of geometric isomerism and ligand substitution in bifunctional dinuclear platinum complexes on binding properties and conformational changes in DNA, Biochemistry 34 (1995) 15480 – 15486. [19] J.W. Lown, A. Begleiter, Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay, Can. J. Biochem. 54 (1976) 110. [20] M. Kharatishvili, M. Mathieson, N. Farrell, Effect of quinoline as ligand in binding of mononuclear and dinuclear platinum complexes to DNA, Inorg. Chim. Acta 255 (1997) 1 – 6. [21] J.B. LePecq, Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes, Methods Biochem. Anal. 20 (1971) 41–86. [22] J.L. Butour, J.P. Macquet, Differentiation of DNA – platinum complexes by fluorescence: the use of an intercalating dye as a probe, Eur. J. Biochem. 78 (1977) 455 – 463. [23] J.L. Butour, J.P. Macquet, Viscosity, nicking, thermal and alkaline denaturation studies on three classes of DNA–platinum complexes, Biochim. Biophys. Acta 653 (1981) 305 – 315. [24] J.L. Butour, A.M. Mazard, J.P. Macquet, Kinetics of the reaction of cis-platinum compounds with DNA in vitro, Biochem. Biophys. Res. Commun. 133 (1985) 347 – 353. [25] F.L. Wimmer, S. Wimmer, P. Castan, S. Cros, N. Johnson, E. Colacio-Rodriguez, The antitumor activity of some palladium(II) complexes with chelating ligands, Anticancer Res. 9 (1989) 791 – 794. [26] B. Malfoy, B. Hartmann, M. Leng, The B Z transition of poly(dGdC) · poly(dG-dC) modified by some platinum derivatives, Nucleic Acids Res. 9 (1981) 5659 – 5669. [27] H.M. Ushay, R.M. Santella, J.P. Caradonna, D. Grunberger, S.J. Lippard, Binding of [(dien)PtCl]Cl to poly(dG-dC) · poly(dG-dC) facilitates the B Z conformational transition, Nucleic Acids Res. 10 (1982) 3573–3588. [28] J.L. Butour, J.P. Macquet, Platinum determination in DNA – platinum complexes by fluorescence spectrophotometry, Anal. Biochem. 89 (1978) 22 – 30. [29] J.P. Macquet, J.L. Butour, Platinum-amine compounds: importance of the labile and inert ligands for their pharmacological activities toward L1210 leukemia cells, J. Natl. Cancer Inst. 70 (1983) 899–905. [30] M. Howe-Grant, K. Wu, W.R. Bauer, S.L. Lippard, Binding of platinum and palladiun metallointercalation reagents, Biochemistry 19 (1976) 4339 – 4346. [31] A. Johnson, Y. Qu, B. Van Houten, N. Farrell, B Z DNA conformational changes induced by a family of dinuclear bis(platinum) complexes, Nucleic Acids Res. 20 (1992) 1697 – 1703. .
Comparison of the mode of action of a dinuclear platinum complex containing a pyridine derivative with its monomeric analog Guanghua Zhao a, Huakoan Lin a,*, Ping Yu b, Hongwei Sun a, Shourong Zhu a, Yunti Chen a b
a Department of Chemistry, Nankai Uni6ersity, Tianjin 300071, PR China Institute of Basic Medical Sciences, Academy of Military Medicine Sciences, Beijing 100850, PR China
Received 4 March 1998; received in revised form 8 July 1998; accepted 23 July 1998
Abstract The DNA binding and interstrand cross-linking properties of the dinuclear platinum complex [{cis-Pt(NH3)2Cl}2bpsu](NO3)2 (bpsu is 4,4%-dipyridyl sulfide) (II) and the mononuclear complex [cis-Pt(NH3)2Cl(4-methylpyridine)]NO3 (I) were compared with those of [{cis-Pt(NH3)2Cl}2H2N(CH2)4NH2](NO3)2 (III) in order to understand the mode of action of complexes I and II. Both compound I and compound II caused significantly different changes of conformation in poly(dG-dC) · poly(dG-dC) than compound III did. Studies of DNA binding, interstrand cross-linking and fluorescence assay suggest that compound I monofunctionally binds to DNA and compound II bifunctionally binds to DNA, that the dinuclear platinum complex II more efficiently interacts with DNA compared to its monomeric analog, and that platinum I and II complexes both interact with DNA in a non-intercalative mode. All the results indicate that the mode of action of the dinuclear complex II is different from that of the mononuclear complex I. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Conformational changes; DNA – metal complexes; Mononuclear or dinuclear complexes; Platinum complexes
* Corresponding author. 0009-2797/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(98)00074-X
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1. Introduction While cis-diamminedichloroplatinum(II) (cisplatin) is efficient against testicular and ovarian cancers and neck and head tumors [1,2], a continuing effort is being made in analog development programs to broaden the spectrum of activity and to improve the therapeutic properties of platinum-based agents. Direct structural analogs of cisplatin have not, however, shown a greatly altered spectrum of clinical efficacy in comparison to the parent drug [3,4]. The explanation for this finding is that all the cisplatin analogs produce a similar array of adducts on DNA. Thus, their biological consequences are also expected to be similar [3,5]. One approach to this problem is to identify new classes of active platinum complexes having structural features that differ from those of the existing cisplatin analogs. In general, the majority of the cisplatin analogs that display antitumor activity are neutral platinum(II) complexes of the form cis-[PtA2X2], in which A is an amine ligand with at least one NH group and X is a moderately strong leaving group such as chloride [6]. The activity of platinum compounds in vivo and in vitro stems from their interaction with DNA. These complexes produce bifunctional lesions on the DNA of the tumor cell that are capable of inhibiting DNA replication and transcription [7,8], whereas the interaction of the platinum compounds with protein is generally believed to be the most likely origin of the several toxic side-effects [9]. However, recently a new class of compounds have been synthesized with the general formula cis-[Pt(NH3)2Cl(N-het)] + (N-het is a heterocyclic amine like
Fig. 1. Structures of the platinum complexes used in this study.
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pyridine), which violates two of the classical rules by being cationic and monofunctional (Fig. 1). These agents show promising antitumor activities [10]. We have also recently synthesized four dinuclear platinum complexes with the general formula [{cis-Pt(NH3)Cl}2L](NO3)2 (L is 4,4%-dipyridyl sulfide or selenide, 4,4%-bis(3-methylpyridyl) sulfide or selenide) [11]. These dinuclear platinum complexes are of special interest because they do not contain a cis-[PtA2X2] unit yet show good antitumor activities in vitro [11] and in vivo (G. Zhao, unpublished results). The structure of the dinuclear platinum complexes has some similarity to that of the above mononuclear complexes. However, the dinuclear platinum complexes are not simple adducts of two monomeric analogs. Some studies indicate that sulfur-containing compounds such as glutathione can antagonize the toxic effects of the drugs [12,13] and an important application of selenium compounds may be their use as chemoprotectors against the toxic side-effects of drugs [14,15]. It is more important that alteration of the structure of dinuclear platinum complexes in comparison to their monomeric analogs may result in alteration of mode of DNA binding, which may result in an altered spectrum of antitumor activity and/or toxicity to non-target cells [3,5,16,17]. Therefore, in the present study, it is of interest to compare binding properties and conformational changes in DNA of the complex [{cisPt(NH3)2Cl}2bpsu](NO3)2 (bpsu is 4,4%-dipyridyl sulfide) (II) with those of its mononuclear analog [cis-Pt(NH3)2Cl(4-methylpyridine)]NO3 (I) which is one of the most active complexes [10].
2. Materials and methods
2.1. Compounds The synthesis of the dinuclear platinum complexes II and III and of the mononuclear platinum complex I has already been described [10,11,18]. Poly(dG-dC) · poly(dG-dC) and calf thymus DNA (CT DNA) were purchased from Pharmacia. Ethidium bromide (EtdBr) was purchased from Sigma; its molar absorbance coefficient in water was 5450 M − 1 cm − 1 at 480 nm. Other chemicals used were of analytical grade or high purity grade.
2.2. Physicaf methods Ultraviolet absorbance were run on a Shimadzu UV-265 UV–Vis recording spectrophotometer. Circular dichroism (CD) spectra were performed on a Jasco J-715 spectropolarimeter. Fluorescence measurements were performed on a Shimadzu RF-540 spectrophotometer. In the experiments, the excitation wavelength was set at 540 nm and the emission at 590 nm. Flameless atomic absorption spectroscopy (FAAS) measurements were carried out on a Perkin-Elmer 560 instrument with a graphite furnace.
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2.3. Determination of relati6e binding (rb) Relative binding, rb, is defined as a number of the compound molecules bound per base pair (bp) of DNA: rb = Cb/CDNA(bp). The procedure is as follows. Poly(dG-dC) · poly(dG-dC) (0.016 mg/ml) was incubated with varying concentrations (10 – 200 mM) of platinum complexes in TEN buffer (10 mM Tris-HCl, 0.1 mM EDTA, 10 mM NaCl, pH 7.2) for 24 h at 37°C, followed by addition of 200 mM NaCl to terminate the reaction. Unbound platinum complexes were removed by extensive dialysis against TEN buffer at 4°C. The CDNA(bp) of the samples was determined by DNA absorption at 260 nm. Cb, the Pt content of the samples, was determined by FAAS by suspending the solutions in 2% nitric acid and heating at 50°C for 24 h to hydrolyze the DNA.
2.4. Measurement of interstrand cross-links Interstrand cross-links formed in CT DNA upon binding of complex I, complex II and [{cis-Pt(NH3)2Cl}2(NH2(CH2)4NH2)](NO3)2 (III), which may be considered as a positive control [18], were studied using an EtdBr fluorescence assay based on that described by Lown and Kharatishvili [19,20]. Ir, the ratio of fluorescence of bound EtdBr of heated samples (Ibd) to that of unheated samples (Ibn), represents the fraction of DNA which has renatured after the ‘temperature jump’.
2.5. Fluorescence spectroscopic measurements EtdBr binding to DNA and DNA–metal complexes (10 mg/ml) was performed using fluorospectrophotometry in 0.3 M KNO3 in order to avoid the second non-intercalative binding site of EtdBr [21]. The results were expressed by the ratio I1/I0, where I1 is the fluorescence intensity of metal–DNA–EtdBr complex minus fluorescence intensity of pure EtdBr and I0 is the fluorescence intensity of DNA– EtdBr complex minus fluorescence intensity of pure EtdBr.
2.6. Kinetics of DNA– metal interaction Two parallel reactions were run at 37°C in the dark and under stirring in 0.3 M KNO3 and 0.3 M NaCl, respectively. At zero time, freshly dissolved compounds in 0.3 M KNO3 or 0.3 M NaCl were added to DNA (25 mg/ml). At various times after initiation, aliquots (1 ml) were added to 1.5 ml EtdBr at a final concentration of 40 mg/ml, corresponding to the saturation of all the intercalation sites of DNA. Fluorescence measurements were performed in the same conditions as described in Section 2.5.
2.7. CD spectroscopy Each Pt – poly(dG-dC) · poly(dG-dC) sample, which was prepared according to the procedure described in Section 2.3, was allowed to warm up to room tempera-
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Fig. 2. Relative binding rb of the mononuclear complex I ( ), the dinuclear complex II () and the dinuclear complex III () with poly(dG-dC) · poly(dG-dC) as a function of dose.
ture (18 91°C) prior to placing a 1-ml sample into the cell for analysis. Each sample was scanned in the range 220–350 nm. A CD spectrum was generated which represented the mean of three scans from which the buffer background had been electronically subtracted.
3. Results The structures of complexes used in this study are shown in Fig. 1. Fig. 2 shows rb values for compound I, compound II and compound III with poly(dG-dC) · poly(dG-dC) plotted versus the initial concentration of the compounds (10 – 200 mM). Both of the dinuclear platinum complexes displayed a plateauing effect at concentrations of approximately 150 mM and this effect was not observed for the mononuclear complex. Under the conditions employed, the two dinuclear complexes showed a similar ability to bind, which was much stronger than that of the mononuclear complex I. Interstrand cross-linking was assayed by the fluorescence methods of Lown and Khavatishvli [19,20]. This assay is useful for the preliminary assessment of crosslinking abilities. Fig. 3 shows the fraction of CT DNA renatured after the temperature jump plotted against the initial dose. Increasing the concentration of the dinuclear compounds significantly increased this fraction. The dinuclear platinum complex III showed the strongest ability to cause cross-links in CT DNA with concentrations as low as 5×10 − 9 M causing a 50% increase in fluorescence. The interstrand cross-linking of another dinuclear complex II containing 4,4%-dipyridyl sulfide, 10 − 8 M, which caused only a 27% increase of fluorescence, was significantly weaker in comparison to the dinuclear complex III bridged by 1,4-butanediamine. For the mononuclear complex I, increasing the concentration induced very little increase in fluorescence.
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Fig. 3. Renaturation of CT DNA complexed with complex I ( ), complex II () amd complex III () upon boiling and fast cooling at pH 11.8 as determined by EtdBr fluorescence. Ir is the ratio of EtdBr fluorescence in heated and cooled samples of DNA treated by compounds against the samples kept at room temperature.
The complexation of a metal to DNA can lead to perturbations of helix DNA which have been revealed by different physicochemical techniques [22,23]. Among these techniques, the use of a fluorescence intercalating agent, EtdBr, offers a useful means of studying the reaction of a compound with DNA. The perturbations induced by complexation of platinum compounds to DNA inhibit the intercalation of EtdBr and lead to a decrease of the fluorescence of the DNA–Pt–EtdBr complex [24,25]. In Fig. 4, the percentage of fluorescence decrease I1/I0 for DNA–cis[Pt(NH3)2Cl(4-methylpyridine)]NO3 and DNA–[{cis-Pt(NH3)Cl}2(bpsu)](NO3)2 complexes was plotted against the concentration. While the dinuclear platinum
Fig. 4. Relationship between the fluorescence decrease (I1/I0) and the concentration of complex I ( ) or complex II. I1 represents the increasing DNA – metal – EtdBr fluorescence above background EtdBr fluorescence and I0 the increasing DNA – EtdBr fluorescence above background EtdBr fluorescence. Final DNA–metal complexes and EtdBr concentrations were 10 and 40 mg/ml, respectively.
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Fig. 5. Kinetics of the interaction between DNA and complex I. The reactions were run at 37°C in 0.3 M NaCl ( ) and 0.3 M KNO3 ( ), respectively. (b) Kinetics of the interaction between DNA and complex II. The reactions were also run at 37°C in 0.3 M NaCl () and 0.3 M KNO3 (), respectively. At the starting point, metal solutions (final concentration 50 mM) were added to the DNA (25 mg/ml). Aliquots (1 ml) were taken at different times and mixed with 1.5 ml of EtdBr (66 mg/ml) in 0.3 M NaCl and 0.3 M KNO3, respectively, and metal – DNA complexes determined.
compound II induced a fluorescence decrease, its monomeric analog I caused very little decrease. Fig. 5a,b show profiles of the kinetics of the interaction between DNA and the metal compounds. With the increase of reaction time, both mononuclear complex I and dinuclear complex II did not induce a decrease in fluorescence when the reactions were run in 0.3 M NaCl. This indicates that the two platinum complexes do not react with DNA in 0.3M NaCl. In contrast, with an increase in reaction time, the dinuclear complex II caused a significant decrease in fluorescence and had a t1/2 of approximately 7.0 (t1/2 is defined as a fluorescence decrease of 50%), and the mononuclear complex I did not cause a significant decrease in fluorescence, when the reactions were run in 0.3 M KNO3.
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Fig. 6a,b represent CD spectra of poly(dG-dC) · poly(dG-dC) which was treated with different concentrations of the two complexes containing an aromatic ligand. Previous studies had indicated that the Z form of poly(dG-dC) · poly(dG-dC) was induced very efficiently upon binding of the dinuclear complex III [18] and mononuclear platinum complex [Pt(dien)Cl] + but not by cis-DDP [26,27] The generality of this feature was examined with the two complexes studied here. Unlike the dinuclear complex III, the dinuclear complex II [{cis-Pt(NH3)2 Cl}2(bpsu)](NO3)2 did not induce a BZ transition as observed earlier for cisplatin. This suggests that the adducts formed by the two dinuclear platinum complexes are structurally distinct. However, the mononuclear platinum complex I shows a significantly different conformation of poly(dG-dC) · poly(dG-dC) in comparison to its dimeric analog. Compound I caused a B Z transition, but it did not induce a complete inversion from B form to Z form even at a concentration of 100 mM.
4. Discussion The DNA binding of the metal complexes studied here is not reversible even upon prolonged dialysis of Pt – DNA samples, indicating that their binding to DNA likely involves covalent interaction. The irreversible binding of metal complex to DNA is strictly true for kinetically inert platinum(II)–DNA complex. However, the binding of complexes I and II to DNA can be completely prevented when the reactions are run in 0 3 M NaCl. Similarly, cisplatin (in its dichloro form) in the
Fig. 6. CD spectra of poly(dG-dC) · poly(dG-dC) modified by complex II (a) and complex I (b). The corresponding rb values are found in Fig. 2.
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presence of at least 10 − 1 M Cl − (the leaving group) does not react with DNA [28]. This indicates that hydrolysis is the first step when the two metal complexes interact with DNA as suggested earlier for the interaction of cisplatin with DNA. At the same time, it suggests that the two metal complexes may bind to DNA by non-intercalation, although both of them contain planar pyridine ring(s). Our previous studies with fluorescence Scatchard assay also suggest that the dinuclear complex II interacts in a non-intercalative mode with DNA [11]. The results of bacterial survival and mutagenesis experiments with Escherichia coli strains show that such an intercalation in the DNA by the pyridine ring moiety of the mononuclear complex I is also unlikely [6]. The mononuclear complex I shows promising antitumor activities [10]. It is expected to behave monofunctionally in reactions with nucleophiles, since the three amine ligands are poor leaving groups. Other related monofunctional compounds such as [Pt(dien)Cl] + and [Pt(NH3)3Cl] + are known to be antitumor inactive [26,27,29]. A possible explanation for the antitumor activity could be the in vivo generation of a bifunctional platinum compound cis-[Pt(NH3)(4-methylpyridine)]2 + , which could then be the active species [10]. In the present work, measurement of interstrand cross-links indicated that complex I shows much weaker ability to cause cross-links in CT DNA in comparison to the reported bifunctional complex cis-[Pt(NH3)(quinoline)Cl2] in which a concentration of 5×10 − 7 M caused a 60% increase in fluorescence [20], and the dinuclear complex III, bifunctionally binding to DNA [18], showed an almost two times stronger ability for DNA binding than that of the monomeric complex I. These results do not support the formation of the bifunctionally active species cis-[Pt(NH3)(4-methylpyridine)]2 + . In addition, like the monofunctional complex [Pt(dien)Cl] + [30], complex I also had no apparent effect on EtdBr binding, further indicating that it monofunctionally binds to DNA. However, the mode of action of complex I with DNA is different from that of [Pt(dien)Cl] + with DNA based on the different changes in conformation in poly(dG-dC) · poly(dG-dC) induced by the two mononuclear complexes. Complex [Pt(dien)Cl] + caused a complete inversion from B form to Z form [26,27] but not complex I. The pyridine ring moiety of complex I may hinder the inversion and, at the same time, it may play an important role in its antitumor activities. This suggests that the conformational changes in DNA induced by platinum complexes are closely related to their antitumor activities. As compared to its monomeric analog, the dinuclear complex II shows a stronger ability for DNA binding and interstrand cross-links, and a more significant decrease in fluorescence with increases in concentration or time. This indicates that it may bifunctionally bind to DNA. From Fig. 2, it is observed that the DNA binding ability of compound II is very similar to that of the bifunctional compound III. This result further supports the conclusion that complex II also bifunctionally interacts with DNA. However, interestingly, complex II has a lower interstrand cross-linking efficiency than compound llI. This may be a reason why complex III induces the BZ transition in poly(dA-dC) · poly(dG-dC) [18] but not complex II which contains the planar pyridine ring ligand, since some studies indicate that interstrand cross-links may be very useful in ‘locking’ the Z conformation [31]. On
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the other hand, due to steric hindrance, complex II possibly causes more intrastrand cross-links than complex III, as suggested for cisplatin [3]. The intrastrand cross-linking interaction of cisplatin with poly(dG-dC) · poly(dG-dC) prevents the rotation of the guanine bases about their glycosyl bonds and prohibits the B Z transition in poly(dG-dC) · poly(dG-dC) [27]. The results presented here suggest that the mononuclear complex I produces monofunctional adducts on DNA. However, the mode of action of complex I with DNA is different than that of [Pt(dien)Cl] + with DNA. The different mode of action of the two mononuclear platinum complexes may be related to their difference in antitumor activities. Compared to its monomeric analog, the dinuclear complex II may produce bifunctional adducts on DNA. The DNA binding of complex II, which is different from that of the dinuclear complex III, is characterized by relatively large numbers of intrastrand cross-links and small numbers of interstrand cross-links. It would be of considerable clinical interest if the novel DNA adducts led to a broader spectrum of antitumor activity.
Acknowledgements This project 29671020 was supported by the National Natural Science Foundation of P.R. China, the Nature Science Foundation of Tianjin and the National Key Laboratory of Coordination Chemistry, Nanjing University.
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