Antitumor platinum(IV) derivatives of oxaliplatin with axial valproato ligands

Article abstract of DOI:10.1016/j.jinorgbio.2014.07.004

We report new anticancer prodrugs, platinum(IV) derivatives of oxaliplatin conjugated with valproic acid (VPA), a well-known drug having histone deacetylase inhibitory activity. Like most platinum(IV) derivatives, the cytotoxicity of the conjugates was lower in cell culture than that of oxaliplatin, but greater than those of its Pt(IV) derivative containing biologically inactive axial ligands in several cancer cell lines. Notably, these conjugates display activity in both cisplatin sensitive- and resistant tumor cells capable of both markedly enhanced accumulation in tumor cells and acting in a dual threat manner, concurrently targeting histone deacetylase and genomic DNA. These results demonstrate the dual targeting strategy to be a valuable route to pursue in the design of platinum agents which may be more effective in cancer types that are typically resistant to therapy by conventional cisplatin. Moreover, platinum(IV) derivatives containing VPA axial ligands seem to be promising dual-targeting candidates for additional preclinical studies.

Full text of DOI:10.1016/j.jinorgbio.2014.07.004

Journal of Inorganic Biochemistry 140 (2014) 72–79
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Antitumor platinum(IV) derivatives of oxaliplatin with axial
valproato ligands
Vojtech Novohradsky a,b, Lenka Zerzankova a, Jana Stepankova a, Oldrich Vrana a, Raji Raveendran c,
Dan Gibson c, Jana Kasparkova b, Viktor Brabec a,
⁎
a
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-61265 Brno, Czech Republic
Department of Biophysics, Faculty of Science, Palacky University, 17. listopadu 12, CZ-77146 Olomouc, Czech Republic
Institute for Drug Research, School of Pharmacy, The Hebrew University, Jerusalem 91120, Israel
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
We report new anticancer prodrugs, platinum(IV) derivatives of oxaliplatin conjugated with valproic acid (VPA),
a well-known drug having histone deacetylase inhibitory activity. Like most platinum(IV) derivatives, the
cytotoxicity of the conjugates was lower in cell culture than that of oxaliplatin, but greater than those of its
Pt(IV) derivative containing biologically inactive axial ligands in several cancer cell lines. Notably, these conju-
gates display activity in both cisplatin sensitive- and resistant tumor cells capable of both markedly enhanced
accumulation in tumor cells and acting in a dual threat manner, concurrently targeting histone deacetylase
and genomic DNA. These results demonstrate the dual targeting strategy to be a valuable route to pursue in
the design of platinum agents which may be more effective in cancer types that are typically resistant to therapy
by conventional cisplatin. Moreover, platinum(IV) derivatives containing VPA axial ligands seem to be promising
dual-targeting candidates for additional preclinical studies.
Received 21 April 2014
Received in revised form 1 June 2014
Accepted 6 July 2014
Available online 15 July 2014
Keywords:
Platinum(IV) prodrugs
Anticancer
Histone deacetylase
Oxaliplatin
Valproic acid
© 2014 Elsevier Inc. All rights reserved.
Dual targeting
1. Introduction
in vitro and in vivo [7–9]. Agents, such as HDACis, that induce
hyperacetylation of histone proteins complexed with DNA could in-
Despite the success of conventional antitumor platinum drugs,
such as cisplatin [cis-diamminedichloridoplatinum(II)], carboplatin
[cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)] or oxaliplatin
[{(1R,2R)-diaminocyclohexane}oxalatoplatinum(II)], the emergence
of drug resistance is a major cause of treatment failure [1,2]. Studies,
performed with the goal to put forth strategies to overcome resistance
of tumor cells to conventional platinum drugs, have revealed that the
mechanisms of resistance of tumor cells to platinum drugs are multifacto-
rial processes. Moreover, these studies have also shown that the inhibi-
tion of single pathways that sustain cisplatin resistance fails to restore
sensitivity to normal levels [3]. These findings gave impetus to design
several conjugated platinum complexes with dual functionality to im-
prove antitumor activity of platinum drugs.
A class of such platinum agents involves bifunctional molecules in
which various histone deacetylase inhibitors (HDACis) were tethered
to a Pt(II)-based DNA binding agent [4–6]. HDACs are key regulators of
chromatin structure and transcription. The design of these conjugated
metallodrugs was inspired by the thesis that combining classic chemo-
therapy by conventional antitumor agents with epigenetic drugs shows
promise as the latter have been proven to have anticancer activity both
crease the accessibility of DNA within chromatin [3,10,11] and conse-
quently potentiate the anticancer activities of metallodrugs, such as
DNA-binding platinum complexes. These facts suggest that simultaneous
HDAC inhibition and DNA damage triggering apoptosis, autophagy and
cell death could be a viable alternative approach in cancer therapy.
As regards Pt(II)-HDACi conjugates [4–6], it was anticipated that
these new conjugated agents after accumulation in tumor cells will
aquate in the cytoplasm releasing simultaneously Pt(II) complex capa-
ble of coordinating to DNA base residues and HDACi which will increase
the accessibility of DNA within chromatin to this Pt(II) agent so that a
higher degree of DNA damage mediated by the Pt(II) agent would be
achieved. Unfortunately, the anticipated enhancement of toxicity of
these Pt(II)-HDACis in tumor cells (due to dual functionality of these
conjugated Pt(II) complexes) has not been evident, apparently since
the covalent linker group negatively impacted these processes [12].
We and others have been exploring strategies to conjugate an HDACi
to Pt(IV) complex. These strategies employ substitutionally inert octa-
hedral Pt(IV) compounds [13], which serve as prodrugs and release
clinically effective levels of the square planar Pt(II) compounds, such
as cisplatin, following cellular uptake and intracellular reduction
[14–16]. Appropriately designed Pt(IV) complexes are less likely to be
deactivated before reaching their cancer cell destination target, can
modulate the lipophilicity (and thus the ability to enter the tumor
⁎
Corresponding author. Tel.: +420 541517148; fax: +420 541240499.
E-mail address: brabec@ibp.cz (V. Brabec).
http://dx.doi.org/10.1016/j.jinorgbio.2014.07.004
0162-0134/© 2014 Elsevier Inc. All rights reserved.

V. Novohradsky et al. / Journal of Inorganic Biochemistry 140 (2014) 72–79
73
cells) and redox properties (the ability to be reduced under the hypoxic
conditions typical of tumor tissues) of the Pt(IV) complexes [17,18].
Moreover, the axial coordination positions serve as binding sites for
other biologically active ligands, which may have synergistic or additive
effects with platinum therapy or may act as carriers for drug targeting
and delivery [16,19–26]. The activity of Pt(IV) complexes generally
involves 2e−reduction with the loss of the axial ligands, affording an
active Pt(II) complex that readily binds to DNA [27]. In addition, an im-
portant property of the pharmacology of the HDACis is their selectivity
for tumor cells as compared to normal cells [28,29]. Hence, the HDACi,
with its known affinity for cancer cells, confers selectivity to the drug
and is expected to deliver the chemotherapeutic DNA binding agent
alongside the HDAC inhibitor to the tumor [30,31]. Thus, also for this
reason HDACi-Pt(IV) conjugates should have a mechanism of action,
biodistribution and toxicity that are different from those of convention-
al Pt(II) drugs and might therefore be active against (i) a broader
spectrum of human cancer cells relative to classical Pt(II) drugs and
(ii) human malignancies that have acquired or intrinsic resistance to
conventional Pt(II)-based therapies.
and DNA and with a view to understand more deeply the effects that
may play an important role in the biological (pharmacological) properties
of this class of platinum conjugates against cancer cells.
2. Experimental
2.1. Chemicals
Oxaliplatin and cisplatin were from AK Scientific, Inc., Union City,
CA, USA and Sigma-Aldrich, Prague, Czech Republic, respectively.
Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), VPA, and 1-
octanol (≥99%) were purchased from Sigma-Aldrich, Prague, Czech
Republic. [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide] (MTT) was from Calbiochem (Darmstadt, Germany). Other
chemicals used to synthesize compounds I, II, and III (vide infra) were
purchased from Sigma-Aldrich, Jerusalem, Israel.
2.2. Synthesis and characterization
Very recently, the bifunctional Pt(IV) conjugate cis,cis,trans-
diamminedichlorido-bis(valproato)platinum(IV) [cisPt(IV)-(VPA)2],
based on the cisplatin square-plane with two axial valproato (2-
propylpentanoate, VPA) ligands, has been synthesized and tested for tox-
icity in tumor cells and for HDAC inhibitory activity [32,33]. VPA is a well-
known drug having HDACi activity, which can potentiate chemotherapy
by enhancing tumor growth suppression and apoptosis [34,35]; it in-
hibits HDAC activity by binding to the catalytic center of HDACs [36,
37]. It has been demonstrated that cisPt(IV)-(VPA)2 exhibits promising
toxicity in several tumor cells which correlated with enhanced cellular
accumulation of this metallodrug [32,33]. On the other hand, the antici-
pated enhanced HDAC inhibitory activity of cisPt(IV)-(VPA)2 in tumor
cells treated with this Pt(IV) complex and consequently enhanced
amount of platinum bound to nuclear cellular DNA due to the increased
accessibility of DNA within chromatin have not been demonstrated.
The activity of cisPt(IV)-(VPA)2 involves its reduction with the loss
of the axial ligands, affording cisplatin that readily binds to DNA. To
overcome the limitations associated with clinical use of cisplatin [38],
platinum derivatives, such as carboplatin and oxaliplatin have been
developed and tested. In particular oxaliplatin demonstrated the activi-
ty in cisplatin-resistant tumors including colorectal xenografts [39,40]
and a lack of cross-resistance with cisplatin and carboplatin [41]. Also
interestingly, the HDAC inhibitor, trichostatin A, was found to exhibit an
additive effect to cytotoxicity of human gastric tumor cells to oxaliplatin
[42]. Therefore, in the present study, we present new Pt(IV) derivatives
of oxaliplatin with two axial hydroxido ligands (I), with one axial
hydroxido and one axial VPA ligand (II) or with two axial VPA ligands
(III) (Fig. 1) in order to introduce more potent bifunctional inhibitors,
HDACi-Pt(IV) conjugates capable of dual targeting of histone deacetylase
2.2.1. [Pt(DACH)(OH)2(ox)] (I) (DACH = trans-(1R,2R)-1,2-
cyclohexanediamine, ox = oxalato)
[Pt(DACH)(OH)2(ox)] was prepared from oxaliplatin by oxidation
with 30% H2O2 as described previously [43]. 195Pt NMR (DMF): 1328
ppm. 1H NMR (D2O): δ ppm 1.2–2.0 (m, 6H), 2.4 (m, 2H), and 2.93
(m, 2H). HPLC: C18 column, isocratic 5% acetonitrile/95% water as an
eluent, retention time: 2.47 min. Purity N 95%. ESIMS: 431.3.
2.2.2. [Pt(DACH) (OH)(VPA)(ox)] (II)
Valproic anhydride was prepared by the addition of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) (0.77 g, 5 mmol) to a solu-
tion of VPA (1.44 g, 10 mmol) in 15 mL dichloromethane and stirring
the reaction overnight at room temperature. The solution was washed
with 3% citric acid (2 × 30 mL), 3% NaHCO3 (2 × 30 mL) and 3% NaCl
(2 × 30 mL). The organic phase was dried over sodium sulfate, filtered
and evaporated to yield the desired compound as a colorless liquid.
Yield: 80%, 860 mg.
To a suspension of [Pt(DACH)(OH)2(ox)] (50 mg, 1.16 × 10−4 mol)
in DMF (1 mL), valproic anhydride (47.02 mg, 1.74 × 10−4 mol) was
added and the reaction mixture was stirred at room temperature over-
night. DMF was evaporated and the residue was dissolved in acetone
and diethyl ether was added to precipitate the compound. The precipi-
tate was collected by centrifuge, washed with ether and purified by
HPLC using acetonitrile and water [0 to 6 min, 30% acetonitrile as an el-
uent (6 to 7 min), 30 to 70% acetonitrile as an eluent (7 to 12 min), 70%
acetonitrile, retention time: 7.1 min]. Yield: 43 mg, 67%. 195Pt NMR
(DMF): 1381 ppm. 1H NMR (D2O): δ ppm 0.8–0.96 (m, 6H), 1.10–1.60
(m, 16H), 2.35 (m, 1H), 2.85 (m, 2H), and 8.74–9.09 (b, 4H) Purity N 98%.
ESIMS: 557.21.
Fig. 1. Schematic representation of structures of compounds used in the present work.

74
V. Novohradsky et al. / Journal of Inorganic Biochemistry 140 (2014) 72–79
2.2.3. [Pt(DACH)(VPA)2(ox)] (III)
complexes in medium during treatment were verified by flameless
To a suspension of [Pt(DACH)(OH)2(ox)] (50 mg, 1.16 × 10−4 mol)
in N,N-DMF (1 mL), valproic anhydride (78.37 mg, 2.88 × 10−4 mol,
2.5 eq) was added. The mixture was stirred at room temperature for
12 h. The solvent was evaporated at the end of the reaction and the res-
idue was redissolved in 5 mL of dichloromethane. Diethyl ether was
added to precipitate the desired compound as a white powder. The pre-
cipitate was separated by centrifugation, washed twice with diethyl
ether and dried under vacuum. Yield: 68 mg, 86%. 195Pt NMR (DMF):
1569 ppm. 1H NMR (DMSO-d6): δ ppm 1.10 (t, 12H), 1.30–1.90
(m, 24H), 2.4–2.8 (m, 4H), and 8.74–9.09 (b, 4H). HPLC: C18 column,
isocratic 60% acetonitrile/40% water as an eluent, retention time:
6.21 min. Purity N 99%. ESIMS 684.3.
atomic absorption spectrometry (FAAS).
2.6. Measurement of the partition coefficient
To determine the partition coefficient (P) the shake flask method
was used. 1-Octanol-saturated water (OSW) and water-saturated 1-
octanol (WSO) were prepared using analytical grade 1-octanol and 0.2
M aqueous NaCl solution (to suppress hydrolysis of the chlorido com-
plexes). Mixing was done by vortexing for 30 min at room temperature
to establish the partition equilibrium. To separate the phases, centrifu-
gation was done at 3000 g for 5 min. The aqueous layer was carefully
separated from the 1-octanol layer for platinum analysis. Pt was quanti-
fied from aliquots taken from the 1-octanol-saturated aqueous samples
before and after partition by FAAS. Partition coefficients of Pt complexes
were calculated using the equation log P = log([Pt]WSO / [Pt]OSW).
2.3. Preparation of stock solutions for cellular studies
Complexes II, III, and oxaliplatin (Sigma-Aldrich, Prague, Czech
Republic) were dissolved in DMF to a final concentration of 25 mM
and serially diluted prior to testing. The final DMF concentration in cul-
ture medium did not exceed 0.1%. To enable the miscibility of VPA with
culture medium, the sodium salt of VPA was prepared by adding one
equivalent of sodium hydroxide to VPA solution. After the application
of VPA/NaOH solution to the cell suspension, the pH was monitored,
not exceeding the range of pH 6.5–8.0. Stock solutions of I and
oxaliplatin were prepared in water and diluted directly into culture
medium.
2.7. Cellular uptake
The A2780 cells were seeded in 150 cm2 culture flasks at a density of
1 × 107 cells/flask (67 000 cells/cm2). After 48 h incubation, the cells
were treated with the compounds (0.35 μM) for 24 h. The attached
cells were washed twice with PBS (4 °C). Cell pellets were digested by
a high pressure microwave digestion system (MARS5, CEM) with hy-
drochloric acid (11 M) to give a fully homogenized solution, and final
platinum content was determined by inductively coupled plasma
mass spectroscopy (ICP-MS). Cellular distribution into the different
cell fractions (cytosol, nucleus, membrane/particulate, and cytoskele-
ton) was performed using a Fraction PREP cell fractionation kit from
BioVision (Mountain View, CA).
2.4. Cell lines
The human ovarian carcinoma cisplatin sensitive A2780 cells,
cisplatin resistant A2780/cisR (cisplatin resistant variant of A2780
cells) and the human breast cancer MCF-7 cells were kindly supplied
by Professor B. Keppler, University of Vienna (Austria). The A2780 and
A2780/cisR cell lines were grown in RPMI 1640 medium (PAA,
2.8. Cellular platinum DNA binding
The cells were seeded in 150 cm2 culture flasks at a density of 1 × 107
cells/flask (67 000 cells/cm2). After 48 h incubation, the cells were
treated with the compounds (0.35 μM) for 24 h. The attached cells
were washed twice with PBS (4 °C). Cells were then lysed in DNAzol
(DNAzol® genomic DNA isolation reagent, MRC) supplemented with
RNAse A (100 μg·mL−1). The genomic DNA was precipitated from the
lysate with ethanol, dried and resuspended in water. The DNA content
in each sample was determined by UV spectrophotometry. To avoid
an affection of high DNA concentration on ICP-MS detection of platinum
in the samples, the DNA samples were digested in the presence of hy-
drochloric acid (11 M) using high pressure microwave mineralization
system (MARS5, CEM). Experiments were performed in triplicate and
the values are the means SD.
Pasching, Austria) supplemented with gentamycin (50 μg·mL−1
,
Serva, Heidelberg, Germany) and 10% heat inactivated fetal bovine
serum (PAA). The acquired resistance of A2780/cisR cells was main-
tained by supplementing the medium with 1 μM cisplatin every second
passage. The MCF-7 (human breast cancer cells) were grown in DMEM
medium (high glucose, 4.5 g·L−1, PAA) supplemented with gentamycin
(50 μg·mL−1) and 10% heat inactivated fetal bovine serum (PAA). The
cells were cultured in a humidified incubator at 37 °C in a 5% CO2 atmo-
sphere and subcultured 2–3 times a week with an appropriate plating
density.
2.5. Cytotoxicity
2.9. Preparation of nuclear extracts
Cell death was evaluated by using a system based on the tetrazo-
lium compound MTT. The cells were seeded in 96-well tissue culture
plates at a density of 104 A2780/cisR cells/well or 6 × 103 MCF-7
cells/well in 100 μL of medium. After overnight incubation (16 h),
the cells were treated with the compounds at the final concentra-
tions in the range of 0 to 40 μM in a final volume of 200 μL/well.
After an additional 72 h, 10 μL of a freshly diluted MTT solution
(2.5 mg·mL−1) was added to each well and the plate was incubated
at 37 °C in a humidified 5% CO2 atmosphere for 4 h. At the end of the
incubation period the medium was removed and the formazan prod-
uct was dissolved in 100 μL of DMSO. The cell viability was evaluated
by measurement of the absorbance of 570 nm, using an Absorbance
Reader SUNRISE TECANS CHOELLER. IC50 values (IC50 value is the
concentration of the agent inhibiting cell growth by 50%) were calculat-
ed from curves constructed by plotting cell survival (%) versus drug
concentration (μM). All experiments were made in triplicate. The
reading values were converted to the percentage of control (% cell sur-
vival). Cytotoxic effects were expressed as IC50. Concentrations of Pt
A2780 cells were seeded in 100 mm tissue culture dishes (3 × 106
cells) and grown for 48 h to 80–90% confluence. The compounds were
added to isotoxic concentrations (IC50; Table 1). Cells were harvested
after 24 h of treatment. Concretely, the dishes were washed two times
Table 1
Cytotoxicity [IC50 mean values μM/mM] obtained for oxaliplatin, I, II, III and VPA.a
A2780
A2780/cisR
RFb
MCF-7
Oxaliplatin (μM)
I (μM)
II (μM)
III (μM)
VPA (mM)
0.52
11.4
5.4
1.3
8.6
0.08
1.8
0.5
0.2
1.9
4.2
22.5
16.1
8.8
1.6
4.6
0.7
2.1
1.5
8.1
2.0
3.0
6.8
0.5
19.6
77.4
31.6
5.7
1.5
15.3
2.1
0.9
1.1
4.3
8.5
a
The experiments were performed in triplicate. The drug-treatment period was 72 h.
SD for three independent samples.
RF — resistance factor is defined as IC50 (resistant, A2780/cisR)/IC50 (sensitive,
A2780).
The results are expressed as mean values
b

V. Novohradsky et al. / Journal of Inorganic Biochemistry 140 (2014) 72–79
75
with ice-cold PBS and the cells were scraped into tubes and centrifuged
for 10 min at 0 °C (200 g). The pellets were resuspended in the lysis
buffer [Tris·HCl (10 mM, pH 8.0), KCl (60 mM), EDTA (1.2 mM), dithio-
threitol (DTT, 1 mM), PMSF (0.1 mM), and NP-40 (0.04%)] for 10 min on
ice and centrifuged for 4 min at 0 °C (800 g). The pellets were rinsed
with the above lysis buffer without PMSF and NP-40 and centrifuged
for another 4 min at 0 °C (300 g). The pellets were resuspended in 50
μL nuclear extraction buffer [Tris·HCl (20 mM, pH 8.0), NaCl (420
mM), MgCl2 (0.7 mM), EDTA (0.25 mM), and glycerol (25%)] and incu-
bated for 30 min at 4 °C, continuously mixed on end-over-end roller,
and then centrifuged for 15 min at 0 °C (13 000 g). Protein concentration
in nuclear extracts was determined by the Bradford assay, and stored
at−80 °C until analysis.
FUJIFILM). Quantification of visualized bands was performed by densi-
tometry using AIDA software (Advanced Image Data Analyzer).
2.13. Cyclic voltammetry
The cyclic voltammetry (CV) was measured on PGSTAT101 (Autolab,
Metrohm) in a range of potentials from +0.5 V to−1.4 V, with a scan
rate of 100 mV·s−1 at 25 °C. KCl (0.1 M, pH 7.0) was used as a back-
ground electrolyte. A basal plane pyrolytic graphite and silver chloride
(Ag/AgCl, 3 M KCl) electrodes were used as working and reference
electrodes, respectively and platinum wire as a counter electrode.
2.14. Other physical methods
NMR spectroscopy: 1H and 195Pt NMR data were recorded on a
Varian Unity-500 MHz spectrometer equipped with a 5-mm switchable
probe or a 5-mm inverse detection probe. Data were processed using
either VnmrJ or MestreNova software. Analytical HPLC for assessing pu-
rity was performed in a Varian ProStar HPLC system equipped with a UV
detector, set at 220 nm, using a RP-C18 column (Phenomenex, Inc. Tor-
rance, CA, USA, Luna, 250 × 4.6 mm, 5 μm, 100 Å). Electrospray ioniza-
tion mass spectrometry was carried out using a Thermo Scientific
triple quadrature mass spectrometer (Quantum Access) in the positive
ion mode. Data were processed using Thermo Scientific's Xcalibur™
software. Absorption spectra were measured with a Beckmann DU-
7400 spectrophotometer. FAAS measurements were carried out with a
Varian AA240Z Zeeman atomic absorption spectrometer equipped
with a GTA 120 graphite tube atomizer. The analysis with the aid of
ICP-MS was performed using Agilent 7500 (Agilent, Japan).
2.10. Histone deacetylase assay
The A2780 nuclear extracts were tested for their HDAC activity
following the protocol recommended by the manufacturer of the Color-
imetric HDAC Activity Assay Kit (BioVision Research Products, Moun-
tain View, CA, USA). The standard curve was constructed using the
standard included in the kit. The absolute amount of deacetylated prod-
uct was calculated from standard curve. The values in the bar chart are
expressed as the concentration (μM) of deacetylated product in the
sample, subtracted the amount of deacetylated product in the control
(untreated) cells. Twenty micrograms of nuclear protein was added to
each sample.
2.11. Acid extraction of histones
Acid extraction of histones was performed as described previously
[12]. Cells were grown on 100 mm Petri dishes for 24 h. The compounds
were added to equitoxic concentrations (IC50), and after another 24 h
the cells were harvested. The dishes were washed three times with
ice-cold PBS and the cells were scraped into tubes and centrifuged for
10 min at 0 °C (1000 g). Cells were then suspended with five volumes
of lysis buffer [HEPES (10 mM, pH 7.9), MgCl2 (1.5 mM), KCl (10
mM), DTT (0.5 mM), and PMSF (1.5 mM)] and hydrochloric acid at a
final concentration of 0.2 M and subsequently lysed on ice for 30 min.
After centrifugation at 11 000 g for 10 min at 4 °C, the supernatant frac-
tion containing the acid-soluble proteins was retained. The supernatant
was dialyzed against acetic acid (0.1 M) twice for 1–2 h each and then
dialyzed against H2O for 1 h, 3 h and overnight. Dialysis was performed
using Spectra/Pore 3 Dialysis Membranes 3500 MWCO. The protein
concentration of the extracts was determined by Bradford assay using
bovine serum albumin as a standard.
3. Results and discussion
3.1. Cytotoxicity
The effect on cell-growth inhibition of Pt(IV) complexes I–III and
free VPA was screened against cisplatin-sensitive and cisplatin-
resistant ovarian cancer cell lines A2780 and A2780/cisR, respectively
and the MCF-7 breast cancer cell line (with inherent cisplatin resistance
[44]). The corresponding IC50 values are reported in Table 1.
The IC50 values found for the Pt(IV) derivative of oxaliplatin I (con-
taining two hydroxido axial ligands) were in the micromolar range
(11–77 μM). The decreased cytotoxicity of I relative to that of oxaliplatin
was expected because of the relative kinetic inertness of the Pt(IV) moi-
ety in I. For example, the IC50 of I in A2780 cells is 11.4 μM after 72 h in-
cubation time, whereas the IC50 for oxaliplatin is 0.52 μM under the
same conditions (IC50 ratio ~22, Table 1). In A2780/cisR and MCF-7
cells, however, the Pt(IV) complex I exhibited cytotoxicity closer to
that of oxaliplatin (IC50 ratios ~5 and ~4, respectively, Table 1). The re-
placement of axial hydroxido groups in I by VPA resulted in a significant
enhancement of toxicity in the cells tested in this work; this enhance-
ment was more pronounced for III, i.e. when both axial hydroxido
groups in I were replaced by VPA (Table 1). The results, at first glance,
might indicate that replacement of biologically inactive axial hydroxido
ligands by the HDACi VPA represents a significant advantage enhancing
its toxic potency in cancer cell lines. Free VPA exhibited toxic effects in
the A2780, A2780/cisR and MCF-7 cells at the concentrations in the mil-
limolar range [IC50 value (Table 1)] in accordance with the literature
[45]. These IC50 values were more than 110–7000-fold higher than
those found for Pt(IV) derivative of oxaliplatin containing two axial
VPA ligands (III).
2.12. Western blot analysis
The extracts containing 10 μg of protein were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (15% polyacryl-
amide gel) in loading buffer [Tris·HCl (50 mM, pH 8.0), SDS (2%),
bromophenol blue (0.1%), and glycerol (10%)] and blotted onto nitrocel-
lulose membrane in a transfer buffer containing Tris·HCl (25 mM,
pH 8.0), glycine (192 mM), and methanol (20%). The detection of acet-
ylated histones H3 was carried out by overnight incubation of the
membrane with primary antibody (Ac-histone H3 (Lys 24):sc-34262;
dilution 1:200). Then the membrane was washed with TBS Tween and
incubated with secondary horse radish peroxidase conjugated antibody
(sc-2004, goat-anti-rabbit Igb-HRP, dilution 1:5000) for 2 h. Equal load-
ing of transferred proteins was verified by the detection of β-actin using
monoclonal anti-β-actin clone AC-15 primary antibody, dilution 1:5000
followed by incubation of the membrane with secondary peroxidase
conjugated goat-antimouse Igb (H + L) (dilution 1:500). The proteins
were visualized using Super Signal West Pico Chemiluminescent
Substrate Kits (Pierce) with luminescent image analyzer (LAS-3000,
It is also important to note that comparisons of the effect on cell-
growth inhibition of I–III screened against cisplatin-sensitive and
cisplatin-resistant cell line A2780/cisR (variant of A2780 cells with ac-
quired resistance to cisplatin [46,47]) yielded interesting information
as well. Transformation of I into its Pt(IV) derivative containing one or
two VPA axial ligands resulted in a marked increase of the killing

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V. Novohradsky et al. / Journal of Inorganic Biochemistry 140 (2014) 72–79
efficiency in both A2780 and A2780/cisR. However, the IC50 value found
for III in A2780 cells was ~9-fold lower than that found for I, whereas
the IC50 value found for III in A2780/cisR cells was only ~3-fold lower
than that found for I. As a result, resistance factor increased as a conse-
quence of the replacement of axial hydroxido groups in I by VPA (the
trend is III N II).
In an effort to understand more deeply the effects that may play an
important role in the biological (pharmacological) properties of this
class of platinum conjugates against cancer cells, it was deemed impor-
tant to elucidate the biochemical and pharmacological factors that may
affect its cytotoxic properties. Hence, our primary objective in the
present study was to understand more fully the molecular and cellular
mechanisms underlying cytostatic effects of the dual function agents II
and III with a view to elucidating why the replacement of axial
hydroxido groups in I by one or two VPA ligands resulted in a significant
enhancement of toxicity in cancer cell lines.
investigate a possible relationship between the cellular uptake, hydro-
phobicity, and in vitro cytotoxicity of the complexes. Cellular concentra-
tions were determined by ICP-MS after 24 h of exposure to the
complexes at 0.35 μM. The results are summarized in Table 2. The re-
placement of axial hydroxido groups in I by VPA resulted in a significant
enhancement of the level of platinum associated with the cells. It is also
interesting to note that as the hydrophobicity increases (III N II N I), the
ratio of the cell associated Pt levels of A2780/A2780cisR increases sug-
gesting that increased lipophilicity increases the cellular accumulation
in the sensitive A2780 much more than in the resistant A2780/cisR
resulting in the higher RF values for II and III compared with I. Thus,
the results revealed that the presence of VPA in the Pt(IV) derivatives
of oxaliplatin significantly facilitated association of these conjugates in
cells. This observation is consistent with the lipophilic character of
VPA in the Pt(IV) derivatives of oxaliplatin.
Cellular uptake studies were also carried out to measure the distri-
bution of platinum species in different subcellular compartments.
A2780 cells were treated with 0.35 μM Pt(IV) derivatives of oxaliplatin
for 24 h and the cells were fractionated into four pools: membrane,
cytosol, nucleus, and cytoskeleton. The platinum levels in each pool
were measured by ICP-MS. The results are presented in Fig. 2.
3.2. Cellular accumulation
The early phases by which Pt compounds exert their anticancer
activity are cellular entry, activation, and DNA binding [48]. Before the
active form of a Pt drug reaches its major pharmacological target in
the cell nucleus, the Pt drug has to first accumulate in cells. It has been
demonstrated that higher accumulation of Pt drugs generally correlates
with enhanced cytotoxicity [49,50].
Partition coefficient (log P) values for 1-octanol/water partition
provide a measure of hydrophobicity that is often a factor relevant
for cell uptake and anticancer activity. For several classes of metallo-
anticancer complexes, a correlation between increased hydrophobicity
and increased cytotoxic activity has been reported [51–55]. The log
P values for I, II and III are shown in Table 2. Complex I is hydrophilic
(partitions preferentially into water ca. 170-fold). In contrast, the re-
placement of axial hydroxido ligands in I by one or two VPA groups
transformed the Pt(IV) derivative of oxaliplatin in hydrophobic
species.
The hydrophobicity (Table 2) and cytotoxicity (Table 1) of the Pt(IV)
derivatives of oxaliplatin correlate in this study. Complex I is hydrophil-
ic and exhibited lowest cytotoxicity. Complexes II and III display hydro-
phobicity (the trend is III N II) and are significantly more cytotoxic (III is
more cytotoxic than II). Thus, the cytotoxicity differences appear to re-
flect these hydrophobicity differences. In addition, it seems reasonable
to suggest that complexes II and III (contrary to I) are hydrophobic
enough to partition efficiently into cells so that it is very likely that
this difference results in efficient cancer cell uptake of II and III and re-
duced uptake of I. Hence, an interesting generalization of these results
might be that replacement of hydroxyl ligands with VPA ligand in I
markedly enhances hydrophobicity and consequently also toxicity in
cells of this class of Pt(IV) complexes.
The extent of accumulation of both VPA conjugated Pt(IV) prodrugs
into the cell membrane fraction was significantly greater than that of I,
accounting for 65% (III), 60% (II), and 29% (I) of the total Pt associated
with the cell. This property could be due to their higher intrinsic lipophi-
licity. Conversely, the extent of accumulation of both VPA conjugated
Pt(IV) prodrugs into the cytosol fraction was significantly lower than
that of I, accounting for 15% (III), 16% (II), and 57% (I) of the total Pt
in the cell. For the two remaining fractions, the extent of accumulation
of all three Pt(IV) derivatives of oxaliplatin still dropped. The amount
of Pt in the nucleus was significant and similar for all three Pt(IV)
prodrugs 13% of the total Pt for III, 9% for I, and 11% for II. The amount
of Pt in the cytoskeleton was also significant and slightly higher for II
in comparison with other two Pt(IV) derivatives of oxaliplatin: 16% of
the total Pt for II, and 5–6% for I or III. Overall, our data suggest that
large amounts of Pt(IV) derivatives of oxaliplatin containing axial VPA
ligand(s) become trapped in cell membrane.
The cellular accumulation of Pt(IV) derivatives of oxaliplatin in the
A2780 and A2780/cisR ovarian cancer cell lines was studied to
Table 2
The log P (1-octanol/water) values obtained by shake flask method, cellular platinum
accumulation in A2780 and A2780/cisR cellsa and platinum content of DNA isolated from
A2780 or A2780/cisR cells exposed to Pt(IV) derivatives of oxaliplatinb.
log P
ng Pt/106 cellsa,c,d
A2780 A2780/cisR
0.19 0.27
pg Pt/μg of DNAb,c,d
A2780
A2780/cisR
I
II
III
−2.38
0.10
0.37
0.08 0.12
0.13 0.40
0.65 0.69
0.02
0.04
1.95
3.00
0.17 1.08
0.65 1.72
0.03 2.07
0.08
0.03
0.07
0.05 1.33
0.08 4.27
0.05 12.12
a
The table shows the uptake of tested compounds into A2780 or A2780/cisR cells.
Platinum content of DNA fraction isolated from A2780 or A2780/cisR cells.
Cells were exposed to tested compounds (0.35 μM) for 24 h.
b
c
Fig. 2. Cell uptake of I, II, or III in A2780 cells. (A) The amount of Pt in cell cytoplasm, mem-
brane, nucleus and cytoskeleton fractions; (B) the percentage of Pt in different fractions
over total uptake. Cells were exposed to 0.35 μM of platinum compounds for 24 h.
d
Results are expressed as mean
SD for three independent samples.

V. Novohradsky et al. / Journal of Inorganic Biochemistry 140 (2014) 72–79
77
3.3. DNA-bound platinum in cells exposed to Pt(IV) derivatives of
oxaliplatin
As for cisplatin and its direct derivatives, DNA is believed to be the
main target also for Pt(IV) prodrugs examined in the present work.
For this reason, DNA from A2780 and A2780/cisR cells after the expo-
sure to 0.35 μM Pt(IV) derivatives of oxaliplatin for 24 h was isolated
and the levels of platinum on DNA were determined by ICP-MS
(Table 2). The platinum content of DNA from A2780 cells treated with
II and III was approximately 1.5- and 6.2-fold greater, respectively,
than that from the cells treated with I. Notably, the platinum content
of DNA from A2780/cisR cells treated with II and III was only approxi-
mately 1.2- and 1.9-fold greater, respectively, than that from the cells
treated with I.
We made a rough estimate of the percentage of the amount of plat-
inum in the DNA fraction of the total amount of platinum not trapped in
A2780 cell membrane considering the data in Table 2 and Fig. 2. Thus, in
these estimations the amount of platinum not trapped in cell membrane
and available for interaction with DNA was calculated as the total
amount of platinum associated with the cells from which the amount
of platinum detected in cell membrane was subtracted. Of the total Pt
inside the A2780 cells (not trapped in cell membrane), a considerably
higher fraction of platinum from III or II was bound to DNA in compar-
ison with the treatment with I, accounting for 7.2% (III), 5.5% (II), and
2.9% (I). In aggregate, the yield of platination of DNA by Pt(IV) deriva-
tives of oxaliplatin not trapped in A2780 cell membrane (normalized
to the same amount of platinum inside the cell) available for reaction
with nuclear DNA was markedly higher for derivatives containing
axial VPA ligand(s) than for I. Hence, notably, an additional mechanism
besides enhanced cell internalization of Pt(IV) conjugates containing
VPA axial ligands had to be involved in a considerably enhanced fraction
of platinum from III or II bound to DNA from A2780 cells in comparison
with that from I.
Fig. 3. Western blot analysis for acetylated histone H3 in A2780 cells treated with the
agents tested in the present work. The cells were treated for 24 h with the equitoxic con-
centrations of the agents corresponding to the IC50 values (Table 1). (A) The proteins were
transferred onto nitrocellulose membranes and stained with antiacetylated histone H3 an-
tibody. β-Actin is shown as equal loading control. Lanes: 1, control, untreated cells; 2, cells
treated with VPA (8.6 mM); 3, cells treated with III (1.3 μM); 4, cells treated with II
(5.4 μM); 5, cells treated with I (11.4 μM). (B) Quantification of the acetylated histone
H3 (H3ac). The levels of H3ac were computed by subtracting the H3ac level measured
for the control untreated cells, from the value measured for the treated cells. The results
are the means of three independent samples, and standard deviations are shown by
error bars.
of Pt(IV) derivatives of oxaliplatin containing one or two axial VPA
ligands was markedly enhanced compared with that of free VPA
(Table 3 and Fig. 4).
The HDAC activity in cells treated with oxaliplatin or I increased,
which is in agreement with the results obtained by Western blot analy-
sis (Fig. 3). Thus, both methods used to assay the HDAC inhibitory ef-
fects showed that Pt(IV) derivatives of oxaliplatin containing one or
two axial VPA ligands (II and III) exhibited a marked inhibitory effect
on HDAC activity in A2780 tumor cells at the concentrations ~1000-
fold lower than that of free VPA exhibiting the same HDAC inhibitory
effect. In aggregate, the presence of the axial VPA ligand(s) in Pt(IV)
derivatives of oxaliplatin considerably enhances one of the dual func-
tionalities anticipated to be effective after the novel Pt(IV) derivatives
of oxaliplatin containing VPA ligand(s) enter the tumor cell.
3.4. Inhibition of histone deacetylase activity
Pt(IV) derivatives of oxaliplatin containing axial VPA ligand(s) were
designed such that their reduction products should bind nuclear DNA
in much the same way as cisplatin or its derivatives. Moreover, their
additional functionality (HDAC inhibitory activity of VPA released
upon intracellular reduction) that induces hyperacetylation of histone
proteins complexed with DNA thereby increasing the accessibility of
DNA within chromatin to DNA-binding agents [3,10,11] could conse-
quently enhance the extent of DNA damage by platinum adducts and
in this way potentiate the anticancer activity. Thus, further studies
were aimed at determining the effect of the treatment of A2780 cells
with Pt(IV) derivatives of oxaliplatin on HDAC activity.
The results shown in Fig. 4A show that III exhibited a marked inhib-
itory effect on HDAC activity in A2780 tumor cells when these cells were
treated with this Pt(IV) complex at the concentration of 1.3 μM. As it is
demonstrated in Fig. 4B and Table 3, free VPA even at a higher
Western blot analysis indicated that the levels of histone H3 acetyla-
tion measured directly in A2780 cells increased compared to the basal
(control) level, when A2780 cells were treated with 8 mM VPA
(Fig. 3). This observation is in good agreement with the well-known
HDAC inhibitory effect of VPA [32,33,56]. The treatment of A2780 cells
with I resulted in a decrease of the level of histone H3 acetylation
below the basal level of acetylated histone H3 in the untreated A2780
cells. Conversely, the treatment of A2780 cells with Pt(IV) derivatives
of oxaliplatin containing one or two axial VPA ligands resulted in the
markedly increased levels of histone H3 acetylation in A2780 cells com-
pared to the basal (control) level. Importantly, the markedly enhanced
levels of histone H3 acetylation were observed at the concentrations
three orders of magnitude lower than the concentration of free VPA
needed to induce the same effects (Fig. 3). Qualitatively identical results
were also obtained when the levels of acetylation of histone H4 were
determined in A2780 cells treated with I, II, and III (not shown).
The results obtained by Western blotting were complemented by
the direct analysis of HDAC activity using a commercial colorimetric
HDAC Activity Assay Kit. The results confirmed that the inhibitory effect
Table 3
Effect of the single agents tested in the present work on HDAC activity in A2780 cells and
comparison with oxaliplatin, cisplatin and trichostatin.
c [μM]a,c,d
c [μM]b,c,d
Control (untreated cells)
VPA
Oxaliplatin
I
II
III
Oxaliplatin + VPA (1:2)
Cisplatin
Trichostatin A
38.3
24.5
42.8
42.3
31.5
29.1
0.8
38.3
36.3
0.8
0.5
1.3
0.4
1.4
1.1
3.9
24.7
34.9
0.4
0.3
43.9
0.07
1.0
0.01
a
The cells were treated for 24 h with the equitoxic concentrations of the agents cor-
responding to the IC50 values (Table 1).
b
The cells were treated for 24 h with the equimolar concentrations of the agents used
as single agents (5 μM) or in the case of oxaliplatin also in its 1:2 mixture with VPA.
c
Results are expressed as mean
Concentration of the deacetylated product.
SD of two independent experiments.
d

78
V. Novohradsky et al. / Journal of Inorganic Biochemistry 140 (2014) 72–79
VPA ligand(s) in I enhances markedly the ease of electrochemical
reduction and also toxicity in tumor cells of this class of Pt(IV) com-
plexes. Hence, the electrochemical reducibility of Pt(IV) derivatives
of oxaliplatin tested in the present work might be another important
determinant in relation to their toxicity in cells.
4. Conclusions
In this study, we synthesized a series of new anticancer prodrugs,
Pt(IV) derivatives of oxaliplatin with one or two axial VPA ligand(s).
This work also presents a systematic investigation of the molecular
pharmacology of these new anticancer prodrugs. We find that these
conjugated compounds represent more potent bifunctional inhibitors
capable of both enhanced accumulation in tumor cells and dual
targeting of histone deacetylase and DNA. We show that the free VPA,
released from the Pt(IV) complexes (as a consequence of their re-
duction in tumor cells), downregulates histone deacetylases lead-
ing to hyperacetylation of histone proteins complexed with DNA.
This hyperacetylation was shown to increase nuclear DNA damage
responsible for enhanced toxic effects toward tumor cells. The results
contribute to understanding more deeply the effects that may play an
important role in the biological (pharmacological) properties of this
class of platinum conjugates against cancer cells and will aid in the de-
sign of the next generation of Pt–HDACi conjugates. In addition, owing
to the fact that compared with conventional cisplatin, oxaliplatin ex-
hibits a different anticancer spectrum and has a more favorable toxicity
and tolerability profile, our findings also indicate that Pt(IV) derivatives
of oxaliplatin with VPA axial ligands seem to be promising candidates
for additional preclinical studies.
Fig. 4. HDAC activity in A2780 cells as indicated in Table 3. The ΔC values were computed
by subtracting the concentration of deacetylated product in the control, the untreated
cells, from the concentration of deacetylated product in the treated cells. The standard de-
viations are shown by error bars. A. The cells were treated for 24 h with the equitoxic
concentrations of the agents corresponding to the IC50 values (Table 1). B. The cells were
treated for 24 h with the equimolar concentrations of the agents (5 μM) used as single
agents (5 μM) or in the case of oxaliplatin also in its 1:2 mixture with VPA; an asterisk
denotes a significant difference (p b 0.006) between the HDAC activity in cells treated
with III and 1:2 mixture of oxaliplatin with VPA.
concentration (5 μM) and the 1:2 mixture of oxaliplatin (5 μM) and VPA
(10 μM) exhibited only a negligible inhibitory effect on HDAC activity
in A2780 tumor cells. However, it is important to realize that in the
case when the cells were treated with III, VPA was delivered to the
cytoplasm via this conjugate; free VPA or VPA in the mixture with
oxaliplatin may be accumulated in the cells differently, presumably
much less. Moreover, it has been shown [57] that the concentrations
at which free VPA efficiently inhibits catalytic activity of HDACs
in vitro are at least ten times higher. Hence, it seems unlikely that solely
a direct inhibition of catalytic activity of HDACs caused by the free VPA
released from III (or II) could be responsible for their total HDAC inhib-
itory effect in A2780 tumor cells (Table 3 and Fig. 4). In other words, an
additional mechanism downregulating HDACs triggered by a relatively
low concentration of VPA (micromolar) delivered by III into A2780
tumor cells had to be involved in the effects of VPA on hyperacetylation
of histone proteins complexed with DNA. The work aimed at revealing
these additional mechanisms is in progress in our laboratories and
will be published in a separate communication.
Abbreviations
I
Pt(IV) derivative of oxaliplatin with two axial hydroxido
ligands
II
Pt(IV) derivative of oxaliplatin with one axial hydroxido and
one axial VPA ligand
III
Pt(IV) derivative of oxaliplatin with two axial VPA ligands
cisplatin cis-diamminedichloridoplatinum (II);
cis-Pt(IV)-(VPA)2
cis,cis,trans-diamminedichlorido-bis(valproato)platinum(IV)
CV
cyclic voltammetry
DACH
DMF
DMSO
FAAS
HPLC
HDAC
HDACi
IC50
trans-(1R,2R)-1,2-cyclohexanediamine
dimethylformamide
dimethyl sulfoxide
flameless atomic absorption spectrometry
high-pressure liquid chromatography
histone deacetylase
histone deacetylase inhibitor
compound concentrations that produce 50% of cell growth
inhibition
3.5. Electrochemistry
The antitumor effects of the Pt(IV) complexes are considered to be a
result of their reduction into cytotoxic Pt(II) species by both extracellu-
lar and intracellular ligands [58]. Hence, it is naturally assumed that a
correlation exists between the reduction potentials of Pt(IV) prodrugs
and the rates of reduction on one hand and overall biological activity
on the other hand. Reduction of Pt(IV) complexes can be achieved elec-
trochemically or chemically. It has been shown that the reduction po-
tentials of Pt(IV) complexes depend not only on the nature of the
equatorial ligands, but also and mainly on the nature of axial ligands
which have a much stronger influence [59,60]. Moreover, several stud-
ies have reported that for Pt(IV) derivatives of cisplatin chemical reduc-
tion rates correlate with electrochemically determined irreversible
reduction peak potentials under conditions of cyclic voltammetry
(CV), although in general correlation of chemical and electrochemical
data is not clear and certain [61].
ICP-MS inductively coupled plasma mass spectroscopy
Ox oxalate
Oxaliplatin {(1R,2R)-diaminocyclohexane}oxalatoplatinum (II)
VPA valproic acid
Acknowledgments
This work was supported by the Czech Science Foundation (Grant
14-21053S). The authors also acknowledge that their participation in
the EU COST Action CM1105 enabled them to exchange regularly the
most recent ideas in the field of platinum anticancer drugs with several
European colleagues. DG acknowledges the support of the Israel Science
Foundation (grant 1332/10). We thank Dr. Ezequiel Wexselblatt for the
useful discussions.
The results of the electrochemical studies (the reduction potentials)
indicate that the ease of reduction follows the trend III N II N I (the re-
duction potentials obtained by CV were−0.58 V, −0.65 V and−1.02
V vs. Ag/AgCl, respectively). It is important to note that the reported
Ep values are not thermodynamic values. An interesting generalization
of these results might be that replacement of hydroxido ligands with

V. Novohradsky et al. / Journal of Inorganic Biochemistry 140 (2014) 72–79
79
[31] J.H. Lee, M.L. Choy, L. Ngo, S.S. Foster, P.A. Marks, Proc. Natl. Acad. Sci. U. S. A. 107
(2010) 14639–14644.
References
[32] J. Yang, X. Sun, W. Mao, M. Sui, J. Tang, Y. Shen, Mol. Pharm. 9 (2012) 2793–2800.
[33] M. Alessio, I. Zanellato, I. Bonarrigo, E. Gabano, M. Ravera, D. Osella, J. Inorg.
Biochem. 129 (2013) 52–57.
[34] E. Jasek, G.J. Lis, M. Jasinska, H. Jurkowska, J.A. Litwin, Anticancer Res. 32 (2012)
2791–2799.
[1] Z.H. Siddik, Oncogene 22 (2003) 7265–7279.
[2] V. Brabec, J. Kasparkova, Drug Resist. Updat. 8 (2005) 131–146.
[3] H.V.K. Diyabalanage, M.L. Granda, J.M. Hooker, Cancer Lett. 329 (2013) 1–8.
[4] D. Griffith, M.P. Morgan, C.J. Marmion, Chem. Commun. (2009) 6735–6737.
[5] D.M. Griffith, B. Duff, K.Y. Suponitsky, K. Kavanagh, M.P. Morgan, D. Egan, C.J.
Marmion, J. Inorg. Biochem. 105 (2011) 793–799.
[6] J.P. Parker, H. Nimir, D.M. Griffith, B. Duff, A.J. Chubb, M.P. Brennan, M.P. Morgan, D.
A. Egan, C.J. Marmion, J. Inorg. Biochem. 124 (2013) 70–77.
[7] B.S. Mann, J.R. Johnson, M.H. Cohen, R. Justice, R. Padzur, Oncologist 12 (2007)
1247–1252.
[8] R. Furumai, A. Matsuyama, N. Kobashi, K.H. Lee, N. Nishiyama, I. Nakajima, A.
Tanaka, Y. Komatsu, N. Nishino, M. Yoshida, S. Horinouchi, Cancer Res. 62 (2002)
4916–4921.
[9] G. Lopez, J. Liu, W. Ren, W. Wei, S. Wang, G. Lahat, Q.S. Zhu, W.G. Bornmann, D.J.
McConkey, R.E. Pollock, D.C. Lev, Clin. Cancer Res. 15 (2009) 3472–3483.
[10] D.C. Marchion, E. Bicaku, A.I. Daud, V. Richon, D.M. Sullivan, P.N. Munster, J. Cell.
Biochem. 92 (2004) 223–237.
[11] M.S. Kim, M. Blake, J.H. Baek, G. Kohlhagen, Y. Pommier, F. Carrier, Cancer Res. 63
(2003) 7291–7300.
[12] V. Brabec, D.M. Griffith, A. Kisova, H. Kostrhunova, L. Zerzankova, C.J. Marmion, J.
Kasparkova, Mol. Pharm. 9 (2012) 1990–1999.
[13] C.M. Giandomenico, M.J. Abrams, B.A. Murrer, J.F. Vollano, M.I. Rheinheimer, S.B.
Wyer, G.E. Bossard, J.D. Higgins, Inorg. Chem. 34 (1995) 1015–1021.
[14] S. Dhar, F.X. Gu, R. Langer, O.C. Farokhzad, S.J. Lippard, Proc. Natl. Acad. Sci. U. S. A.
105 (2008) 17356–17361.
[15] S. Dhar, Z. Liu, J. Thomale, H.J. Dai, S.J. Lippard, J. Am. Chem. Soc. 130 (2008)
11467–11476.
[35] B. Tsolmongyn, N. Koide, E. Odkhuu, A. Haque, Y. Naiki, T. Komatsu, T. Yoshida, T.
Yokochi, Cell. Immunol. 282 (2013) 100–105.
[36] M. Gottlicher, S. Minucci, P. Zhu, O.H. Kramer, A. Schimpf, S. Giavara, J.P. Sleeman, F.
Lo Coco, C. Nervi, P.G. Pelicci, T. Heinzel, EMBO J. 20 (2001) 6969–6978.
[37] J.A. Bermudez-Lugo, O. Perez-Gonzalez, M.C. Rosales-Hernandez, I. Ilizaliturri-Flores,
J. Trujillo-Ferrara, J. Correa-Basurto, J. Mol. Model. 18 (2012) 2301–2310.
[38] R.B. Weiss, M.C. Christian, Drugs 46 (1993) 360–377.
[39] E. Raymond, C. Buquet-Fagot, S. Djelloul, J. Mester, E. Cvitkovic, P. Allain, C. Louvet, C.
Gespach, Anti-Cancer Drugs 8 (1997) 876–885.
[40] E. Raymond, S. Faivre, J.M. Woynarowski, S.G. Chaney, Semin. Oncol. 25 (1998)
4–12.
[41] A.M. Di Francesco, A. Ruggiero, R. Riccardi, Cell. Mol. Life Sci. 59 (2002) 1914–1927.
[42] X. Zhang, M. Yashiro, J. Ren, K. Hirakawa, Oncol. Rep. 16 (2006) 563–568.
[43] M.D. Hall, C.T. Dillon, M. Zhang, P. Beale, Z. Cai, B. Lai, A.P.J. Stampfl, T.W. Hambley, J.
Biol. Inorg. Chem. 8 (2003) 726–732.
[44] S.J. Fan, M.L. Smith, D.J. Rivet, D. Duba, Q.M. Zhan, K.W. Kohn, A.J. Fornace, P.M.
O'Connor, Cancer Res. 55 (1995) 1649–1654.
[45] I. Zanellato, I. Bonarrigo, E. Gabano, M. Ravera, N. Margiotta, P.G. Betta, D. Osella,
Inorg. Chim. Acta 393 (2012) 64–74.
[46] L.R. Kelland, C.F.J. Barnard, K.J. Mellish, M. Jones, P.M. Goddard, M. Valenti, A. Bryant,
B.A. Murrer, K.R. Harrap, Cancer Res. 54 (1994) 5618–5622.
[47] L.R. Kelland, S.Y. Sharp, C.F. O'Neill, F.I. Raynaud, P.J. Beale, I.R. Judson, J. Inorg.
Biochem. 77 (1999) 111–115.
[16] S. Mukhopadhyay, C.M. Barnes, A. Haskel, S.M. Short, K.R. Barnes, S.J. Lippard,
Bioconjug. Chem. 19 (2008) 39–49.
[17] M.D. Hall, H.R. Mellor, R. Callaghan, T.W. Hambley, J. Med. Chem. 50 (2007)
3403–3411.
[18] A.M. Montana, C. Batalla, Curr. Med. Chem. 16 (2009) 2235–2260.
[19] K.R. Barnes, A. Kutikov, S.J. Lippard, Chem. Biol. 11 (2004) 557–564.
[20] W.H. Ang, I. Khalaila, C.S. Allardyce, L. Juillerat-Jeanneret, P.J. Dyson, J. Am. Chem.
Soc. 127 (2005) 1382–1383.
[48] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307–320.
[49] A.L. Harris, X. Yang, A. Hegmans, L. Povirk, J.J. Ryan, L. Kelland, N.P. Farrell, Inorg.
Chem. 44 (2005) 9598–9600.
[50] J. Kasparkova, O. Novakova, O. Vrana, F. Intini, G. Natile, V. Brabec, Mol. Pharmacol.
70 (2006) 1708–1719.
[51] S.Y. Loh, P. Mistry, L.R. Kelland, G. Abel, K.R. Harrap, Br. J. Cancer 66 (1992)
1109–1115.
[52] S.P. Oldfield, M.D. Hall, J.A. Platts, J. Med. Chem. 50 (2007) 5227–5237.
[53] M.-G. Mendoza-Ferri, C.G. Hartinger, R.E. Eichinger, N. Stolyarova, K. Severin, M.A.
Jakupec, A.A. Nazarov, B.K. Keppler, Organometallics 27 (2008) 2405–2407.
[54] P. Gramatica, E. Papa, M. Luini, E. Monti, M. Gariboldi, M. Ravera, E. Gabano, L.
Gaviglio, D. Osella, J. Biol. Inorg. Chem. 15 (2010) 1157–1169.
[55] Z. Liu, L. Salassa, A. Habtemariam, A.M. Pizarro, G.J. Clarkson, P.J. Sadler, Inorg. Chem.
50 (2011) 5777–5783.
[56] M.J. Lee, Y.S. Kim, S. Kummar, G. Giaccone, J.B. Trepel, Curr. Opin. Oncol. 20 (2008)
639–649.
[57] D.M. Fass, R. Shah, B. Ghosh, K. Hennig, S. Norton, W.-N. Zhao, S.A. Reis, P.S. Klein, R.
Mazitschek, R.L. Maglathlin, T.A. Lewis, S.J. Haggarty, ACS Med. Chem. Lett. 2 (2011)
39–42.
[58] M.D. Hall, T.W. Hambley, Coord. Chem. Rev. 232 (2002) 49–67.
[59] L.T. Ellis, H.M. Er, T.W. Hambley, Aust. J. Chem. 48 (1995) 793–806.
[60] T.W. Hambley, A.R. Battle, G.B. Deacon, E.T. Lawrenz, G.D. Fallon, B.M. Gatehouse, L.
K. Webster, S. Rainone, J. Inorg. Biochem. 77 (1999) 3–12.
[61] J.Z. Zhang, E. Wexselblatt, T.W. Hambley, D. Gibson, Chem. Commun. 48 (2012)
847–849.
[21] M.R. Reithofer, S.M. Valiahdi, M.A. Jakupec, V.B. Arion, A. Egger, M. Galanski, B.K.
Keppler, J. Med. Chem. 50 (2007) 6692–6699.
[22] S. Dhar, W.L. Daniel, D.A. Giljohann, C.A. Mirkin, S.J. Lippard, J. Am. Chem. Soc. 131
(2009) 14652–14653.
[23] I. Zanellato, I. Bonarrigo, M. Sardi, M. Alessio, E. Gabano, M. Ravera, D. Osella,
ChemMedChem 6 (2011) 2287–2293.
[24] L.J. Parker, L.C. Italiano, C.J. Morton, N.C. Hancock, D.B. Ascher, J.B. Aitken, H.H. Harris,
P. Campomanes, U. Rothlisberger, A. De Luca, M. Lo Bello, W.H. Ang, P.J. Dyson, M.W.
Parker, Chem. Eur. J. 17 (2011) 7806–7816.
[25] L. Gaviglio, A. Gross, N. Metzler-Nolte, M. Ravera, Metallomics 4 (2012) 260–266.
[26] N. Graf, T.E. Mokhtari, I.A. Papayannopoulos, S.J. Lippard, J. Inorg. Biochem. 110
(2012) 58–63.
[27] Y. Shi, S.-A. Liu, D.J. Kerwood, J. Goodisman, J.C. Dabrowiak, J. Inorg. Biochem. 107
(2012) 6–14.
[28] M. Dokmanovic, G. Perez, W.S. Xu, L. Ngo, C. Clarke, R.B. Parmigiani, P.A. Marks, Mol.
Cancer Ther. 6 (2007) 2525–2534.
[29] H. Hess-Stumpp, T.U. Bracker, D. Henderson, O. Politz, Int. J. Biochem. Cell Biol. 39
(2007) 1388–1405.
[30] N. Takai, H. Narahara, Obstet. Gynecol. Int. 2010 (2010) 1–8.