Cisplatin and its dibromido analogue: a comparison of chemical and biological profiles

Article abstract of DOI:10.1007/s10534-016-9934-4

The dibromido analogue of cisplatin, cis-PtBr₂(NH₃)₂ (cisPtBr₂ hereafter), has been prepared and characterised. Its solution behaviour in standard phosphate buffer, at pH 7.4, was investigated spectrophotometrically and found to reproduce quite closely that of cisplatin; indeed, progressive sequential release of the two halide ligands typically occurs as in the case of cisplatin, with a roughly similar kinetics. Afterward, patterns of reactivity toward model proteins and standard ctDNA were explored and the nature of the resulting interactions elucidated. The antiproliferative properties were then evaluated in four representative cancer cell lines, namely A549 (human lung cancer), HCT116 (human colon cancer), IGROV-1 (human ovarian cancer) and FLG 29.1 (human acute myeloid leukaemia). Cytotoxic properties in line with those of cisplatin were highlighted. From these studies an overall chemical and biological profile emerges for cisPtBr₂ closely matching that of cisplatin; the few slight, but meaningful differences that were underscored might be advantageously exploited for clinical application.

Full text of DOI:10.1007/s10534-016-9934-4

Biometals
DOI 10.1007/s10534-016-9934-4
Cisplatin and its dibromido analogue: a comparison
of chemical and biological profiles
.
Tiziano Marzo Gianluca Bartoli Chiara Gabbiani Gennaro Pescitelli
.
.
.
.
.
.
Mirko Severi Serena Pillozzi Elena Michelucci Benedetta Fiorini
.
.
Annarosa Arcangeli Adoracion G. Quiroga Luigi Messori
.
´
Received: 1 April 2016 / Accepted: 5 April 2016
Ó Springer Science+Business Media New York 2016
Abstract The dibromido analogue of cisplatin, cis-
PtBr2(NH3)2 (cisPtBr2 hereafter), has been prepared
and characterised. Its solution behaviour in standard
phosphate buffer, at pH 7.4, was investigated spec-
trophotometrically and found to reproduce quite
closely that of cisplatin; indeed, progressive sequential
release of the two halide ligands typically occurs as in
the case of cisplatin, with a roughly similar kinetics.
Afterward, patterns of reactivity toward model pro-
teins and standard ctDNA were explored and the
nature of the resulting interactions elucidated. The
antiproliferative properties were then evaluated in four
representative cancer cell lines, namely A549 (human
lung cancer), HCT116 (human colon cancer), IGROV-
1 (human ovarian cancer) and FLG 29.1 (human acute
myeloid leukaemia). Cytotoxic properties in line with
those of cisplatin were highlighted. From these studies
an overall chemical and biological profile emerges for
cisPtBr2 closely matching that of cisplatin; the few
slight, but meaningful differences that were under-
scored might be advantageously exploited for clinical
application.
Keywords Cancer Á Platinum Á Cisplatin analogues Á
Mass spectrometry Á Circular dichroism
Electronic supplementary material The online version of
this article (doi:10.1007/s10534-016-9934-4) contains supple-
mentary material, which is available to authorized users.
T. Marzo (&) Á B. Fiorini Á L. Messori (&)
Laboratory of Metals in Medicine (MetMed), Department
of Chemistry, University of Florence, Via della Lastruccia
3, 50019 Sesto Fiorentino, Italy
M. Severi
Department of Chemistry, University of Florence, Via
della Lastruccia 3, 50019 Sesto Fiorentino, Italy
e-mail: tiziano.marzo@unifi.it
E. Michelucci
Mass Spectrometry Centre (CISM), University of
Florence, Via U. Schiff 6, 50019 Sesto Fiorentino, Italy
L. Messori
e-mail: luigi.messori@unifi.it
A. G. Quiroga
Department of Inorganic Chemistry, Universidad
T. Marzo Á C. Gabbiani Á G. Pescitelli
Department of Chemistry and Industrial Chemistry,
University of Pisa, via Moruzzi, 3, 56124 Pisa, Italy
´
Autonoma de Madrid, C/Francisco Tomas y Valiente 7,
28049 Madrid, Spain
´
G. Bartoli Á S. Pillozzi Á A. Arcangeli
Department of Experimental and Clinical Medicine,
University of Florence, Viale GB Morgagni 50,
50134 Florence, Italy
123

Biometals
Introduction
(2.4 mmol) of KI in 3 mL of water was added to an
aqueous solution (5 mL) of 250 mg of K2[PtCl4]
(0.6 mmol) which was quantitatively converted into a
dark solution containing K2[PtI4] after five minutes of
stirring at room temperature. Then the addition of two
equivalents of ammonium hydroxide as a 40 %
solution results in the separation of cis-PtI2(NH3)2 as
a bright yellow compound, leaving behind a colorless
solution. The solid was filtered off and thoroughly
washed with water (yield 90 %).
Cisplatin is a leading and established anticancer
compound in widespread clinical use. It is very
effective against a few cancer types such as testicular
and ovarian cancer but scarcely active against other
important and more frequent solid tumors such as
colorectal, ovarian and lung cancer (De simone et al.
1986; Wang and Wu 2014; Rosell et al. 2002). While
thousands of analogues of cisplatin have been pre-
pared and tested so far, quite surprisingly the imme-
diate parent Pt compounds that are obtained through
simple replacement of the two chlorides with different
halides as metal ligands (in particular the diiodido and
dibromido derivatives) were poorly investigated.
Most likely, this situation arises from the early
misconception and/or generalisation that chloride
replacement with other halides may result into
substantial loss of the anticancer activity (Wilson
and Lippard 2014; Johnstone et al. 2014; Cleare and
Hoeschele 1973). These arguments led us to explore
this kind of modification in a more systematic way and
analyse its chemical and biological consequences.
Recently, we demonstrated that the diiodido ana-
logue of cisplatin manifests truly interesting biological
properties and warrants a more extensive pharmaco-
logical evaluation (Marzo et al. 2015). This encour-
aging result prompted us to prepare and evaluate even
the dibromido analogue of cisplatin (Fig. 1). Accord-
ingly, we report here the synthesis and chemical
characterisation of cisPtBr2 as well as an initial and
comparative assessment of its biological and pharma-
cological profile.
A
suspension of 200 mg of cis-PtI2(NH3)2
(0.41 mmol) in water (5 mL) was mixed with a
solution of AgNO3 (0.82 mmol) in water (1,5 mL)
and stirred in the dark (40 °C) until a pale yellow
solution was formed over the suspension. The AgI
formed was then filtered off using neutral celite over a
solution of two equivalents of KBr in 7 mL of water.
The colorless solution was stored until orange crys-
talline precipitation was observed. The bright orange
crystals of cis-PtBr2(NH3)2 were filtered off, washed
with water and dried in air. Yield was 25 %. Purity of
the product was assessed through elemental analysis of
C, N and H [calculated C: 0 %, H: 1.45 %, N: 7.20 %,
experimental: C: 0.54 %, H: 1.45 %, N: 7.18 %], 1H,
195Pt NMR and IR analysis (see SI). Cis geometry was
checked as described in (Nakamoto 1997). Solution
behaviour of cis-PtBr2(NH3)2 was assessed through
spectrophotometric experiments performed with a
Varian Cary 50 Bio UV–Vis spectrophotometer
(1 cm pathlength quartz cell) in buffered solutions
without the use of DMSO and NaCl. A solution of the
complex (10-4 M) was prepared in 50 mM phosphate
buffer at pH 7.4. The absorbance was monitored in the
wavelength range between 200 and 800 nm for 72 h at
25 °C.
Materials and methods
Log P determination
Chemistry of cis-PtBr2(NH3)2, synthesis
and characterisation
The octanol–water partition coefficients for cisPtBr2
was determined by modification of the reported shake
flask method (Marzo et al. 2015). Water (50 mL,
distilled after Milli-Q purification) and n-octanol
(50 mL) were shaken together for 72 h to allow
saturation of both phases. Solution of the complex was
prepared in the aqueous phase (3 9 10-3 M) and an
equal volume of octanol was added. Biphasic solutions
were mixed for ten minutes and then centrifuged for
five minutes at 6000 rpm to allow separation. Con-
centration in both phases was determined by UV–Vis.
The synthesis of this Pt complex was performed
through a slight modification of Dhara’s synthesis for
cisplatin (Dhara 1970). A solution of 400 mg
Fig. 1 Structure of cisplatin and its dibromido analogue
123

Biometals
Cellular studies
Reported logP is defined as log [complex]oct/[com-
plex]wat. Final value was reported as the mean of three
determinations.
Cell cultures
A549 cells were cultured in DMEM High glucose
(Euroclone; Milan, Italy) with 10 % Fetal Bovine
Serum (FBS) (Euroclone Defined; Euroclone; Milan,
Italy). HCT-116, FLG 29.1 and IGROV-1 were
cultured in RPMI 1640 (Euroclone; Milan, Italy) with
10 % Fetal Bovine Serum (FBS) (Euroclone Defined;
Euroclone; Milan, Italy). HCT116 cells were kindly
provided by Dr R. Falcioni (Regina Elena Cancer
Institute, Roma). All the cell lines were cultured at
37 °C in humidified atmosphere containing 5 % CO2
in air.
Electrospray mass spectrometry, interaction
with lysozyme
A solution of cis-PtBr2(NH3)2 (3 x 10-4 M) was
incubated with lysozyme (3:1 metal/protein ratio) for
72 h at 37 °C in 20 mmol L-1 ammonium acetate at
pH 6.8. ESI MS spectra were recorded after 24 and
72 h. After a 20-fold dilution with water, ESI MS
spectra have been recorded by direct introduction at
5 ll min-1 flow rate in an Orbitrap high-resolution
mass spectrometer (Thermo, San Jose, CA, USA),
equipped with a conventional ESI source. The work-
ing conditions were the following: spray voltage
3.1 kV, capillary voltage 45 V, capillary temperature
220 °C, tube lens voltage 230 V. The sheath and the
auxiliary gases were set, respectively, at 17 (arbitrary
units) and 1 (arbitrary units). For acquisition, Xcalibur
2.0. software (Thermo) was used and monoisotopic
and average deconvoluted masses were obtained by
using the integrated -Xtract tool. For spectrum acqui-
sition, a nominal resolution (at m/z 400) of 100,000
was used.
Pharmacology experiments
Cells were seeded in 96-well flat-bottomed plates
(Corning-Costar, Corning, NY, USA) at a cell density
of 1 9 104 cells per well in either RPMI or DMEM
complete medium. Drugs were used, after solubiliza-
tion in water, without using DMSO, at concentrations
ranging from 0 to 200 lM. After 24 h, viable cells
(determined by Trypan blue exclusion test) were
counted in triplicate using a haemocytometer. Each
experimental point represents the mean of a single
experiment carried out in triplicate.
Circular dichroism experiments, interaction
with ctDNA
CD spectra were measured with a Jasco J-715
spectropolarimeter with the following conditions:
scan speed 50 nm/min; response 1 s; data pitch
0.1 nm; bandwidth 2.0 nm; 8 accumulations. Calf
thymus DNA (ctDNA) 52.0 lM in phosphate buffer
50 mM, pH 7.4 was additioned with cis-PtBr2(NH3)2
2.3 mM in H2O or cisplatin 1.0 mM in H2O, to 0.5:1
and 1:1 molar ratio directly in a 1-cm quartz cylindri-
cal cell, volume 2.5 ml. Concentrations and molar
ratios are indicated per base. All samples (except one,
see results) were incubated at room temperature
(22 °C) for 72 h before measurement.
The ctDNA had a base-pair length of ca. 800,
obtained with a standardised procedure by sonication
(Biver et al. 2003). The DNA concentration was
checked by absorbance measurement at 260 nm (0.34
u.A., 1 cm cell). Phosphate buffer was used in all case
to measure the blank spectrum.
Trypan blue exclusion test
Cells viability was assessed by the Trypan blue
exclusion assay. In brief, 10 lL of a 0.4 % trypan
blue solution were added to 10 lL of cell suspensions
in culture medium. The suspension was gently mixed
and transferred to a haemocytometer. Viable and dead
cells were identified and counted under a light
microscope. Blue cells failing to exclude the dye were
considered non viable, and transparent cells were
considered viable. The percentage of viable cells was
calculated on the basis of the total number of cells
(viable plus non viable). The IC50 value (i.e. the dose
that caused apoptosis of 50 % of cells) was calculated
by fitting the data points (after 24 h of incubation) with
a sigmoidal curve using Calcusyn software (Biosoft,
Cambridge, UK).
123

Biometals
Cell cycle analysis
The effect of cisplatin and cisPt(NH3)2Br2 on cell
cycle distribution was assessed by flow cytometry
after staining the cells with propidium iodide (PI).
Briefly, the cells (5 9 105 cells per mL) were analysed
prior to and after treatment with IC50s of both drugs for
24 h. The cells were harvested, washed with PBS and
resuspended in 300 lL of propidium iodide staining
solution and incubated at 4 °C in the dark for 20 min.
DNA content of the cells was measured by BD
FACSCanto (Becton–Dickinson, Franklin Lakes, NJ,
USA) Flow Cytometer and the cell population residing
in each phase cell cycle phase was determined using
ModFit LT 3.0 analysis software (Verity Software
House, Topsham, ME USA).
Fig. 2 Time course spectra of cisPtBr2 10-4 M in 50 mM
phosphate buffer recorded for 24 h, RT
Annexin/PI assay
cisplatin analogs (Marzo et al. 2015). After dissolution
of the study compound in the reference buffer,
progressive spectral changes are observed that are
ascribed to the progressive release of the two bromide
ligands. The nature of the process and its kinetics are
similar to what observed in the case of cisplatin despite
release of halides in the case of cisPtBr2 appears to be
slight slower than in case of cisplatin (see SI for details
and numerical data on the hydrolysis processes). By
comparative inspection of the spectral profiles of
cisPtBr2 and cisplatin, monitored over 24 h, their
progressive convergence was noticed, indicating that
the final species are the same for both drugs (see SI for
further details).
The effects of the two compounds on FLG 29.1 cells
was investigated through the Annexin V/propidium
iodide test (Annexin-VFLUOS staining kit; Roche
Diagnostics, Mennheim, Germany). Cells were seeded
at 2 9 105/well in 6-well plates and incubated with
either drug for 24 h. Cells were then harvested,
washed with PBS, re-suspended in 100 lL of binding
buffer and incubated with FITC-conjugated Annexin
V and propidium iodide for 15 min. Flow cytometry
analysis was performed with BD FACSCanto (Bec-
ton–Dickinson, Franklin Lakes, NJ, USA) and anal-
ysed with BD FACSDiva Software 6.1.3. Each
experiment was performed in triplicate.
Furthermore, log P determination was carried out
on cisPtBr2 and a value of -1.04 was found.
Remarkably this value is intermediate between those
previously measured for cisplatin (-2.4) and cis-
PtI2(NH3)2 (-0.13) (Marzo et al. 2015), thus, being
the dibromido analogue of cisplatin far more lipophilic
than cisplatin itself, it is well conceivable that this
difference may have some appreciable effect in terms
of pharmacological activity.
Results and discussion
Solution behaviour of cis-PtBr2(NH3)2, activation
profile and logP determination
cisPtBr2 shows an acceptable solubility in the refer-
ence phosphate buffer, at physiological pH; this allows
to carry out experiments in the absence of DMSO or
other organic solvents, thus avoiding potential inter-
ferences. Freshly prepared solutions of cis-PtBr2(-
NH3)2 manifest typical absorption bands in the UV–
Visible, with a main band at 330 nm and a pronounced
shoulder at 300 nm (Fig. 2a).
Mechanistic studies: biomolecular interactions
To investigate the possible interactions occurring
between cisPtBr2 and its potential biomolecular
targets we first performed a few ESI MS experiments
for samples where the study complex was incubated
These bands are tentatively assigned to a d–d and a
LMCT transition, respectively, in analogy with other
123

Biometals
CD experiments, interaction with ctDNA
with the model protein lysozyme for increasing time
intervals (Fig. 3).
Since nuclear DNA is believed to be the primary target
for the pharmacological effect of cisplatin, (Siddik
2003) we next characterized the interaction of cisPtBr2
with calf thymus DNA through CD experiments, and
compared the results with those of cisplatin.
First attempts to characterise the protein metalation
process through ESI MS detection were performed by
analysing samples after 24 h of incubation, but no
clear evidence of adduct formation was gained.
However, upon increasing the incubation time up to
72 h, under identical solution conditions, a number of
peaks of higher molecular weight than the native
protein, were instead observed that were straightfor-
wardly assigned to metallodrug-lysozyme adducts.
From careful inspection of the spectrum, it is
evident that Pt coordination occurs through a mech-
anism that involves preferential detachment of halide
ligands and full retention of ammonia ligands;
assignments were validated by theoretical calculations
(see SI for comparison between experimental and
theoretical peaks). This kind of protein metalation
strictly resembles that produced by cisplatin resulting
in an adduct characterised by selective Pt coordination
to His15 following the release of the two chloride
ligands (Casini et al. 2007).
The CD spectrum of ctDNA, before addition of the
drugs is in keeping with literature (Tamburro et al.
1977) (Fig. 4). Addition of 0.5 equiv of cis-PtBr2(-
NH3)2 did not lead to any immediate change in the CD
spectrum (data not shown). However, after incubation
at 22 °C for 72 h, the spectrum changed appreciably
showing clear differences on the whole wavelength
range (Fig. 5). In particular, both CD bands at 280 and
250 nm increased in intensity, the first by about 15 %.
Furthermore, no significant spectral changes were
found after increasing the amount of complexes up to 1
equiv, being the two spectral profiles at 0.5 or 1 equiv
superimposable (Fig. 4). Evidence that cisPtBr2 inter-
acts with ctDNA similarly to cisplatin derives from the
comparative analysis of the CD spectra of ctDNA after
incubation with 1 equivalent of cisPtBr2 or cisplatin
(Fig. 5).
Remarkably, this behaviour being in accord with
cisplatin is in contrast with that of cisPtI2, where
metalation of lysozyme occurs through protein bind-
ing of metal fragments resulting from preferential
release of the ammonia ligands (Messori et al. 2013).
Modification in the CD spectral profiles are well
detectable, consistent and superimposable for the two
complexes. A direct comparison with previous CD
investigations on the ctDNA/cisplatin system (Tamburro
5
ctDNA
4
ctDNA + 0.5 equiv cis-PtBr2(NH3)2
ctDNA + 1 equiv cis-PtBr2(NH3)2
3
2
1
0
-1
-2
-3
200
250
300
350
λ / nm
Fig. 3 Deconvoluted ESI MS spectra of lysozyme treated with
3 9 10-4 mol L-1 of cisPtBr2 recorded after 72 h of incubation
at 37 °C, metal: protein ratio = 3:1; ammonium acetate buffer
20 mmol L-1 at pH 6.8
Fig. 4 CD spectra of ctDNA without and with cis-PtBr2(NH3)2,
0.5 and 1 equiv, after 72 h incubation
123

Biometals
5
4
Table 1 Evaluation of IC50 on a panel of tumor cell lines
ctDNA
IC50 Cisplatin (lM)
mean SD
IC50 cis-PtBr2(NH3)2
(lM)
mean SD
ctDNA + 1 equiv cis-PtBr2(NH3)2
ctDNA + 1 equiv cisplatin
3
A549
8.61 1.26
24.33 1.30
20.04 1.65
24.12 0.85
10.08 1.26
14.66 1.10
28.28 5.39
25.63 0.43
2
FLG 29.1
HCT-116
IGROV-1
1
A549,FLG,HCT-116 and IGROV-1 cell lines were exposed to
IC50 concentrations of cisplatin and cisPtBr2 for 24 h and cell
viability was evaluated via Trypan Blue exclusion assay.
Means and SDs are relative to one experiment mounted in
triplicate
0
-1
-2
-3
leukaemia cells. These encouraging results led us to
deepen our investigation.
200
250
300
350
λ / nm
The effects of cisPtBr2 on cell cycle and apoptosis
were further evaluated in FLG 29.1 cells. Upon
treatment with cis-PtBr2(NH3)2 a reduction in the
percentage of cells in the G0/G1 phase accompanied
by an increase in G2/M cells was observed. Such effect
was more evident than that produced by cisplatin (see
Fig. 6). We next evaluated the effect of cis-PtBr2(-
NH3)2 on the induction of apoptosis by Annexin/PI
test. As reported in Fig. 7 cis-PtBr2(NH3)2 determined
a more evident pro-apoptotic effect compared to
cisplatin, in FLG 29.1 cells (percentage of
Annexin?/PI- cells: f 51.85 4.5 vs. 41.15
2.75, respectively). Finally, we determined the effects
of either cisPtBr2 or cisplatin (both at 19 lM concen-
tration) on proliferation of FLG 29.1 cells. Live cell
number was monitored for 72 h. As shown in Fig. 8
the dibromido analogue impaired leukaemia cell
proliferation even better than cisplatin, especially at
longer incubation times.
Fig. 5 CD spectra of ctDNA with cis-PtBr2(NH3)2 and
cisplatin, 1 equivalent, after 72 h incubation
et al. 1977; Sristava et al. 1978) is complicated by
relevant differences in the applied solution conditions
and in the nature of ctDNA samples (non-sonicated vs.
sonicated). Qualitatively speaking, the differences
observed upon binding occur in the same spectral
regions and to a similar extent. However, our
fragmented ctDNA (800 bp) shows a full enhance-
ment of the main CD bands, already with 0.5
equivalent of ligand per base.
Cellular effects
We then investigated the effects of cisPtBr2 on cell
vitality of a small panel of human cancer cell lines,
applying the Trypan blue exclusion test according to
the method described in the experimental section.
The cancer cell panel included the following lines:
A549 (human lung cancer), HCT116 (human colon
cancer), IGROV-1 (human ovarian cancer) and FLG
29.1 (human acute myeloid leukaemia). These cells
were exposed to increasing concentrations of the drug,
in the 0–200 lM range, and treated for 24 h; IC50
values were determined (Table 1). It is evident that
cis-PtBr2(NH3)2 produces cytotoxic effects almost
superimposable to those of cisplatin in all the cancer
cell lines. A slightly greater effect of cisPtBr2
compared to cisplatin was observed in FLG 29.1
Discussion and conclusions
Cisplatin is one of the oldest and most used Pt-based
anticancer drug with a long success story in the clinics
since 1979. Yet, cisplatin shows some severe draw-
backs that have triggered a lot of attention in the
preparation and identification of new and hopefully
more active and less toxic anticancer Pt drugs.
Despite the intense investigations carried out in the
field of Pt-based drugs during the last three decades,
the closest analogues of cisplatin, e.g. Pt complexes
123

Biometals
Fig. 6 Effect of Cisplatin and cisPtBr2 on cell cycle of FLG
cells (a = untreated). Effect of cisplatin (b) and of cisPtBr2
(c) given at their IC50s on cell cycle of FLG cells after 24 h of
treatment. The means and SEMs relative to two independent
experiments are reported next to each representative panel
Fig. 7 Effect of cisplatin and of cisPtBr2 given at their IC50s on the induction of apoptosis on FLG cells after 24 h of treatment. The
means and SEMs relative to two independent experiments are reported in each representative panel. a untreated, b cisplatin, c cisPtBr2
differing only in the chemical nature of the two halide
ligands, were poorly studied. We have recently
analysed the behaviour of the diiodido analogue of
cisplatin (Marzo et al. 2015). Very interesting chem-
ical and biological features of this compound emerged.
Notably, the diiodido analogue turned out to have
strong cytotoxic effects on different cancer cell lines
in vitro, especially on those more resistant to cisplatin
effects. Some peculiarities in the nature of the reaction
of cisPtI2 with model proteins were also highlighted
(Marzo et al. 2015). Here, we extended our investi-
gation to the dibromido analogue, in order to have a
deeper insight and a more complete description of this
topic.
cisPtBr2 could be prepared quite easily according to
Fig. 8 Effects of cisplatin (black dots) and cisPtBr2 (black
triangles) on FLG 29.1 cell line proliferation during the passage
of time given as the number of Trypan Blue negative cells.
Drugs were added once a t = 0. Data are mean SD of two
independent experiments
the classical synthetic procedure of Dhara, with slight
modifications. Its solution chemistry was explored in a
standard physiological buffer and a behaviour very
close to cisplatin emerged. Indeed, activation of cis-
123

Biometals
Cancro, 3-years Fellowship for Italy Project Code: 18044).
CIRCMSB is also acknowledged.
PtBr2(NH3)2 occurs through progressive release of the
two bromide ligands.
When tested with a model protein (lysozyme) a
platination pattern similar to cisplatin emerged, with
protein coordination of [cisPt(NH3)2]2? fragments.
Even the process of platination of standard ctDNA
(800 bp) was highly reminiscent of that of cisplatin
and CD spectroscopy clearly proved the similar nature
of those interactions. One equivalent of ligand (per
base pair, 0.5 equiv per base) turned out to achieve full
binding. The binding is similar for cis-PtBr2(NH3)2
and cisplatin. Using our fragmented ctDNA, the
effects of cisplatin are slightly different from those
reported in the literature for non-sonicated ctDNA
(Tamburro et al. 1977; Sristava et al. 1978).
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might be exploited for further preclinical studies of
this analogue and for a full assessment of its pharma-
cological profile with respect to cisplatin. Indeed, as
the two compounds are very similar in their overall
chemical and biological profile, the slight differences
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Acknowledgments We gratefully acknowledge Beneficentia
Stiftung, Ente Cassa Risparmio Firenze (ECR), COST Action
J
Biol Chem
`
CM1105 and Universita di Pisa (PRA2015-0055) for financial
support, CISM (University of Florence) for recording ESI–MS
spectra, and Dr. Tarita Biver for a sample of fragmented ctDNA.
T.M. thanks AIRC-FIRC (Fondazione Italiana per la Ricerca sul
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