ORIGINAL PAPER
Reaction of human metallothionein-3 with cisplatin
and transplatin
Andrei V. Karotki Æ Milan Vasˇa´k
Received: 27 March 2009 / Accepted: 8 June 2009 / Published online: 18 June 2009

SBIC 2009
Abstract Human metallothioneins, small cysteine- and
metal-rich proteins, play an important role in the acquired
resistance to platinum-based anticancer drugs. These
proteins contain a M(II)4(CysS)11 cluster and a
M(II)3(CysS)9 cluster localized in the a-domain and the
b-domain, respectively. The noninducible isoform metal-
lothionein-3 (Zn7MT-3) is mainly expressed in the brain,
but was found overexpressed in a number of cancer tis-
sues. Since the structural properties of this isoform sub-
stantially differ from those of the ubiquitously occurring
Zn7MT-1/Zn7MT-2 isoforms, the reactions of cis-dia-
mminedichloridoplatinum(II) (cisplatin) and trans-dia-
mminedichloridoplatinum(II) (transplatin) with human
Zn7MT-3 were investigated and the products character-
ized. A comparison of the reaction kinetics revealed that
transplatin reacts with cysteine ligands of Zn7MT-3 faster
than cisplatin. In both binding processes, stoichiometric
amounts of Zn(II) were released from the protein. Marked
differences between the reaction rates of cisplatin and
transplatin binding to Zn7MT-3 and the formation of the
Pt–S bonds suggest that the binding of both Pt(II) com-
pounds is a complex process, involving at least two sub-
sequent binding steps. The electrospray ionization mass
spectrometry characterization of the products showed that
whereas all ligands in cisplatin were replaced by cysteine
thiolates, transplatin retained its carrier ammine ligands.
The 113Cd NMR studies of Pt1
113Cd6MT-3 revealed that
cisplatin binds preferentially to the b-domain of the pro-
tein. The rates of reaction of cisplatin and transplatin with
Zn7MT-3 were much faster than those of cisplatin and
transplatin with Zn7MT-2. The biological consequences of
a substantially higher reactivity of cisplatin toward
Zn7MT-3 than Zn7MT-2 in the acquired resistance to
platinum-based drugs are discussed.
Keywords Metallothionein-3
Cisplatin
Transplatin

NMR
Mass spectrometry
Abbreviations
Cisplatin cis-Diamminedichloridoplatinum(II)
GSH Glutathione
MT Metallothionein
Transplatin trans-Diamminedichloridoplatinum(II)
Zincon 2-Carboxy-2’-hydroxy-5’-
sulfoformazylbenzene sodium salt
Introduction
cis-Diamminedichloridoplatinum(II) (cisplatin) is one of
the most potent antitumor agents known, displaying clini-
cal activity against a wide variety of solid tumors, such as
ovarian, testicular, and bladder tumors. Cisplatin is also
used in combination with radiotherapy or surgical treat-
ment [1]. The cytotoxic mode of action is mediated by its
binding mainly to two adjacent purine bases of nuclear
DNA, forming primarily intrastrand cross-links. This DNA
modification distorts the DNA structure such that transla-
tion and excision repair is strongly inhibited, directing cells
into apoptosis or necrosis [2, 3].
A. V. Karotki
M. Vasˇa´k (&)
Department of Biochemistry,
University of Zurich,
Winterthurerstrasse 190,
8057 Zurich, Switzerland
e-mail: mvasak@bioc.uzh.ch
123
J Biol Inorg Chem (2009) 14:1129–1138
DOI 10.1007/s00775-009-0557-x

A geometrical isomer of cisplatin, trans-diamminedi-
chloridoplatinum(II) (transplatin), is clinically inactive.
This is believed to be due mainly to its inability to form
similar DNA adducts as cisplatin [4]. The resistance of
cancer cells to Pt(II) drugs can be either intrinsic or acquired
following Pt(II) administration [1]. The acquired resistance
after initial treatment is the major drawback of these che-
motherapeutics. Because of the strong reactivity of Pt(II)
compounds toward sulfur-donor molecules and the forma-
tion of very stable Pt–S bonds, intracellular thiols through
their competition with DNA confer resistance to antitumor
platinum drugs. The abundant intracellular thiols involved
in the drug resistance are glutathione (GSH) and metallo-
thionein (MT) [1, 5, 6]. The four MT isoforms expressed in
humans (designated MT-1 through MT-4) consist of 61–68
amino acid residues, of which 20 are conserved cysteines.
The structural studies revealed that MTs bind seven Zn(II) or
Cd(II) through cysteine thiolates forming two metal–thiolate
clusters: a M(II)3S9 cluster located in the b-domain and a
M(II)4S11 cluster in the a-domain of the protein [7]. The
ubiquitously occurring MT-1 and MT-2 isoforms are
inducible by a variety of compounds, including hormones,
cytokines, and metal ions, including Pt(II) drugs. These
proteins are involved in the zinc and copper homeostasis,
heavy metal detoxification, immune system function, and
protection from apoptosis [8–11]. Although GSH effectively
deactivates cisplatin and in the certain cancer cell lines its
cellular concentration increases upon exposure to Pt(II)
drugs, thiols of MTs react faster with cisplatin compared
with GSH [12, 13]. In cancer cells basal MT-1/MT-2
expression levels are often significantly increased, resulting
in an even stronger Pt(II) scavenging effect [14, 15]. The
overexpression of MT-1/MT-2 isoforms in response to Pt(II)
drug administration is responsible for most of the cases of
acquired resistance to cisplatin [15–17]. In our previous
studies we showed that the rates of Zn7MT-2 reaction with
cis-[Pt(N-donor)2Cl2] and trans-[Pt(N-donor)2Cl2] com-
pounds depend on the nature of the coordinated ligands and
that trans-[Pt(N-donor)2Cl2] compounds react faster. In this
reaction, owing to the high affinity of Pt(II) for cysteine
thiolates, exceeding that of Zn(II) 107 times, Zn(II) is
substituted by Pt(II) [18, 19]. The characterization of the
products showed that whereas all ligands in cis-Pt(II) com-
pounds were replaced by cysteine thiolates, trans-Pt(II)
compounds retained their nitrogen-donor ligands.
The MT-3 isoform (Zn7MT-3), also termed ‘‘neuronal
growth inhibitory factor,’’ occurs intra- and extracellularly
and shows neuroinhibitory activity in vitro that distinguishes
it from the widely expressed Zn7MT-1 and Zn7MT-2
isoforms. MT-3 shows a brain-specific expression, mainly
in glutamatergic neurons [20–23]. This isoform is unres-
ponsive to the inducers of MT-1 and MT-2 expression
mentioned above [24]. However, MT-3 was found
overexpressed in a number of cancers, such as prostatic
adenocarcinoma, esophageal cancer, and breast cancer,
where its presence positively correlates with the poor sur-
vival prognosis after platinum chemotherapy [22, 25–27].
Furthermore, since MT-3 is absent in normal bladder tissues
but is present in large amounts in bladder cancer, the use of
this isoform as a marker of this disease has been suggested
[28]. The changes in the primary structure of MT-3, which
include a unique C(6)PCP(9) motif in the b-domain and an
acidic hexapeptide insert in the a-domain (Fig. 1), translate
into widely different structural properties of Zn7MT-3
compared with Zn7MT-1/Zn7MT-2 [7, 29]. Thus, the
Zn7MT-3 structure is highly dynamic and the thiolate
ligands possess a substantially higher nucleophilicity, indi-
cated by their reactivity toward a number of compounds,
including 5,50-dithiobis(2-nitrobenzoic acid) and S-nitroso-
thiols [30–32]. However, the reactivity of Zn7MT-3 toward
cisplatin and the products formed is not known.
In the present work, the reactions of Zn7MT-3 with cis-
platin and transplatin were investigated and the products
characterized by electrospray ionization mass spectrometry
(ESI-MS) and 113Cd NMR. The studies were conducted
under nativelike conditions regarding pH, ionic strength, and
a Pt(II) to Zn7MT ratio of 2:1 relevant to in vivo studies
[18, 33]. The results obtained were compared with those
obtained with Zn7MT-2. The biological implications of these
studies for the acquired resistance to platinum-based drugs
are discussed.
Materials and methods
Expression and purification of MTs
Recombinant human MT-3 and MT-2 were expressed in
Escherichia coli as Cd(II) proteins and purified essentially
as described in [18, 34]. The correctness of the protein
Fig. 1 Alignment of amino acid sequences of human metallothionein-2 (MT-2) and metallothionein-3 (MT-3) isoforms used in this study.
Cysteine residues are highlighted in gray
1130 J Biol Inorg Chem (2009) 14:1129–1138
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