Cellular trafficking, accumulation and DNA platination of a series of cisplatin-based dicarboxylato Pt(IV) prodrugs

Article abstract of DOI:10.1016/j.jinorgbio.2015.05.012

A series of Pt(IV) anticancer prodrug candidates, having the equatorial arrangement of cisplatin and bearing two aliphatic carboxylato axial ligands, has been investigated to prove the relationship between lipophilicity, cellular accumulation, DNA platination and antiproliferative activity on the cisplatin-sensitive A2780 ovarian cancer cell line. Unlike cisplatin, no facilitated influx/efflux mechanism appears to operate in the case of the Pt(IV) complexes under investigation, thus indicating that they enter by passive diffusion. While Pt(IV) complexes having lipophilicity comparable to that of cisplatin (negative values of log P_o/w) exhibit a cellular accumulation similar to that of cisplatin, the most lipophilic complexes of the series show much higher cellular accumulation (stemming from enhanced passive diffusion), accompanied by greater DNA platination and cell growth inhibition. Even if the Pt(IV) complexes are removed from the culture medium in the recovery process, the level of DNA platination remains very high and persistent in time, indicating efficient storing of the complexes and poor detoxification efficiency.

Full text of DOI:10.1016/j.jinorgbio.2015.05.012

Journal of Inorganic Biochemistry 150 (2015) 1–8
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Cellular trafficking, accumulation and DNA platination of a series of
cisplatin-based dicarboxylato Pt(IV) prodrugs
Mauro Ravera a, Elisabetta Gabano a, Ilaria Zanellato a, Ilaria Bonarrigo a, Manuela Alessio a, Fabio Arnesano b,
Angela Galliani b, Giovanni Natile b, Domenico Osella a,
⁎
a
Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, Viale T. Michel 11, 15121 Alessandria, Italy
b
Dipartimento di Chimica, Università di Bari “Aldo Moro”, Via E. Orabona, 4, 70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Pt(IV) anticancer prodrug candidates, having the equatorial arrangement of cisplatin and bearing two
aliphatic carboxylato axial ligands, has been investigated to prove the relationship between lipophilicity, cellular
accumulation, DNA platination and antiproliferative activity on the cisplatin-sensitive A2780 ovarian cancer cell
line. Unlike cisplatin, no facilitated influx/efflux mechanism appears to operate in the case of the Pt(IV) com-
plexes under investigation, thus indicating that they enter by passive diffusion. While Pt(IV) complexes having
lipophilicity comparable to that of cisplatin (negative values of log Po/w) exhibit a cellular accumulation similar
to that of cisplatin, the most lipophilic complexes of the series show much higher cellular accumulation (stem-
ming from enhanced passive diffusion), accompanied by greater DNA platination and cell growth inhibition.
Even if the Pt(IV) complexes are removed from the culture medium in the recovery process, the level of DNA
platination remains very high and persistent in time, indicating efficient storing of the complexes and poor detox-
ification efficiency.
Received 9 January 2015
Received in revised form 19 May 2015
Accepted 21 May 2015
Available online 27 May 2015
Keywords:
Pt(IV) complexes
Lipophilicity
Cellular accumulation
DNA platination
Cell growth inhibition
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
the overall lipophilicity and substantially determine the reduction po-
tential and/or reduction kinetics.
It is widely accepted that the antitumor activity of Pt(II) drugs is
related to their ability to form adducts with DNA, which generally
correlates with their potency [1].
In previous studies on large series of Pt(IV) complexes, a clear rela-
tionship between reduction potential and lipophilicity on the one
hand and cytotoxicity on the other hand has been established: “the eas-
ier the reduction and the higher the lipophilicity, the greater is the cyto-
toxicity” [10–16]. However, this paradigm was not fully satisfied for
other Pt(IV) compounds, indicating the difficulty to simply relate the
chemico-physical parameters with the biological activity of such com-
plexes acting with an intricate sequence of reactions [13,17,18].
DNA platination should parallel the trend of cellular Pt accumula-
tion, with a latency time given by the kinetics of Pt(IV) → Pt(II)
reduction in addition to that of aquation of Pt(II) metabolites to pro-
duce reactive electrophiles. Moreover, removal of Pt-DNA adducts
and efflux of the corresponding Pt derivatives can occur more or
less efficiently, depending upon the type of cell line and its status.
Thus, the final amount of DNA platination at a given time point is a
consequence of an intricate sequence of events. For cisplatin, the
IC50 value (half maximal inhibition concentration) inversely corre-
lates with the amount of Pt-DNA adducts [19], but it is not granted
that the same relationship holds also for Pt(IV) complexes. Equimo-
lar concentrations of cisplatin and lipophilic satraplatin (i.e. (OC-6-
43)-bis(acetato)amminedichlorido(cyclohexylamine)platinum(IV))
resulted in comparable amount of DNA platination, but the cytotoxic
potency was different [20]. It is important to recall that the metabo-
lites obtained from reduction of cisplatin and satraplatin are
Octahedral Pt(IV) complexes are considered as prodrugs [2]. They
are kinetically quite inert and should reach the tumor cells intact.
Here they undergo reduction by biomolecules, whose low molecular
weight (MW) prototypes are ascorbic acid (AA) and reduced glutathi-
one (GSH) [3–5]. However, the reduction of Pt(IV) complexes by a still
unidentified high MW cellular component plays an important role [6].
Such a reduction is usually accompanied by loss of the two axial ligands
generating the corresponding cytotoxic Pt(II) species (Fig. 1). Some-
times scrambling between axial ligands (acetates) and equatorial
leaving groups (chlorides) may occur during the reduction process,
and this phenomenon seems to befall in the presence of bulky carrier
groups such as adamantylamine, cyclohexylamine, n-butylamine,
isopropylamine [7], and picoline [8]. The equatorial carrier ligands,
which always remain coordinated to Pt core during the reduction,
should influence the antiproliferative activity on different tumor cell
lines in the same manner they do in their Pt(II) counterparts [9]. As a
rule of thumb, the axial ligands affect the cellular accumulation of the
complex by virtue of their contribution, among the other ligands, to
⁎
Corresponding author.
E-mail address: domenico.osella@uniupo.it (D. Osella).
http://dx.doi.org/10.1016/j.jinorgbio.2015.05.012
0162-0134/© 2015 Elsevier Inc. All rights reserved.

2
M. Ravera et al. / Journal of Inorganic Biochemistry 150 (2015) 1–8
Fig. 1. Sketch of the dicarboxylato Pt(IV) complexes under investigation and their reduction reaction.
structurally different, therefore the comparison between the activi-
ties of the two drugs is not straightforward.
presence of an excess of the reducing agent dithiothreitol (DTT) to pre-
serve the protein in its active form. Protein purity was determined by
SDS-PAGE and ESI-MS. To remove the buffer and to reduce the excess
of DTT, the apoprotein was extensively washed with deoxygenated
H2O by using Centricon filters (3 kDa molecular weight cutoff) and
under strictly anaerobic condition. The freshly prepared protein sample,
handled under inert N2 atmosphere inside a glove-box, was immediate-
ly used for the incubations with the Pt complexes.
Therefore, up to now, the relationship between lipophilicity, cell
accumulation, DNA platination and antiproliferative activity of
Pt(IV) complexes is not completely assessed. To answer this ques-
tion, a series of dicarboxylato-platinum(IV) complexes, having
the equatorial arrangement of cisplatin and bearing two aliphatic
carboxylato axial ligands of different chain lengths, namely
trans,cis,cis-[Pt(carboxylato)2Cl2(NH3)2], where carboxylato =
CH3(CH2)nCOO− (n = 0, 1; n = 2, 2; n = 4, 3; n = 6, 4, Fig. 1) has
been investigated having cisplatin as a reference. The interaction
with copper transport proteins, the cellular accumulation, the
DNA platination, and the antiproliferative activity of these com-
plexes have been studied on the cisplatin-sensitive A2780 ovarian
cancer cell line.
The reduction of Pt(IV) complexes by GSH (in phosphate buffer, PB)
or sodium ascorbate, as well as the reactions between the Pt(IV) or Pt(II)
complexes and the Mets7/Mnk1 (1:1 molar ratio) were analyzed with a
dual electrospray interface and a 6530 Series Accurate-Mass Quadru-
pole Time-of-Flight LC/MS (Agilent, USA). ESI-MS spectrometry, due to
the soft ionization, is generally accepted as an efficient method for the
characterization of metal complexes in solution, albeit care must be
used when correlating gas phase ions to species present in solution
[24]. The reaction mixtures were kept stirring under anaerobic condi-
tions at 37 or 40 °C and, from time to time, aliquots were removed,
five-fold diluted with deoxygenated water, and injected in the mass
spectrometer. Acetic acid (1% v/v) was added before injection to obtain
a good volatilization. When the reaction was performed in the presence
of PB, the samples for spectrometric analysis were desalted by Zip-
TipC18 pipette tips (Millipore, USA). Binding and washing of the
protein or peptide on the Zip-Tip resin were performed using 0.1%
trifluoroacetic acid (TFA) in water, while the elution was performed
with 0.1% TFA in acetonitrile/water (50/50 v/v). This sample was further
diluted with pure water and then injected. Ionization was achieved in
the positive and negative ion modes by application of +3.5 kV at the en-
trance of the capillary; the pressure of the nebulizer gas was 35 psi. The
drying gas was heated to 300 °C at a flow rate of 8 L min−1. Full scan
mass spectra were recorded in the mass/charge (m/z) range of
100–3000. For all relevant peaks, it was checked that the experimental
isotopic pattern corresponded to the theoretical one.
2. Experimental section
2.1. Synthesis of the Pt(IV) complexes 1–4
Compounds 1–4 were prepared according to published proce-
dures [21,22]. Characterization and determination of the purity of
the complexes were performed by means of the usual analytical
techniques: elemental analysis, determination of Pt content by induc-
tively coupled plasma–optical emission spectroscopy (ICP–OES), HPLC,
electrospray ionization-mass spectrometry (ESI-MS) and multinuclear
NMR.
2.2. Reduction of 1 and interaction with model proteins
Complex 1 was dissolved in water at 1 mM concentration. The
solution was vortexed and sonicated at 40 °C for 15 min and the exact
Pt concentration was determined by atomic absorption spectroscopy
using a Varian 880Z instrument. Sodium ascorbate stock solution
(45 mM) was prepared from ascorbic acid (AA, 80 mg, 0.225 mmol) dis-
solved in H2O (10 mL) and neutralized with NaOH until pH 7.1. Mets7
peptide (MTGMKGMS) was purchased from GenScript (USA) and
was dissolved, immediately prior to use, in pure water at a 1 mM con-
centration. Mnk1 protein was recombinantly expressed by standard
biotechnological techniques, using Escherichia coli BL21DE3-Gold cells
(Stratagene, USA) and a pET vector under the control of the IPTG
(isopropyl-β-D-1-thiogalactopyranoside) inducible lac (lactose) pro-
moter (Novagen, USA). Purification was achieved by combining
immobilized metal affinity and size-exclusion chromatography, as pre-
viously described [23]. All steps of purification were carried out in the
2.3. Cell culture and viability tests
All the compounds under investigation were tested on the
human ovarian carcinoma cell line A2780, purchased from ECACC
(European Collection of Cell Cultures, UK). The cells were grown
in RPMI1640 medium supplemented with L-glutamine (2 mM),
penicillin (100 IU mL−1), streptomycin (100 mg L−1) and 10% fetal
bovine serum (FBS). Cell culture and the treatments were carried on
at 37 °C in a 5% CO2 humidified chamber. Cells were treated with the
compounds under investigation according to different schedules of con-
tact time (CT, the time for which cells are challenged with the drug

M. Ravera et al. / Journal of Inorganic Biochemistry 150 (2015) 1–8
3
under investigation dissolved in the culture medium) and recovery
(R, the time in which the cells are incubated in fresh, drug-free medi-
um) as indicated in the tables. Cisplatin was dissolved in 0.9% w/v
NaCl aqueous solution brought to pH 3 with HCl (final stock concentra-
tion 1 mM). All Pt(IV) complexes were dissolved in absolute ethanol
(final stock concentration 5 mM) and stored at −66 °C. The mother
solutions were diluted in complete medium, to the required concentra-
tion range. In the case of co-solvent the total absolute ethanol concen-
tration never exceeded 0.2% (this concentration was found to be non-
toxic to the tested cell). To assess the growth inhibition of the
compounds under investigation, a cell viability test, i.e. the resazurin re-
duction assay, was used [25]. Briefly, cells were seeded in black sterile
tissue-culture treated 96-well plates. At the end of the treatment, viabil-
ity was assayed by 10 μg mL−1 resazurin (Acros Chemicals, France) in
fresh medium for 1 h at 37 °C, and the amount of the reduced product,
resorufin, was measured by means of fluorescence (excitation λ =
535 nm, emission λ = 595 nm) with a Tecan Infinite F200Pro plate
reader (Tecan, Austria). In each experiment, cells were challenged
with the drug candidates at different concentrations and the final data
were calculated from at least three replicates of the same experiment
performed in triplicate. The fluorescence of 8 wells containing the me-
dium without cells was used as blank. Fluorescence data were normal-
ized to 100% cell viability for untreated (NT) cells. Half inhibiting
concentration (IC50), defined as the concentration of the drug reducing
cell viability by 50%, was obtained from the dose–response sigmoid
using Origin Pro (version 8, Microcal Software, Inc., Northampton, MA,
USA).
with a commercial kit (PerfectPure Cultured Mammalian Cells,
5Prime-Eppendorf), following the manufacturer's instructions. Briefly,
during cell lysis, DNA was purified by RNAse A and proteinase K
treatment, then extracted on silica-based centrifugation columns.
After washing, DNA was eluted in 300 μL of elution buffer. An amount
of 8 μL of sample or elution buffer (used as blank) was diluted in TE buff-
er (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) to 80 μL, corresponding to a
0.5 cm path length in UV-transparent microplate half-area wells (UV-
Star®, Greiner Bio-one). Absorbance at 260 nm (A260, relative to nucleic
acids) and 280 nm (A280, relative to proteins) was recorded from tripli-
cate wells with a Tecan Infinite F200Pro plate reader (Tecan, Austria).
For each well, A260 and A280 were corrected by subtraction of the back-
ground, then the purity of the samples was verified by means of the A260
to A280 ratio, that always resulted N2. After the subtraction of mean A260
of blank wells, the DNA concentration was computed from the corrected
A260 by means of a calibration curve obtained with calf thymus DNA.
Under these conditions an absorbance of 1 unit at 260 nm corresponds
to 100 μg of DNA per mL. The remaining amount of DNA elution buffer
was transferred into a borosilicate glass tube and its precise volume
determined by means of weight to compute the total amount of DNA,
then stored at −20 °C until mineralization. Mineralization and Pt
content determination were performed as above reported for the
whole cells.
The level of Pt found in cells after drug treatment and normalized
upon the cell number (cellular Pt accumulation) was expressed as ng
Pt per 106 cells. The mean cellular volume, calculated from the actual
mean cell diameter measured for every sample, was used to obtain
the cellular Pt concentration. The ratio between the cellular and actual
extracellular concentrations, is defined as accumulation ratio, AR [11].
The amount of Pt bound to DNA (experimental PDNA) was expressed
as pg of Pt per μg of DNA experimentally found. Unfortunately the DNA
recovery by the commercial kits is never quantitative especially when a
large number of cells is processed (in the actual case the yield was eval-
uated about 70%). This discrepancy can be overcome by taking into ac-
count that one million of human female diploid nuclei contain 6.55 μg
of DNA [26] and then calculating the overall amount of DNA-bound Pt
per million cells by multiplying experimental PDNA by 6.55:
2.4. Cellular accumulation and DNA platination
A2780 cells were seeded in 10 mm Petri dishes or 175 cm2 flasks and
treated with the complexes under investigations (10 μM) with the fol-
lowing schedules: 4 h CT, 24 h CT, or 4 h CT followed by 20 h R (4 h
CT + 20 h R). At the end of the exposure, cells were washed three
times with phosphate buffered saline (PBS), detached from the Petri
dishes using 0.05% Trypsin 1X + 2% EDTA (HyClone, Thermo Fisher)
and harvested in fresh complete medium. An automatic cell counting
device (Countess®, Life Technologies), was used to measure the num-
ber and the mean diameter from every cell count. From the same sam-
ple, about 5 × 106 cells were taken out for cellular accumulation
analysis, while about 20 × 106 cells were taken out for the DNA
platination analysis. Moreover, 100 μL of medium was taken out from
each sample at time zero to check the extracellular Pt concentration.
For the cellular Pt accumulation analysis, the cells were transferred
into a borosilicate glass tube and centrifuged at 1100 rpm for 5 min
at room temperature. The supernatant was carefully removed by
aspiration, while about 200 μL of the supernatant was left in order to
limit the cellular loss. Cellular pellets were stored at −80 °C until
mineralization.
Platinum content determination was performed by ICP-MS (Thermo
Optek X Series 2). Instrumental settings were optimized in order to
yield maximum sensitivity for platinum. For quantitative determina-
tion, the most abundant isotopes of platinum and indium (used as
internal standard) were measured at m/z 195 and 115, respectively.
Mineralization was performed by the addition of 70% w/w HNO3 to
each sample (after defrosting), followed by incubation for 1 h at 60 °C
in an ultrasonic bath. Before the ICP-MS measurement, the HNO3 was
diluted to a final 1% concentration.
ꢀ
ꢁ
ꢀ
ꢁ
pg Pt
106 cells
pg Pt
μg DNA
overall PDNA
¼ experimental PDNA
ꢀ
ꢁ
μg DNA
ꢀ 6:55
:
106 cells
Therefore, the percent ratio of Pt bound to DNA (PDNA %) is given by
the ratio between overall PDNA (in pg per 106 cells) and cellular Pt
accumulation (in ng per 106 cells) as follows:
ꢀ
ꢁ
pg Pt
overall PDNA
106 cells
ꢀ
ꢁ
P
DNA% ¼
ꢀ 100:
ng Pt
106 cells
1000 ꢀ cellular Pt accumulation
3. Results and discussion
3.1. Interaction of 1–4 complexes with copper transporters
Recently several publications have highlighted the role of trans-
porters in the active translocation of Pt(II) drugs across biological mem-
branes [27]. In particular at least 16 membrane transporter proteins
belonging to different families have been recognized as possible deter-
minants in the transport of platinum-based anticancer compounds.
Copper transporter 1 (Ctr1) is the major copper carrier operating via
facilitated diffusion or secondary active transport in mammals with an
important role in copper homeostasis. The protein contains two extra-
cellular, N-terminal, domains rich in methionines and one intracellular,
The cellular Pt accumulation was referred as ng Pt per 106 cells. In
order to obtain the Pt cellular concentration, the total cellular volume
of each sample was obtained considering the mean cell diameter and
cell number.
For the DNA platination analysis, the cells were transferred into a
plastic tube and centrifuged at 1100 rpm for 5 min at room temperature.
The supernatant was carefully removed by aspiration, and the cell pel-
lets were stored at −20 °C until the total genomic DNA was extracted

4
M. Ravera et al. / Journal of Inorganic Biochemistry 150 (2015) 1–8
C-terminal, domain rich in histidines which can act as metal binding
sites [28–30]. Many recombinant studies have provided strong evidence
for the role of Ctr1 in both platinum cellular accumulation and corre-
sponding antiproliferative activity [31–33].
increasing the absolute concentration of the reactants (0.75 mM
concentration of 1 and 7.5 mM concentration of reducing agent)
and the temperature (40 °C) [44]. The reaction of 1 with sodium
ascorbate was monitored by ESI-MS: the intensity of the peaks of
the Pt(IV) species decreased with time, until complete disappearance
after 24 h, while a rather weak peak at m/z 323.94, corresponding to
{[PtCl2(NH3)2] + Na}+, witnessed the formation of cisplatin. Thus the
Pt(IV) → Pt(II) reduction has occurred without any detectable scram-
bling of axial acetates with equatorial chlorides, as previously reported
for cisplatin-based Pt(IV) derivatives [7,18].
After reduction of 1, the solutions were treated with apoMets7 or
apoMnk1 to reach a Pt complex to protein ratio of 1:1 and a concentra-
tion of 100 μM for each reactant. The reaction solutions were kept at
37 °C and monitored by ESI-MS. After the addition of apoMets7, a new
peak corresponding to the doubly charged species {Mets7H+ + PtCl+}
(m/z 557.69) was observed right from the beginning. After 24 h, such
a species became prevalent together with the {Mets7 + Pt2+} cation
(m/z 539.71), meanwhile the intensity of the apo peptide peak greatly
decreased (Fig. 2). It is noteworthy that the Pt(II) species loses from
the early beginning both ammine ligands, which are replaced by methi-
onine residues of Mets7. The final adducts are identical to those ob-
served when Mets7 was reacted directly with cisplatin [32,45].
When apoMnk1 was incubated with a solution of 1 previously re-
duced with sodium ascorbate, it started to precipitate, causing the
On the contrary, ATP7A and ATP7B are membrane transporters that
translocate copper into the Trans-Golgi network and are involved in the
copper export achieved by relocation of the transporter on the cell
membrane [34]. The overexpression of ATP7A/B in tumor cells increases
their resistance to Pt(II) drugs [35]. The literature data about the
possible involvement of copper transporters in the mechanism of
influx/efflux of Pt(IV) complexes are limited to the observation that
[PtCl6]2− ion interacts with Ctr1 only upon reduction to Pt(II) [32].
To test the ability of Ctr1 and ATP7A to react directly with the Pt(IV)
complexes under investigation, two model proteins (i.e. Mets7, an octa-
peptide resembling one of the methionine-rich motifs present on the
extracellular N-terminal domain of Ctr1 [36], and Mnk1, the first cyto-
plasmic N-terminal domain of ATP7A [37]) were incubated with 1, cho-
sen as model because of its acceptable water solubility. The solution of
each protein was incubated with an equimolar amount of 1 (100 μM
final concentration) at 37 °C and the reaction course monitored by
ESI-MS. Soon after mixing complex 1 with apoMets7 or apoMnk1, the
ESI-MS spectra recorded in positive ion mode showed only the apo
species (peak of doubly charged peptide at m/z 442.73 and series of
multiply charged states of apoMnk1 at the expected m/z values, respec-
tively), whereas the spectra recorded in negative ion mode displayed
the peak at m/z 417.02 corresponding to unreacted complex {1-H}−
.
The reactions were monitored for 5 days and in no case there were
changes in the ESI-MS spectra which could witness the formation of ad-
ducts between Mets7 or Mnk1 and 1.
Although apoMets7 and apoMnk1 contain methionine or cysteine
residues, which have good coordinating ability towards platinum, they
are unable to react with 1 nor they are able to reduce Pt(IV) to Pt(II)
complexes, while such a reduction takes place readily with isoelectronic
Au(III) complexes [38,39]. Therefore, the interaction with model pro-
teins was repeated in the presence of GSH and AA, the biomolecules
usually employed to test the reduction of Pt(IV) prodrugs to Pt(II) spe-
cies [17,32,40].
AA is known to be the primary, low molecular weight, antioxidant
present in living organisms. Considering its two pKa values (pK1
=
3.95 and pK2 = 11.24) at physiological pH the prevailing species
(99.9%) is the ascorbate monoanion. This can undergo two one-
electron losses leading to the formation of dehydroascorbic acid [41].
Ascorbate is present at lower concentration in the blood plasma
(40–80 μM) than in the cytoplasm of human neutrophils (about
1 mM). The tripeptide glutathione, γ-glutamylcysteinylglycine, is the
most relevant non-protein, thiol found in all mammalian tissues. Gluta-
thione exists in the thiol-reduced (GSH) and disulfide-oxidized (GSSG)
forms. GSH is the predominant form, existing in millimolar concentra-
tions in most cells (0.5–10 mM), whereas GSSG content is less than 1%
of GSH [42]. About 85–90% of cellular GSH is in the cytosol, 10% is in mi-
tochondria and a small percent is in the endoplasmic reticulum. GSH, a
one electron reducing agent, is one of the primary defenses against
toxins and oxidants in the cell, and can deactivate electrophilic drugs,
including Pt drugs [43].
Since the octapeptide apoMets7 resembles the methionine-rich
motif of the extracellular N-terminal domains of Ctr1, the interaction
with 1 (100 μM) under reducing conditions was performed by incuba-
tion with a 10-fold excess of sodium ascorbate. On the contrary, in the
case of apoMnk1, the interaction with 1 under reducing conditions
was performed using a 10-fold excess of GSH. The peptide/protein to
complex ratio was always 1:1 and the reactions were performed at
37 °C in 25 mM neutral PB and monitored over time with ESI-MS.
Despite the presence of a reducing agent, no reaction took place even
after 5 days of incubation. ESI-MS spectra showed only the peaks
relative to apoMets7 or apoMnk1 (positive ions mode) and 1 (negative
ions mode). Therefore, the reaction conditions were forced by
Fig. 2. ESI-MS spectra of apoMets7 treated with 1 (previously reduced with sodium ascor-
bate) recorded after 1 h (A) and 24 h (B) of incubation.

M. Ravera et al. / Journal of Inorganic Biochemistry 150 (2015) 1–8
5
progressive disappearance of its ESI-MS peaks, without the appearance
of new peaks indicative of the formation of soluble adducts with Pt.
The formation of adducts with “naked” Pt was already observed in
the case of direct reaction between cisplatin and apoMets7 or apoMnk1
[32,46]. In contrast, in the case of oxaliplatin, that contains the more sta-
ble chelating cyclohexane-1R,2R-diamine (dach) ligand, the “Pt(dach)”
moiety was maintained intact after interaction with the model proteins
[47]. For this reason a complex similar to 1, but containing the dach
ligand in place of the two ammine molecules (namely trans,cis,cis-
[Pt(acetato)2Cl2(dach)]) was tested in a similar experiment. Similarly
to the case of 1, no direct reaction took place with both model proteins.
However, after reduction with sodium ascorbate, the reaction with
apoMets7 showed decrease of the starting apo protein (complete
disappearance after 48 h) and concomitant increase of peaks corre-
sponding to {Mets7 + Pt(dach)2+} (doubly charged adduct, m/z
596.71) and {Mets7H+ + Pt(dach)2+} (triply charged adduct, m/z
398.15) (Fig. S1, see the Supplementary data). When Mnk1 was reacted
with trans,cis,cis-[Pt(acetato)2Cl2(dach)], previously reduced with
sodium ascorbate, a slight precipitation of protein occurred. However,
in this case, it was possible to observe, already after about 3 h of incuba-
tion, the appearance of new signals corresponding to adduct formation
between the protein and {Pt(dach)Cl}+ or {Pt(dach)}2+ moieties. With
time the intensity of signals corresponding to the monodentate
adduct {Mnk1–[Pt(dach)Cl]+} {Mnk1–[Pt(dach)Cl]+} (m/z 1097.55)
decreased while the signals corresponding to the bidentate adduct
{Mnk1–[Pt(dach)]2+} (m/z 1101.78) increased (Fig. S2, see the Supple-
mentary data). This two-step reaction mechanism is peculiar of Mnk1
[46].
pinocytosis. Cellular accumulation affects the activity of cisplatin: a de-
crease in Pt accumulation, due either to decrease in uptake or increase
in efflux, is one of the main causes of chemoresistance [51,52].
Instead of using the cellular Pt accumulation (ng of Pt per 106 cells),
the accumulation ratio (AR), a concept similar to the “accumulation
grade of factor” previously defined by Gust et al. [53], was employed
in the present paper. AR is the dimensionless ratio between the cellular
Pt concentration (taking into account the experimentally measured cell
volume) and the extracellular Pt concentration (i.e. the actual concen-
tration of Pt in the culture medium) [11].
The AR value of 1–4 and cisplatin (all employed at 10 μM concentra-
tion) was measured in A2780 cells after i) 4 h CT, ii) 24 h CT, and iii) 4 h
CT followed by 20 h R in fresh, drug-free complete medium. In the case
of Pt(IV) complexes, all AR values increase with log Po/w, as well as with
the length of the axial carbon chains, as expected for passively diffusing
molecules [40].
As shown in Table 1, the AR of cisplatin increased from 4 h CT to 24 h
CT. However, cisplatin accumulation significantly dropped when, after
4 h CT, the incubation was prolonged in drug-free medium for further
20 h (4 h CT + 20 h R), indicating an extensive, albeit incomplete, efflux
process.
Also in the case of complexes 1–3, the AR progressively increased
when the CT was prolonged up to 24 h. On the contrary, the AR of 4
was quite similar (p N 0.05, two-sample t-test) in the three experimen-
tal conditions (i.e. AR ≅ 50). This saturation plateau suggests a limit in
the storing of 4 within the viable cells.
The effect of recovery is different for the four Pt(IV) complexes. Sim-
ilarly to cisplatin, the less lipophilic compounds 1 and 2 show a signifi-
cant decrease in AR upon incubation in drug-free medium, whereas the
AR value is substantially maintained for 3 and 4.
Data in Table 1 also show that, for a given incubation time, the AR in-
creases in the following order: 1 b cisplatin ≤ 2 b 3 b 4. The diacetato 1
and the dibutanoato 2 exhibit an AR lower than and equal to that of cis-
platin, respectively. It is possible to hypothesize that for 1 and 2 the lack
of influx protein-mediated transport (e.g. Ctr1, operating for cisplatin
[32], but not for intact Pt(IV) complexes) is not fully compensated by
the increase in passive diffusion (the only uptake process occurring for
Pt(IV) derivatives) due to their modest lipophilicity, whereas the oppo-
site is true for 3 and, especially, for 4.
The decrease in AR observed for cisplatin during recovery is related
to the already described efflux transporters. Previous studies demon-
strated that intact cisplatin is the predominant species in the cytosolic
low molecular weight fraction, but it is nearly completely cleared
upon 1 h of recovery by means of extrusion through the P-type
copper-transporters [54]. The remaining fraction is mainly sequestered
by the sulfur proteins, GSH [55], and by other high MW molecules (i.e.
RNA). However, since platinum adducts are slowly extruded from the
cells [56,57], the total amount of accumulated Pt increases with the du-
ration of the contact time (from 4 to 24 h CT). Moreover, a significant
amount of Pt remains inside the cells also during the recovery period.
Since Pt(IV) complexes act as prodrugs for cisplatin [17], it is
intended that reduction products undergo the same fate as cisplatin it-
self. As far as Pt(IV) complexes are not a substrate for the extruding cop-
per transporter, the accumulated total Pt should be the net result
between the Pt(IV) uptake and the efflux of the Pt(II) metabolites. Ac-
cording to previous 13C NMR and X-ray absorption near edge spectros-
copy (XANES) studies performed on A2780 cells for 1 [6,58], Pt(IV)
complexes are significantly reduced to cisplatin within 4 h and
completely reduced after 24 h. Complexes 2–4 showed similar reduc-
tion kinetics than 1 when challenged with AA at physiological pH [40].
Therefore, it is conceivable that during the recovery period, platinum
detoxification mainly involves the Pt(II) metabolites. Unfortunately,
ICP-MS determines only the total amount of Pt, without distinguishing
between its oxidation states. Noteworthy, when the initial AR of
Pt(IV) compounds was similar to that of cisplatin, also the efflux had a
similar efficiency; on the contrary, for compounds with high AR (i.e. 3
In conclusion, the reported experiments suggest that Pt(IV) com-
plexes, differently from Pt(II) complexes, are not substrates for copper
transporters strengthening the idea that they enter the cells by passive
diffusion.
3.2. Lipophilicity and cellular accumulation of 1–4 complexes on A2780 cell
line
Cellular accumulation represents the balance at a given time be-
tween cellular influx and efflux and is an important factor determining
the biological activity of any drug [48]. The lipophilicity of a drug is a key
feature directly related to its ability to passively cross cellular mem-
branes [49,50]. Generally, the shake-flask log Po/w, in which n-octanol
is taken as a model of the lipid bilayer of a cell membrane and water
mimics the extracellular environment, is used to represent the lipophi-
licity of a molecule. The log Po/w values of complexes 1–4 are reported
in Table 1, along with that of cisplatin. Even for such a limited number
of compounds, it is possible to observe a linear relationship between
log Po/w and the number of carbon atoms in the chain of axial ligands
[16,21].
The mechanism by which the prototype cisplatin permeates cells is
certainly multifactorial, as a result of a series of different processes
such as passive diffusion, carrier-mediated transport and, possibly,
Table 1
AR of A2780 cells treated with 10 μM concentration of the platinum complexes. Data are
means standard deviations of at least 3 independent replicates and were compared to
those obtained for each drug after 4 h CT by means of the two sample t-test (*p b
0.05;**p b 0.01; ***p b 0.001; NS = value not statistically different from control sample).
Compound
log Po/w
Accumulation ratio, AR
4 h CT
4 h CT + 20 h R
24 h CT
Cisplatin
−2.27a
−1.92b
−0.39b
1.14b
1.4 0.6
0.3 0.1
2.3 0.8
20.7 4.6
41.0 8.5
0.7 0.2 (*)
0.08 0.02 (**)
1.8 0.2 (*)
15.2 3.0 (NS)
46.8 9.1 (NS)
4.5 1.1 (***)
0.7 0.2 (**)
16.3 5.6 (***)
53.0 16.9 (***)
59.6 14.7 (NS)
1
2
3
4
4.1b
a
From Ref. [22].
From Ref. [40].
b

6
M. Ravera et al. / Journal of Inorganic Biochemistry 150 (2015) 1–8
Table 3
and 4) the detoxification process became less effective. Thus, unlike
complexes 1 and 2 for which the excretion from A2780 cells during
the recovery period was around 70% and 20%, respectively, the overall
efflux was almost negligible in the case of compounds 3 and 4.
% of Pt bound to DNA (PDNA %, i.e. the ratio between PDNA and cellular Pt accumulation mul-
tiplied by 100). Data are means standard deviations of at least 3 independent replicates.
Compound
PDNA %
4 h CT
4 h CT + 20 h R
24 h CT
3.3. DNA platination of A2780 cells treated with complexes 1–4
Cisplatin
4.6 1.4
1.0 0.3
0.7 0.3
1.6 0.4
1.9 0.2
ND
0.8 0.1
8.0 4.0
1.4 0.1
0.6 0.2
1.4 0.6
10.9 3.7
2
3
4
To determine the level of DNA platination (PDNA) after treatment of
A2780 cells with compounds 1–4 and cisplatin, each sample was divid-
ed into two aliquots: one was used for AR determination and the other
for DNA extraction. PDNA was measured by means of ICP-MS and
expressed as pg of Pt per μg of DNA (Table 2). Statistically significant
values of PDNA were not obtained in the case of 1 for which the Pt
amount was found similar to that of the untreated control (NT), in
spite of using a large number of cells. Cisplatin gave the same PDNA
(p N 0.05) at 4 and 24 h CT, but the PDNA dropped to lower values
when, after incubation with the drug for 4 h, cells were incubated in
drug-free medium for further 20 h (4 h CT + 20 h R).
ND = not determined since PDNA for this sample is not statistically different from the un-
treated control.
PDNA % again around 1%. In contrast, the most lipophilic compound 4 ex-
hibited the highest PDNA %, (more than 10%). Interestingly, 4 is poorly
detoxified by the cells and maintains a high platination level even
after drug removal from the culture medium (PDNA % = 8%).
3.4. In vitro antiproliferative activity
It has been previously shown that PDNA of cisplatin is proportional to
the external concentration, but not to the duration of the incubation
[59]. Accordingly, the present data confirm that PDNA caused by cisplatin
was similar for 4 and 24 h CT (Table 2); the found value is similar to that
previously reported for ovarian cancer cells treated with 7 μM cisplatin
(241 Pt atoms per 106 nucleotides correspond to 16 pg of Pt per μg of
DNA) [55]. Thus, PDNA represents the result, at a given time, of the equi-
librium between DNA platination and DNA repair. The removal of Pt-
DNA adducts starts within 2 h [55], a time-lapse comparable to the
first aquation reaction, which is the rate-delimiting step for platination
by cisplatin [60]. Moreover, as previously reported [61], an efficient re-
moval of the Pt-DNA adducts is observed after post-incubation in drug-
free medium for 20 h (Table 2). Pt(IV) complexes generate the same
metabolites as cisplatin, however their formation is delayed by the re-
duction kinetics. In general, the trend of PDNA parallels that of AR
(Tables 1 and 2). Accordingly, PDNA by 2–4 significantly increased from
4 to 24 h CT. The recovery period in drug-free medium had different ef-
fects, PDNA was significantly lowered for complex 2 (probably as a result
of the combination between Pt efflux and DNA repair); on the contrary,
PDNA was unchanged in the case of 3 and even increased in the case of 4.
The percent ratio between overall PDNA and cellular Pt accumulation
(both referred to one million cells, see Experimental section) is reported
as PDNA % (Table 3). PDNA % strongly depends on the cell line employed
and its status, the compound under study, its concentration, and the
time scale of the experiment. Therefore, for a meaningful comparison
all the experiments were performed in strictly analogous conditions.
For cisplatin, the amount of DNA-bound platinum remains almost
unchanged, while the cellular Pt content increases with the treatment
time, mainly for sequestration of cisplatin by off-target biomolecules
such as S-proteins and RNA, and consequently PDNA % decreases with
time. In our experimental conditions (i.e. 24 h CT), PDNA % for cisplatin
was around 1%, in agreement with the common belief that approxi-
mately 1% of the cellular Pt reacts with genomic DNA [62–64]. As far
as the Pt(IV) complexes are concerned, 2 and 3 exhibited values of
In the attempt to correlate the biological effects with the AR and
PDNA values, the residual cell viability was evaluated under strictly anal-
ogous conditions. A2780 cells were treated with cisplatin and 1–4 for
4 h CT, 4 h CT + 20 h R, or 24 h CT and the residual viability was mea-
sured at the end of the experiments by means of a cell viability assay
(Fig. 3). After 4 h CT no significant changes were observed in terms of
viability as expected for genotoxic agents, that require considerable
amount of time to manifest their action. On the contrary, the two longer
treatment schedules lowered the viability.
The biological effect (growth inhibition) of Pt-based drugs depends
both on the amount of DNA platination and activation of DNA repair sys-
tems [65], that lead to different cellular fates [66,67]. It is found that for
different Pt(IV) complexes and different treatment times the residual
viability of A2780 cells decreases exponentially with the increase of
DNA platination (Fig. 4).
4. Conclusions
In the present work, a direct relationship between lipophilicity, cel-
lular accumulation, DNA platination and antiproliferative activity in a
series of Pt(IV) candidate prodrugs has been found. Unlike cisplatin,
Pt(IV) complexes are not substrates for copper transporter systems,
therefore Pt(IV) prodrugs (1–4) are internalized by cells through pas-
sive diffusion and their efflux is unaided by transport proteins [32,68,
69]. For complexes 1 and 2, having log Po/w similar to cisplatin, the
lack of protein-mediated transport is not fully compensated by the
Table 2
Overall Pt bound to DNA (PDNA) of A2780 cells treated with 10 μM concentration of the
platinum complexes for 4 h CT, 4 h CT + 20 h R and 24 h CT. Data are means
standard deviations of at least 3 independent replicates and were compared to those ob-
tained for each drug after 4 h CT by means of the two sample t-test (*p b 0.05;**p b
0.01; ***p b 0.001; NS = value not statistically different from control sample).
Compound
PDNA [pg of Pt per μg of DNA]
4 h CT
4 h CT + 20 h R
24 h CT
15.1 3.3 (NS)
22.3 10.9 (NS)
156.3 81.0 (**)
Cisplatin
21.4 10.9
4.9 1.1
38.5 21.7
113.0 41.4
4.8 1.1 (**)
1.9 0.1 (**)
36.4 0.5 (NS)
506.1 114.4 (***)
Fig. 3. Viability (%) of A2780 cells treated with 10 μM of the platinum complexes. The re-
sidual cell number was recorded after 4 h CT (black bars), 4 h CT + 20 h R (gray bars); and
2
3
4
24 h CT (white bars). Data are means
replicates.
standard deviations of at least 3 independent
1697.6 571.8 (***)

M. Ravera et al. / Journal of Inorganic Biochemistry 150 (2015) 1–8
7
R
recovery in fresh, drug-free medium
RPMI1640 Roswell Park Memorial Institute medium
TE buffer Tris–HCl/EDTA buffer
TFA
trifluoroacetic acid
Acknowledgments
This research was carried out within the framework of the European
Union CMST COST Action CM1105 “Functional metal complexes that
bind to biomolecules” and the Inter-University Consortium for Research
on the Chemistry of Metal Ions in Biological Systems (CIRCMSB, Bari,
Italy). We thank Fondazione CRA (Alessandria) and Fondazione CRT
(Torino) for the financial support within the project “Approcci
chemioterapici innovativi per il mesotelioma maligno della pleura”.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the
online version, at http://dx.doi.org/10.1016/j.jinorgbio.2015.05.012.
These data include: ESI-MS spectra of apoMets7 and apoMnk1 treated
with [Pt(acetato)2Cl2(dach)].
Fig. 4. Viability (%) of A2780 cells versus log PDNA of 2 (24 h CT), 3 and 4 (4 h CT + 20 h R
and 24 h CT). Four parameter logistic dose–response curve fit, R2 = 0.995, data are
means standard deviations of at least 3 independent replicates.
increase in passive diffusion. This results in lower/similar AR and hence
lower/similar biological activity with respect to cisplatin. On the con-
trary, for 3 and, especially, for 4 the high lipophilicity enormously favors
the passive diffusion resulting in very high AR, PDNA and cell growth
inhibition.
Interestingly, for compound 4 PDNA increases steeply with the con-
tact time and remains almost constant with post-incubation in drug-
free medium, indicating efficient storing and poor detoxification. A sim-
ilar result was recently obtained during the treatment of multicellular
tumor spheroids with complexes 1–4 [40]. This suggests that sequestra-
tion inside cells, possibly in some lipophilic subcellular compartments,
may be not per se a negative effect on the antiproliferative activity [70,
71]. Thus, the most lipophilic complexes of the title series appear to be
ideally suited for bypassing cisplatin resistance, when it relies on re-
duced cellular accumulation [72].
References
[1] C.M. Sorenson, A. Eastman, Cancer Res. 48 (1998) 4484–4488.
[2] M.D. Hall, H.R. Mellor, R. Callaghan, T.W. Hambley, J. Med. Chem. 50 (2007)
3403–3411.
[3] M.D. Hall, R.A. Alderden, M. Zhang, P.J. Beale, Z. Cai, B. Lai, A.P.J. Stampfl, T.W.
Hambley, J. Struct. Biol. 155 (2006) 38–44.
[4] E. New, J.R. Duan, J.Z. Zhang, T.W. Hambley, Dalton Trans. (2009) 3092–3101.
[5] Y. Song, K. Suntharalingam, J.S. Yeung, M. Royzen, S.J. Lippard, Bioconjug. Chem. 24
(2013) 1733–1740.
[6] A. Nemirovski, Y. Kasherman, Y. Tzaraf, D. Gibson, J. Med. Chem. 50 (2007)
5554–5556.
[7] A. Nemirovski, I. Vinograd, K. Takrouri, A. Mijovilovich, A. Rompel, D. Gibson, Chem.
Commun. (2010) 1842–1844.
[8] M. Ravera, E. Gabano, I. Zanellato, I. Bonarrigo, E. Escribano, V. Moreno, M. Font-
Bardia, T. Calvet, D. Osella, Dalton Trans. 41 (2012) 3313–3320.
[9] T. Fojo, N. Farrell, W. Ortuzar, H. Tanimura, J. Weinstein, T.G. Myers, Crit. Rev. Oncol.
Hematol. 53 (2005) 25–34.
[10] J.A. Platts, D.E. Hibbs, T.W. Hambley, M.D. Hall, J. Med. Chem. 44 (2001) 472–474.
[11] A.R. Ghezzi, M. Aceto, C. Cassino, E. Gabano, D. Osella, J. Inorg. Biochem. 98 (2004)
73–78.
This study confirms the feasibility of obtaining highly lipophilic
Pt(IV) anticancer prodrug candidates by using long-chain aliphatic car-
boxylates as axial ligands. On the contrary, in satraplatin and LA-12, the
Pt(IV) prodrugs under clinical trials at the moment, is the equatorial
amine (i.e. cyclohexylamine and adamantylamine, respectively) that
guarantees a high lipophilicity.
[12] M.D. Hall, S. Amjadi, M. Zhang, P.J. Beale, T.W. Hambley, J. Inorg. Biochem. 98 (2004)
1614–1624.
[13] W.H. Ang, S. Pilet, R. Scopelliti, F. Bussy, L. Juillerat-Jeanneret, P.J. Dyson, J. Med.
Chem. 48 (2005) 8060–8069.
[14] E. Gabano, D. Colangelo, A.R. Ghezzi, D. Osella, J. Inorg. Biochem. 102 (2008)
629–635.
[15] A.M. Montaña, C. Batalla, Curr. Med. Chem. 16 (2009) 2235–2260.
[16] P. Gramatica, E. Papa, M. Luini, E. Monti, M.B. Gariboldi, M. Ravera, E. Gabano, L.
Gaviglio, D. Osella, J. Biol. Inorg. Chem. 15 (2010) 1157–1169.
[17] E. Wexselblatt, D. Gibson, J. Inorg. Biochem. 117 (2012) 220–229.
[18] M. Ravera, E. Gabano, S. Bianco, G. Ermondi, G. Caron, M. Vallaro, G. Pelosi, I.
Zanellato, I. Bonarrigo, C. Cassino, D. Osella, Inorg. Chim. Acta 432 (2015) 115–127.
[19] S.R. Martens-de Kemp, S.U. Dalm, F.M.J. Wijnolts, A. Brink, R.J. Honeywell, G.J. Peters,
B.J.M. Braakhuis, R.H. Brakenhoff, PLoS ONE 8 (2013) e61555.
[20] L. Martelli, F. Di Mario, P. Botti, E. Ragazzi, M. Martelli, L. Kelland, Biochem.
Pharmacol. 74 (2007) 20–27.
Abbreviations
AA
ascorbic acid
AR
BSA
CT
dach
DTT
accumulation ratio
bovine serum albumin
contact time with the drug
cyclohexane-1R,2R-diamine
dithiothreitol
[21] J.A. Platts, G. Ermondi, G. Caron, M. Ravera, E. Gabano, L. Gaviglio, G. Pelosi, D. Osella,
J. Biol. Inorg. Chem. 16 (2011) 361–372.
[22] J.A. Platts, S.P. Oldfield, M.M. Reif, A. Palmucci, E. Gabano, D. Osella, J. Inorg. Biochem.
100 (2006) 1199–1207.
[23] A. Galliani, M. Losacco, A. Lasorsa, G. Natile, F. Arnesano, J. Biol. Inorg. Chem. 19
ESI-MS electrospray ionization-mass spectrometry
FBS
fetal bovine serum
GSH
GSSG
IC50
reduced glutathione, γ-glutamylcysteinylglycine
disulfide-oxidized form of GSH
half maximal inhibition concentration
(2014) 705–714.
[24] V.B. Di Marco, G.G. Bombi, Mass Spectrom. Rev. 25 (2006) 347–379.
[25] E. Magnani, E. Bettini, Brain Res. Protocol. 5 (2000) 266–272.
[26] J. Dolezel, J. Bartos, H. Voglmayr, J. Greilhuber, Cytometry A 51 (2003) 127–128.
[27] J.J. Liu, J. Lu, M.J. McKeage, Curr. Cancer Drug Targets 12 (2012) 962–986.
[28] B. Zhou, J. Gitschier, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 7481–7486.
[29] J. Lee, M.M.O. Pena, Y. Nose, D.J. Thiele, J. Biol. Chem. 277 (2002) 4380–4387.
[30] A.K. Holzer, N.M. Varki, Q.T. Le, M.A. Gibson, P. Naredi, S.B. Howell, J. Histochem.
Cytochem. 54 (2006) 1041–1049.
ICP-MS inductively coupled plasma-mass spectrometry
ICP-OES inductively coupled plasma–optical emission spectroscopy
Po/w
NT
n-octanol/water partition coefficient
untreated cells
PB
phosphate buffer
[31] A.K. Holzer, G.H. Manorek, S.B. Howell, Mol. Pharmacol. 70 (2006) 1390–1394.
[32] F. Arnesano, S. Scintilla, G. Natile, Angew. Chem. Int. Ed. 119 (2007) 9220–9222.
[33] C.A. Larson, B.G. Balir, R. Safaei, S.B. Howell, Mol. Pharmacol. 75 (2009) 324–330.
[34] S. Lutsenko, N.L. Barnes, M.Y. Bartee, O.Y. Dmitriev, Physiol. Rev. 87 (2007)
1011–1046.
PBS
PDNA
PDNA
phosphate buffered saline
Pt bound to DNA (platination)
percentage of Pt bound to DNA
%

8
M. Ravera et al. / Journal of Inorganic Biochemistry 150 (2015) 1–8
[35] C.A. Rabik, E.B. Maryon, K. Kasza, J.T. Shafer, C.M. Bartnik, M.E. Dolan, Cancer
Chemother. Pharmacol. 64 (2009) 133–142.
[36] J. Jiang, I.A. Nadas, M.A. Kim, K.J. Franz, Inorg. Chem. 44 (2005) 9787–9794.
[37] T.M. DeSilva, G. Veglia, S.J. Opella, Proteins 61 (2005) 1038–1049.
[38] G. Annibale, L. Canovese, L. Cattalini, G. Natile, J. Chem. Soc. Dalton Trans. (1980)
1017–1021.
[55] P.D. Sadowitz, B.A. Hubbard, J.C. Dabrowiak, J. Goodisman, K.A. Tacka, M.K. Aktas,
M.J. Cunningham, R.L. Dubowy, A.-K. Souid, Drug Metab. Dispos. 30 (2002)
183–190.
[56] S.C. Mann, P.A. Andrews, S.B. Howell, Cancer Chemother. Pharmacol. 25 (1990)
236–240.
[57] R.P. Sharma, I.R. Edwards, Biochem. Pharmacol. 32 (1983) 2665–2669.
[58] M.D. Hall, G.J. Foran, M. Zhang, P.J. Beale, T.W. Hambley, J. Am. Chem. Soc. 125
(2003) 7524–7525.
[39] E. Bordignon, L. Cattalini, G. Natile, A. Scatturin, J. Chem. Soc. Chem. Commun.
(1973) 878–879.
[40] I. Zanellato, I. Bonarrigo, D. Colangelo, E. Gabano, M. Ravera, M. Alessio, D. Osella,
J. Inorg. Biochem. 140 (2014) 219–227.
[59] M. Akaboshi, K. Kawai, H. Maki, K. Akuta, Y. Ujeno, T. Miyahara, Jpn. J. Cancer Res. 83
(1992) 522–526.
[41] M.D. Hall, T.W. Hambley, Coord. Chem. Rev. 232 (2002) 49–67.
[42] T.P. Akerboom, M. Bilzer, H. Sies, J. Biol. Chem. 257 (1982) 4248–4252.
[43] G.Y. Wu, Y.Z. Fang, S. Yang, J.R. Lupton, N.D. Turner, J. Nutr. 134 (2004) 489–492.
[44] S. Choi, C. Filotto, M. Bisanzo, S. Delaney, D. Lagasee, J.L. Whitworth, A. Jusko, C.R. Li,
N.A. Wood, J. Willingham, A. Schwenker, K. Spaulding, Inorg. Chem. 37 (1998)
2500–2504.
[45] S.E. Crider, R.J. Holbrook, K.J. Franz, Metallomics 2 (2010) 74–83.
[46] F. Tadini-Buoninsegni, G. Bartolommei, M.R. Moncelli, G. Inesi, A. Galliani, M. Sinisi,
M. Losacco, G. Natile, F. Arnesano, Angew. Chem. Int. Ed. 53 (2014) 1297–1301.
[47] F. Arnesano, M. Losacco, G. Natile, Eur. J. Inorg. Chem. (2013) 2701–2711.
[48] E. Lindauer, E. Holler, Biochem. Pharmacol. 52 (1996) 7–14.
[49] G. Caron, G. Ermondi, Lipophilicity: chemical nature and biological relevance, in: R.
Mannhold (Ed.), , Molecular Drug Properties Measurement and Prediction, Methods
and Principles in Medicinal Chemistry, vol. 37, Wiley-VCH, Weinheim, 2008
(ch. 12).
[60] M.S. Davies, S.J. Berners-Price, T.W. Hambley, Inorg. Chem. 39 (2000) 5603–5613.
[61] A.M. Fichtinger-Schepman, S.D. van der Velde-Visser, H.C. van Dijk-Knijnenburg,
A.T. van Oosterom, R.A. Baan, F. Berends, Cancer Res. 50 (1990) 7887–7894.
[62] A. Eastman, N. Schulte, Biochemistry 27 (1988) 4730–4734.
[63] A. Eastman, N. Schulte, N. Sheibani, C.M. Sorenson, Mechanisms of resistance to
platinum drugs, in: M. Nicolini (Ed.), Platinum and Other Metal Coordination
Compounds in Cancer Chemotherapy, Martinus Nijhoff Publishing, Boston 1988,
pp. 178–196.
[64] C.K. Chen, J.Z. Zhang, J.B. Aitken, T.W. Hambley, J. Med. Chem. 56 (2013) 8757–8764.
[65] D. Fu, J.A. Calvo, L.D. Samson, Nat. Rev. Cancer 12 (2012) 104–120.
[66] S. Nowsheen, E.S. Yang, Exp. Oncol. 34 (2012) 243–254.
[67] A. Biroccio, M. Porru, A. Rizzo, E. Salvati, C. D'Angelo, A. Orlandi, D. Passeri, M.
Franceschin, M.F.G. Stevens, E. Gilson, G. Beretta, G. Zupi, C. Pisano, F. Zunino, C.
Leonetti, Clin. Cancer Res. 17 (2011) 2227–2236.
[68] P. Mistry, L.R. Kelland, S.Y. Loh, G. Abel, B.A. Murrer, K.R. Harrap, Cancer Res. 52
[50] X. Liu, B. Testa, A. Fahr, Pharm. Res. 28 (2011) 962–977.
[51] E. Gabano, M. Ravera, D. Colangelo, D. Osella, Curr. Chem. Biol. 1 (2007) 278–289.
[52] P. Heffeter, U. Jungwirth, M. Jakupec, C. Hartinger, M. Galanski, L. Elbling, M.
Micksche, B. Keppler, W. Berger, Drug Resist. Updat. 11 (2008) 1–16.
[53] T. Kapp, A. Dullin, R. Gust, Bioconjug. Chem. 21 (2010) 328–337.
[54] G. Hermann, P. Heffeter, T. Falta, W. Berger, S. Hann, G. Koellensperger, Metallomics
5 (2013) 636–647.
(1992) 6188–6193.
[69] S.Y. Sharp, P.M. Rogers, L.R. Kelland, Clin. Cancer Res. 1 (1995) 981–989.
[70] K. Suntharalingam, Y. Song, S.J. Lippard, Chem. Commun. 50 (2014) 2465–2468.
[71] V. Novohradsky, L. Zerzankova, J. Stepankova, O. Vrana, R. Raveendran, D. Gibson, J.
Kasparkova, V. Brabec, J. Inorg. Biochem. 140 (2014) 72–79.
[72] D.-W. Shen, L.M. Pouliot, M.D. Hall, M.M. Gottesman, Pharmacol. Rev. 64 (2012)
706–721.