ABSTRACT
It has been shown recently that some analogues of clinically ineffective
trans
-diamminedichloroplatinum(II) (transplatin) exhibit antitumor activity. This finding has inverted the
empirical structure-antitumor activity relationships delineated for platinum(II) complexes,
according to which only the
cis
geometry of leaving ligands in the bifunctional platinum complexes is
therapeutically active. As a result, interactions of
trans
platinum compounds with DNA, which is the main pharmacological target of
platinum anticancer drugs, are of great interest. The present paper describes
the DNA binding of antitumor
trans
-[PtCl
2
(
E
-imino ether)
2
] complex (
trans-EE
) in a cell-free medium, which has been investigated using three experimental
approaches. They involve thiourea as a probe of monofunctional DNA adducts of
platinum(II) complexes with two leaving ligands in the
trans
configuration, ethidium bromide as a probe for distinguishing between
monofunctional and bifunctional DNA adducts of platinum complexes and HPLC
analysis of the platinated DNA enzymatically digested to nucleosides. The
results show that bifunctional
trans-EE
preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a
relatively long time (48 h at 37
o
C in 10 mM NaClO
4
). It implies that antitumor
trans-EE
modifies DNA in a different way than clinically ineffective transplatin, which
forms prevalent amount of bifunctional DNA adducts after 48 h. This result has
been interpreted to mean that the major adduct of
trans-EE
, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the
different properties of the adducts formed on DNA by transplatin and
trans-EE
are relevant to their distinct clinical efficacy.
The standard
cis
/
trans
structure-activity relationship of platinum antitumor complexes, exemplified by
cis
-diamminedichloroplatinum(II) (cisplatin), is that only
cis
geometry is therapeutically active. The observed inactivity of the
trans
isomer,
trans
-diamminedichloroplatinum(II) (transplatin), has had a major influence on
both the synthesis of new platinum antitumor agents and the mechanistic
interpretation of the antitumor activity of cisplatin. It is generally accepted
that cisplatin manifests its biological activity through coordination to DNA (
1
-
3
). Possible explanations for the different biological activity of the
cis
and
trans
isomers are that
cis
compounds form platinum-DNA adducts which inhibit DNA replication or transcription to a greater
extent than those formed by transplatin (
5
,
6
), and alternatively, that DNA adducts formed by
trans
compounds may be repaired more efficiently (
7
).
The nature of the DNA adducts formed by cisplatin and its
trans
isomer has also been explored to explain the differences in biological activity between the two isomers. Cisplatin produces a range of
adducts on DNA including bidentate intrastrand cross-links such as 1,2-GG or AG and 1,3-GNG (
1
-
3
,
8
,
9
) and interstrand cross-links at the GC sequence (
10
). These lesions result in conformational alterations in DNA and represent
blocks to DNA and RNA polymerases (
10
-
13
).
Transplatin shows a distinct sequence preference upon binding to DNA, different
from that of cisplatin (
11
,
14
). Transplatin also produces bidentate intrastrand cross-links, such as 1,3-GNG and interstrand cross-links between complementary guanine and cytosine residues (
8
,
14
-
16
). These lesions of transplatin, however, induce in DNA different conformational
alterations than cisplatin (
1
-
4
,
17
,
18
).
It has been reported in recent contributions that
trans
-[PtCl
2
(
E
-imino ether)
2
] complex (
trans-EE
) (imino ether=HN=C(OCH
3
)- CH
3
; it can have either
E
or
Z
configuration depending on the relative position of OCH
3
and N-bonded Pt with respect to the C=N double bond,
cis
in the
Z
isomer and
trans
in the
E
isomer) is not only more cytotoxic than its congener
cis
-[PtCl
2
(E-imino ether)
2
] (
cis-EE
), but is also endowed with significant antitumor activity (
19
,
20
). These results strongly imply a new mechanism of action for
trans-EE
. By analogy with the diamminedichloroplatinum(II) isomers, the inhibition of
DNA synthesis by
cis-EE
and
trans-EE
(
19
) implies a role for DNA binding in the mechanism of action. The presence of the
imino ether group may result in altered hydrogen bonding and steric effects
affecting the kinetics of DNA binding, the structures and/or stability of the
adducts formed and resulting local conformational alterations in DNA.
The breaking of the paradigm for structure-activity relationships of platinum antitumor complexes poses new fundamental questions on the mechanism of antitumor activity of platinum complexes. To explain
the cytotoxicity of imino ether derivatives, it could be important to examine
in detail the DNA binding of the new complexes and compare these results with
those previously obtained for other diamminedichloro isomers. Thus, these
results could lead to the development of new strategies for the systematic
design of platinum antitumor complexes acting by mechanisms different from the
presently used agents, and eventually having a different clinical profile of
antitumor activity.
This paper describes the results of studies of DNA binding of
trans
-
EE
with respect to characterization of preferential binding sites in double-helical DNA and the types of the resulting adducts.
Cisplatin, transplatin and chlorodiethylenetriamineplatinum(II) chloride,
[Pt(dien)Cl]Cl, were from Lachema (Brno, Czech Republic).
cis
-EE and
trans
-EE were synthesized as previously described (
21
). Calf thymus DNA (the content of guanine + cytosine is 42%) was purchased from
Sigma and used without further purification. Deoxyriboguanosine (dGuo), DNase I
from bovine pancreas, nuclease P1 from
Penicillium citrinum
and alkaline phosphatase from calf intestine were also from Sigma. Thiourea and
ethidium bromide (EtBr) were from Merck. Thiourea (
3
H) was from Amersham.
The standards were prepared by reaction of dGuo with the mononitratomonochloro
and dinitrato platinum-iminoether species. Mononitrato platinum-iminoether derivative,
trans
- [PtCl(NO
3
)(
E
-imino ether)
2
] was prepared by dissolving 169 mg (0.41 mmol) of
trans-EE
in 40 ml acetone and treating it with an equivalent amount of AgNO
3
(70 mg, 0.41 mmol). After stirring in the dark for 1 h, the reaction mixture
was filtered through celite, the solution was dried, the solid residue
extracted with ethyl ether and the solution again filtered. By evaporation of
the solvent a solid of the desired compound was obtained in ~70% yield. Anal. Calcd for C
6
H
14
ClN
3
O
5
Pt: C, 16.4; H, 3.2; N, 9.6%. Found: C, 17.0; H, 4.4; N, 9.3%. The dinitrato
analogue,
trans
-[Pt(NO
3
)
2
(
E
-imino ether)
2
], was prepared by reaction of a suspension of 99 mg (0.24 mmol) of
trans-EE
in 30 ml of water with two equivalents of AgNO
3
(82 mg, 0.48 mmol). After stirring for 16 h in the dark at room temperature (25-28oC), the reaction mixture was filtered through celite, the solvent
evaporated and the solid residue redissolved in a minimum volume of methanol
and kept to crystallize in a deep freezer. The colorless crystals were
separated by filtration of the mother liquor and dried. The desired compound was obtained
in ~60% yield. Anal. Calcd for C
6
H
14
N
4
O
8
Pt: C, 15.5; H, 3.0; N, 12.0%. Found: C, 15.4; H, 3.0; N, 12.0%.
Preparation of
trans
-[PtCl(dGuo)(
E
-imino ether)
2
](NO
3
) was done by dissolving 0.043 g (0.1 mmol) of
trans
-[PtCl(NO
3
)(
E
-imino ether)
2
] in 50 ml of water to which a stoichiometric amount of dGuo (26 mg, 0.1 mmol)
was added. The solution was stirred at room temperature in the dark for 2 days.
After this time the solvent was evaporated under a reduced pressure and
trans
-[PtCl(dGuo)(
E
-imino ether)
2
](NO
3
) was recovered quantitatively. Anal.Calcd for C
16
H
27
N
8
O
10
Pt.H
2
O: C, 25.9; H, 3.9; N, 15.1%. Found: C, 25.9; H, 3.8; N, 14.6%.
trans
-[Pt(dGuo)
2
(
E
-imino ether)
2
](NO
3
)
2
was prepared by suspending 0.015 g (0.032 mmol) of
trans
-[Pt(NO
3
)
2
(
E
-imino ether)
2
] in 20 ml of water to which 2 eq of dGuo (17 mg, 0.064 mmol) were added. The
suspension was stirred at room temperature in the dark for 2 days. After this
time a clear solution was obtained, the solvent was evaporated under a reduced
pressure and
trans
-[Pt(dGuo)
2
(
E
-imino ether)
2
](NO
3
)
2
was recovered quantitatively. Anal. Calcd for C
26
H
40
N
14
O18Pt.2H
2
O: C, 29.2; H, 4.1; N, 18.4%. Found: C, 29.8; H, 4.2; N, 18.5%.
Ultraviolet spectra were collected on a Beckmann DU-8 spectrophotometer. Fluorescence measurements of DNA modified by platinum in the
presence of EtBr were performed using a Perkin-Elmer LS 5B spectrofluorimeter. The excitation wavelength was 546 nm and the emitted fluorescence was measured at 590 nm. The fluorescence was measured at 25oC in 0.4 M NaCl to avoid the second fixation site of EtBr to DNA (
22
). The concentrations were 0.01 mg/ml for DNA and 0.04 mg/ml for EtBr, which
corresponded to the saturation of all intercalation sites of EtBr in DNA (
22
,
23
). Flameless atomic absorption spectroscopy (FAAS) measurements were carried out on a Perkin-Elmer 560 instrument with a graphite furnace. For FAAS analysis, DNA was
precipitated with ethanol and dissolved in 0.1 M HNO
3
. High-pressure liquid chromatographic (HPLC) analyses were performed by using a
Perkin-Elmer Series 4 liquid chromatograph equipped with a LCI-100 computing integrator and a Waters [mu]Bondapak C
18
column. Gradient was 0-60% methanol in 0.02 M ammonium acetate, pH 5.5; flow rate was 1 ml/min. Spectra (
1
H NMR) were obtained with a Bruker AM 300 spectrometer. pH measurements were
performed with a CRISON micropH 2002 apparatus.
If not stated otherwise, the platination reactions were performed in 10 mM NaClO
4
at 37oC in the dark. If required, the reactions were terminated by adding
thiourea to 10 mM and incubating at 37oC for 10 min. The ratio of platinum atoms fixed per nucleotide residue (r
b
) was determined by FAAS. Enzymatic digestions of DNA modified by platinum were
carried out by using DNase I, nuclease P1, and alkaline phosphatase (
8
). In a typical experiment, samples (45 [mu]g of DNA) were allowed to react with 72 U of DNase I at 37oC. After 4 h nuclease P1 (40 [mu]g) was added, and the reaction was allowed to continue at 37oC for 18 h. Finally, alkaline phosphatase (39 U) was added and
the incubation continued for additional 4 h at 37oC. The samples were then heated for 2 min at 80oC, centrifuged and the supernatant analyzed by HPLC.
Calf thymus DNA (double-stranded or thermally denatured) at the concentration of 0.16 mg/ml was
incubated with
trans-EE
at various r
i
values (r
i
is defined as the molar ratio of free metal complex to nucleotide-phosphates at the onset of incubation). At each concentration of
trans-EE
, the aliquots were withdrawn at 10 min, 2, 24 and 48 h time intervals. After
the withdrawal, the unbound platinum was immediately removed by a
centrifugation (1500 r.p.m., 30 s) through a column of Sephadex G25 (coarse).
The r
i
values were chosen in the way that the values of r
b
determined after this separation step by FAAS were 0.01, 0.05 and 0.2 for each
incubation time. Of these solutions, 0.15 ml was added to 0.15 ml of 0.9 mM
3
H labeled thiourea, prepared as described previously (
24
), having specific radioactivity of 77 MBq/mmol. After 10 min incubation at 37oC, 0.8 ml of 0.15 M NaCl, pH 7.0 was added and 1.0 ml of the resulting solution was layered on a nitrocellulose filter having pores of 0.4 mm in diameter (Synpor, VCHZ Syntezia, Pardubice). In order to remove the
unreacted thiourea the filter was washed with 15 ml of 5% trichloroacetic acid.
The filters were dried under an infrared lamp, transferred to glass tubes to
which 5 ml of toluene scintillator was added. The radioactivity was measured on
a LKB Wallac1410 Betaspectrometer (Finland).
Cisplatin, transplatin and analogous bifunctional platinum compounds bind to DNA in a two-step process, forming first monofunctional adducts, preferentially at
guanine residues, which subsequently close to bifunctional lesions (
1
-
4
,
8
,
9
). Thus, monofunctional adducts are formed in DNA at an early stage of the
reaction. Thiourea is successfully used to labilize monofunctional transplatin coordination to DNA (
25
). The displacement of transplatin takes place via coordination of thiourea
trans
to the base residue. Because of the strong
trans
effect of sulphur, the nitrogen-platinum bond is weakened so that it becomes susceptible to substitution.
Importantly, whereas thiourea effectively
trans
labilizes transplatin in monofunctional DNA adducts, bifunctional adducts of
this platinum complex are resistant to the thiourea treatment (
25
).
The initial experiment aimed at characterization of DNA adducts of
trans
-
EE
was conducted employing thiourea as a probe of DNA monofunctional adducts of
trans
-dichloroplatinum(II) complexes. Double-stranded and thermally denatured DNAs were incubated with
transplatin or
trans
-
EE
at a formal drug to nucleotide ratio r
i
= 0.05. At various times the reaction was stopped by ethanol precipitation of
the DNA. In parallel tubes, the reactions were stopped by addition of thiourea
to 10 mM. These samples were incubated for 10 min at 37oC and then precipitated by ethanol. The DNA was redissolved, and the
platinum content was determined by FAAS (Fig.
1
).
In order to differentiate between the latter two eventualities we have employed
EtBr as a fluorescent probe. This probe can be used to distinguish between
perturbations induced in DNA by monofunctional and bifunctional adducts of
platinum compounds (
22
,
23
). Binding of EtBr to DNA by intercalation is blocked in a stoichiometric manner
by formation of the bifunctional adducts of a series of platinum complexes
including cisplatin and transplatin, which results in a loss of fluorescence
intensity (
22
). On the other hand, modification of DNA by monodentate platinum complexes
(having only one leaving ligand) results in only a slight decrease of EtBr
fluorescence intensity as compared with non-platinated DNA-EtBr complex.
Double-helical DNA was first modified by cisplatin, transplatin,
cis-EE
,
trans-EE
and by monodentate [Pt(dien)Cl]Cl for 48 h. The levels of the modification corresponded to the values of r
b
in the range between 0 and 0.1. Modification of DNA by all platinum complexes
resulted in a decrease of EtBr fluorescence (Fig.
2
). In accordance with the results published earlier (
22
,
23
), monodentate [Pt(dien)Cl]Cl decreased the fluorescence only to a small extent,
whereas the decrease induced by the DNA adducts of cisplatin, transplatin and
cis-EE
was significantly more pronounced. The decrease induced by the adducts of
trans-EE
was markedly less pronounced than that induced by the DNA adducts of other
bidentate complexes tested in this work. In fact, the fluorescence intensity
was only slightly lower than the fluorescence intensity of DNA modified by the
monodentate [Pt(dien)Cl]Cl. This result suggests that
trans-EE
complex forms the DNA adducts which resemble, from the viewpoint of their
capability to inhibit EtBr fluorescence, those formed by monofunctional
platinum complexes. Importantly, the DNA adducts of
cis-EE
inhibited EtBr fluorescence almost to the same extent as cisplatin. Taken
together, the fluorescent analysis is consistent with the idea and supports the
postulate that the major DNA adducts of
trans-EE
are monofunctional lesions even after long incubations of DNA with this platinum
complex (48 h). On the other hand, under comparable conditions
cis-EE
forms on DNA mainly bifunctional adducts similar to those formed by cisplatin.
To characterize further the coordination mode of
trans-EE
, DNA modified by this platinum complex at r
b
of 0.08 for 48 h was enzymatically digested to mononucleosides and analyzed by
reversed-phase HPLC. Cisplatin or transplatin exhibit a strong preference for
binding to guanine residues in DNA (they also bind in a much smaller extent to
other base residues) (
1
-
4
,
8
,
9
). Therefore, we first characterized the products of the reactions of
trans-EE
with monomeric dGuo by NMR spectroscopy and then used these products as HPLC
standards. The
1
H NMR spectral data are shown in Table
1
. In the case of both standards containing
trans-EE
coordinated to one or two dGuo molecules,
trans
- [PtCl(dGuo)(
E
-imino ether)
2
]
+
or
trans
-[Pt(dGuo)
2
(
E
-imino ether)
2
]
2+
respectively, a major shift was noticed for the H8 protons (0.4-0.6 p.p.m. downfield), which is typical of the N7 coordinated guanines (
26
,
27
). The N7 coordination was also supported by the pH dependence of chemical
shifts (Fig.
3
) and platinum coupling constants of H8 proton (30 and 26 Hz for the mono- and bis-dGuo complexes, respectively).
Table 1
The purpose of this study was to examine the effect of substitution of imino
ether group for NH
3
in dichloroplatinum(II) complexes on their DNA-binding properties. The results presented here show that imino ether
ligands do not alter radically the first step of the binding of bifunctional
platinum complexes to DNA, i.e., the formation of monofunctional adducts at N7
position of guanine residues. Importantly, the imino ether substitution,
especially in the
trans
geometry, results in a greatly reduced rate of closure of the monofunctional
platinum lesions in bifunctional adducts. This result implies an important
difference in the nature and frequency of the DNA adducts of
trans-EE
and clinically ineffective transplatin.
Recently, some
trans
-[PtCl
2
(amine)
2
] complexes containing sterically hindered planar ligands instead of simple NH
3
groups were reported (
28
,
29
). These complexes exhibit greatly enhanced cytotoxicity in tumor cells in
comparison with transplatin and in several cases their cytotoxicity was
equivalent to that of the clinically used cisplatin. These results along with
those obtained for the imino ether derivatives invert the standard
cis
/
trans
structure-pharmacological activity relationships observed previously for [PtCl
2
(NH
3
)
2
] complexes. The presence of bulky planar amine ligands has been found to
enhance strongly DNA interstrand cross-linking capability of the complexes with
trans
geometry. It has been suggested that this enhanced interstrand cross-linking efficiency of
trans
-[PtCl
2
(amine)
2
] complexes along with specific conformational changes in DNA could be relevant
to their enhanced cytotoxicity in tumor cells.
The
trans-EE
complex also exhibits cytotoxicity in tumor cells which was much more
pronounced than that of its
cis
congener. However, in contrast with the
trans
complexes of planar amine ligands and transplatin,
trans-EE
appears to form markedly lower amount of bifunctional DNA adducts. Thus, an
important feature for biological activity of
trans-EE
is its capability to form in DNA relatively stable monofunctional adducts.
Further investigations of conformational alterations induced in DNA by
trans-EE
are warranted to unravel the origin of antitumor activity of platinum complexes
with leaving ligands in the
trans
configuration.
This work was supported in part by the Internal Grant Agency of the Academy of
Sciences of the Czech Republic (grant nos 504406 and 404101), Grant Agency of
the Czech Republic (grant no. 203/93/0092) and the Internal Grant Agency of the
Ministry of Health of the Czech Republic (grant no. 1893-3). This joint research is also a part of the European Cooperation in the
field of Scientific and Technical Research (COST) network (COST projects
D1/0002/92 and D1/0001/95). One of the authors (V.B.) is grateful to the
Consiglio Nazionale delle Ricerche in Italy for the NATO Guest Fellowship.
Compound
H8
C1'H
C2'H
2
C3'H
2
C4'H
2
C5'H
2
O-CH
3
C-CH
3
dGuo
7.98
6.30
2.79
4.62
4.13
3.79
2.51
trans
-[Pt(dGuo)
2
(
E
-imino ether)
2
](NO
3
)
2
8.57
6.35
2.78
4.64
4.16
3.81
3.75
2.13
2.59
trans
-[PtCl(dGuo)(
E
-imino ether)
2
](NO
3
)
8.39
6.32
2.75
4.63
4.15
3.82
3.77
2.44
2.59
trans
-[Pt(NO
3
)
2
(
E
-imino ether)
2
]
a
3.87
2.66
trans
-[PtCl(NO
3
)(
E
-imino ether)
2
]
a
3.84
2.63
REFERENCES
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