Synthesis, in-vitro cytotoxic activity and DNA interactions of new dicarboxylatoplatinum(II) complexes with 2-hydroxymethylbenzimidazole as carrier ligands

Article abstract of DOI:10.1111/jphp.12290

Objectives The aim of this study was to investigate the in-vitro cytotoxic activity of new platinum(II) complexes on the human HeLa (ER-), MCF-7 (ER+) and MDA-MB 231 (ER-) cell lines. Furthermore, we investigated plasmid DNA interactions and inhibition of BamHI and HindIII restriction enzyme activity of the complex 1-4,7. Methods Platinum(II) complexes were synthesised from precursor complexes of [PtL₂Cl₂] and [PtL₂I₂]. Their cytotoxic activity was tested by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Their plasmid DNA interactions and restriction enzyme activities were also investigated using the agarose gel electrophoresis method. Key findings The growth inhibitory effect results showed that the cytotoxicity of complex 2 was found to be the most active complex among the synthesised complexes. Conclusions The MTT results showed that complex 2 was found to be cytotoxic equal to cisplatin and higher than carboplatin against the MCF-7 and MDA-MB-231 cell lines. Furthermore, the estrogen or progesterone co-treatment slightly increased the cytotoxicity of complex 2, the cisplatin and carboplatin compared with the complex 2 tested alone in 50μm concentration. According to plasmid DNA interaction and the restriction studies, complexes 1-4,7 modified the tertiary structure of pBR322 plasmid DNA, and complexes 2-4 prevented enzyme digestion at high concentrations.

Full text of DOI:10.1111/jphp.12290

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Research Paper
Journal of Pharmacy
And Pharmacology
Synthesis, in-vitro cytotoxic activity and DNA interactions
of new dicarboxylatoplatinum(II) complexes with
2-hydroxymethylbenzimidazole as carrier ligands
Semra Utkua, Azime Berna Özçelikb, Fatma Gümüs¸b, S¸ükran Yılmazc, Taibe Arsoyc, Leyla Açıkd and
Ayten Çelebi Keskine
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin, bDepartment of Pharmaceutical Chemistry, Faculty of
d
c
Pharmacy, Department of Biology, Faculty of Science, Gazi University, Cell and Virus Bank Department, Foot and Mouth Disease Institute, Ankara
e
and Department of Biology, Faculty of Art and Science, Kırıkkale University, Kırıkkale, Turkey
Keywords
benzimidazole; cytotoxic activity; DNA
Abstract
Objectives The aim of this study was to investigate the in-vitro cytotoxic activity
of new platinum(II) complexes on the human HeLa (ER−), MCF-7 (ER+) and
MDA-MB 231 (ER−) cell lines. Furthermore, we investigated plasmid DNA inter-
actions and inhibition of BamHI and HindIII restriction enzyme activity of the
complex 1–4,7.
Methods Platinum(II) complexes were synthesised from precursor complexes of
[PtL2Cl2] and [PtL2I2]. Their cytotoxic activity was tested by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Their plasmid
DNA interactions and restriction enzyme activities were also investigated using
the agarose gel electrophoresis method.
binding; gel electrophoresis; platinum(II)
complexes
Correspondence
Fatma Gümüs¸, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Gazi
University, Ankara 06330, Turkey.
E-mail: fgumus@gazi.edu.tr or
gumus53@gmail.com
Received March 19, 2014
Accepted June 29, 2014
Key findings The growth inhibitory effect results showed that the cytotoxicity of
complex 2 was found to be the most active complex among the synthesised
complexes.
doi: 10.1111/jphp.12290
Conclusions The MTT results showed that complex 2 was found to be cytotoxic
equal to cisplatin and higher than carboplatin against the MCF-7 and MDA-MB-
231 cell lines. Furthermore, the estrogen or progesterone co-treatment slightly
increased the cytotoxicity of complex 2, the cisplatin and carboplatin compared
with the complex 2 tested alone in 50 μm concentration. According to plasmid
DNA interaction and the restriction studies, complexes 1–4,7 modified the ter-
tiary structure of pBR322 plasmid DNA, and complexes 2–4 prevented enzyme
digestion at high concentrations.
with modifications of the carrier ligands, resulting in differ-
ent efficacy and a different spectrum of activity.[5–7]
Introduction
Cisplatin (cis-diamminedichloroplatinum(II)) and its
analogues carboplatin (cis-diammine-1,1-cyclobutanedicar
The choice of leaving groups also plays a role in the anti-
tumour properties of the Pt(II) complexes. Modification of
chloride leaving groups of cisplatin generally leads to differ-
ent pharmacokinetic properties. The lability of Pt-X bonds
is crucial for the delivery of platinum to DNA without prior
deactivation by other biological nucleophiles.[8]
Carboplatin is a second-generation Pt(II) compound
that differs from cisplatin by the presence of a bidentate
dicarboxylate ligand as its leaving group instead of the more
label cisplatin’s chlorides. This renders the complex activa-
tion kinetics much slower relative to cisplatin, and conse-
quently leads to a significantly lower toxicity, while yielding
the same type of DNA adducts.[9,10]
boxylateplatinum(II)),
and
oxaliplatin
([(1R,2R)-
cyclohexane-1,2-diamine](ethanedioato-O,O’)platinum(II))
are routinely used worldwide in clinical practice.[1]
The clinical efficacy of these anticancer drugs is diminished
by intrinsic and acquired tumour resistance and side
effects.[2,3]
In the search for new platinum compounds avoiding tox-
icity and resistance, special attention has been paid to the
replacement of one or both NH3 ligands of cisplatin by
other N-donor ligand(s).[4] A large number of mononuclear
platinum compounds have been designed over the years
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1593–1605
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Semra Utku et al.
Cisplatin is highly effective against testicular, ovarian and
lung cancers but has limited efficacy as monotherapy in
breast cancer patients. Breast cancer is the most frequently
diagnosed cancer in women and is frequently hormone-
dependent. Many breast tumours express a high level of
estrogen receptor or/and progesterone receptor, and these
cancers would be good candidates for cisplatin/carboplatin
treatment in conjunction with hormone therapy.[11]
Cisplatin and its analogues bind to various cellular com-
ponents like DNA, RNA, proteins and membrane phospho-
lipids. Although targets other than DNA may also be
important for these agents, it is generally accepted that
DNA is the primary in-vivo cytotoxic target of these com-
pounds. The structural distortions of DNA are recognised
by various structure-specific DNA-binding proteins, for
example DNA repair and high-mobility group (HMG)
domain proteins.[12]
breast cancer cell lines. For this purpose, oxalate, malonate,
methylmalonate, ethylmalonate, DL-malate and D-malate
groups were chosen as leaving ligands and synthesised novel
platinum(II) complexes in this study. The plasmid DNA
interactions and inhibition of the BamHI and HindIII
restriction enzyme activity of the complexes were also
studied.
The aim of this study was also to investigate whether
estrogen or/and progesterone in combination with the most
active Pt(II)complex among the complexes tested in this
study could influence the cytotoxic activity of the complex,
particularly in MCF-7 (ER+) and MDA-MB 231 (ER−)
breast cancer cell lines.
Materials and Methods
Chemistry
All chemicals and solvents used in the synthesis were pur-
chased from Merck (Darmstadt, Germany) or Aldrich
(Steinheim, Germany). Infrared (IR) spectra were obtained
using a Perkin Elmer Spectrum FT-IR/NIR spectrometer
(Waltham, MA, USA) equipped with a Universal ATR Sam-
pling Accessory at Mersin University Advanced Technology,
Training, Research and Application Center (Mersin, Turkey).
Elemental analyses were performed with LECO 932 CHNS
Sex steroid hormones, such as estrogen and progesterone,
are known to induce an estrogen receptor (−) (ER−) medi-
ated overexpression of HMG1, with enhanced cytotoxic
potential of cisplatin or carboplatin in estrogen receptor (+)
(ER+) breast cancer MCF-7 cell line.[11] These findings
suggest that cells with high levels of HMGB1 may sensitise
tumour cells to cisplatin by shielding its major DNA
adducts from nucleotide excision repair.[8] Another hypoth-
esis is that cisplatin-modified DNA may sequester HMGB1,
inhibiting its roles in the progression of cancer. In this
model, cisplatin acts as an anti-HMG1 agent in a way that
may potentiate its anticancer drug activity. The two hypoth-
eses arise from the dual nature of HMGB1. It acts both as
transcription factor in the nucleus and also as a cytokine in
the cytosol.[8]
1
analyser, and H nuclear magnetic resonance (NMR) spectra
were recorded in dimethylsulfoxide (DMSO)-d6 (Merck) on a
Varian Mercury 400 MHz FT-NMR spectrometer (Palo Alto,
CA, USA) using tetramethylsilane as the internal standard at
the Central Laboratuvary of the Faculty of Pharmacy, Ankara
University (Ankara, Turkey). All chemical shifts were recorded
as δ (ppm). Thin-layer chromatography was performed on
pre-coated aluminium plates (Merck) Silica Gel 60 F254. Plates
were visualised by ultraviolet light, Dragendorff reagent or
iodine vapour.
In MCF-7 breast cancer cells, upregulation of HMGB1 in
response to estrogen exposure functions as an enhancer of
cisplatin-induced cytotoxicity.[11]
In previous studies, with the consideration that variations
in the chemical structure of the ammine groups of cisplatin
might have a significant effect on the cytotoxic activity and
toxicity of platinum complexes, and with the aim of deter-
mining the role of the substituents on position 2 of the benz-
imidazole carrier ligands of Pt(II) complexes on cytotoxic
properties, we synthesised some Pt(II) complexes with
2-substituted benzimidazole ligands.[13–23] It was determined
that some of these platinum complexes have in-vitro cytotoxic
activity on RD,[14] HeLa,[17,19–22] MCF-7,[15,17,19–22] HEp-2,[21]
MDA-MB231[22] and SK-Hep1[23] cell lines.
In this study, as an extension of the investigation on the
probable anticancer activity of the platinum complexes with
2-substituted benzimidazole ligands, it was aimed to inves-
tigate the influence of the bidentate dicarboxylate leaving
ligands on the in-vitro cytotoxic properties of the
2-hydroxymethylbenzimidazole Pt(II) complexes against
HeLa (ER-) cervix, MCF-7 (ER+) and MDA-MB 231 (ER−)
Synthesis of carrier ligand
2-Hydroxymethylbenzimidazole (L) used as carrier ligand
in the structure of 1–7 was prepared according to the Phil-
lips method,[24] as shown in Scheme 1.
Yield 59%, mp 172°C (171°C[25]). 1H NMR (DMSO-d6): δ
(ppm) 12.17 (s, 1H, NH, exchangeable with D2O), 7.45–7.47
(m, 2H, ArH), 7.07–7.16 (m, 2H, ArH), 5.53 (broad s, 1H,
-OH exchangeable with D2O), 4.68 (s, 2H,-CH2-). IR (ATR):
υ (cm−1) 3400-2500 (NH, OH, = CH, -CH).
Synthesis of [dichloro-bis(2-
hydroxymethylbenzimidazole)platinum(II)]
[PtL2Cl2] and [diiodo-bis(2-
hydroxymethylbenzimidazole)platinum(II)]
[PtL2I2]
Complexes of [PtL2Cl2] and [PtL2I2] were synthesised as
described previously[19] by the reaction of L carrier ligand
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Semra Utku et al.
Cytotoxic activity of platinum(II) complexes
NH2
NH2
N
5 N HCl
+
HOCH2COOH
CH2OH
N
H
L
CH2OH
N
2-
Cl
Cl
PtCl2
+ 2KCl
HN
+
2K+
Pt
2L
Cl
Cl
. 3H2O
2
K2[PtCl4]
K2[Ptl4]
KCl4
+
Kl
+
CH2OH
N
HN
Ptl2 + 2Kl
K2[Ptl4]
+
2L
2
Scheme 1 Synthesis of carrier ligand and precursor platinum(II) complexes.
with K2PtCl4 and K2PtI4, respectively, in ethanol/water solu-
tion, as presented in Scheme 1.
16.00 Hz, 1H, -CH2). IR (ATR): υ (cm−1) 3442-2952 (NH,
OH, = CH, -CH). Anal. Calcd. for C16H16I2N4O2Pt: C, 25.79;
H, 2.16; N, 7.52. Found: C, 26.19; H, 2.03; N, 7.38.
[Dichloro-bis(2-
hydroxymethylbenzimidazole)platinum(II)].
3H2O [PtL2Cl2].3H2O
Synthesis of [monosulfat-monoaqua-bis(2-
hydroxymethylbenzimidazole)platin(II)].
2H2O (1)
Yield 25%. 1H NMR (DMSO-d6): δ (ppm) 13.41 (s, 2H, 2×
NH, exchangeable with D2O), 8.14 (d, J = 8.97 Hz, 2H,
ArH), 7.57-7.37 (m, 6H, ArH), 6.28 (s, 2H, 2× OH,
exchangeable with D2O), 5.48 (dd, J = 4.00 and 16.40 Hz,
2H, -CH2-), 5.07-4.92 (m, 2H, -CH2-). IR (ATR): υ (cm−1)
3443-2924 (NH, OH, = CH, -CH) cm−1, 330-318 (Pt-Cl).
Anal. Calcd. for C16H16Cl2N4O2Pt.3H2O: C, 31.18; H, 3.60;
N, 9.09. Found: C, 31.12; H, 3.42; N, 9.30.
To a suspension of [PtL2I2] (0.201 mg, 0.27 mmol) in 12 ml
of water was added an aqueous solution of AgSO4
(77.94 mg, 0.25 mmol) in 5-ml acetone. The reaction
mixture was then continuously stirred in the dark for 11
days at room temperature. The precipitate of silver iodide
was filtered off, and the filtrate was evaporated to dryness
under reduced pressure and dried in vacuo, as presented in
Scheme 2.
[Diiodo-bis(2-hydroxymethylbenzimidazole)
platinum(II)] [PtL2I2]
General procedure for synthesis of
dicarboxylatoplatinum(II) complexes
Yield 36%. 1H NMR (DMSO-d6): δ (ppm) 13.18 (s, 2H, 2×
NH, exchangeable with D2O), 8.13-8.00 (m, 2H, ArH), 7.36-
7.12 (m, 6H, ArH), 6.18 (t, J = 5.4 Hz, 1H, OH, exchange-
able with D2O), 6.10 (t, J = 5.6 Hz, 1H, OH, exchangeable
with D2O), 5.27-5.21 (m, 2H, -CH2-), 5.00 (dd, J = 5.3 Hz,
and 16.00 Hz, 1H, -CH2-), 4.54 (dd, J = 5.7 Hz, and
Preparation of disilver salts of dicarboxylic acids
Disilver salts of dicarboxylic acids were synthesised accord-
ing to the reported methods.[26,27] Appropriate dicarboxylic
acids (20 mmol) and AgNO3 (40 mmol) were added to a
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1593–1605
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Semra Utku et al.
H
N
CH2OH
CH2OH
OSO3H
N
N
AgSO4
+
HN
N
Ptl2
2 Agl
+
Pt
H2O
.2H2O
CH2OH
N
H
2
1
O
CH2OH
N
O
CH2OH
O
O
AgO
Pt
(CHR)n
O
+
2 AgCl
HN
HN
N
PtCl2
(CHR)n
O
+
AgO
.× H2O
2
2
Complex
2
R
n
0
X
−
1.5
1
3
4
5
−H
1
1
1
−CH3
2
−CH2CH3
1
O
O
CH2OH
N
CH2OH
O
AgO
CH2
HC
+
2 Agl
CH2
Ptl2
Pt
HN
HN
N
+
OH
HC
OH
O
AgO
O
O
.× H2O
2
2
DL or D
Complex
6 (DL)
X
2
7 (DL)
0.5
Scheme 2 Synthesis of benzimidazole-platinum(II) complexes 1–7.
1596
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Cytotoxic activity of platinum(II) complexes
1 N NaOH solution (40 ml) by stirring. The mixture was
stirred at room temperature in the dark overnight. The
resulting white silver salts were filtered and washed twice
with water and then dried thoroughly in vacuo.
7.50 (m, 2H, 2× ArH), 7.28 (m, 4H, 2× ArH), 6.24 (s, 2H, 2×
OH ), 4.95(s, 4H, 2× -CH2-). IR (ATR): υ (cm−1) 3394-2982
(NH, OH, = CH, -CH), 1642 (C = O), 1394 (C-O), 548 (Pt-
O). Anal. Calcd. for C19H18N4O6Pt.H2O: C, 37.32; H, 3.30; N,
9.16. Found: C, 37.25; H, 3.03; N, 9.38.
Synthesis of [PtL2(oxalato)] (2), [PtL2(malonato)]
(3), [PtL2(methylmalonato)] (4) and [PtL2
(ethylmalonato)] (5)
[Methylmalonato-di
(2-hydroxymethylbenzimidazole)platinum(II)].
2H2O [PtL2 (methylmalonato)].2H2O (4)
A suspension of [PtL2Cl2] (1 mmol) and disilver salt of
dicarboxylic acid (oxalic, malonic, methylmalonic or
ethylmalonic acid) (1 mmol) in water (15–20 ml) were
stirred at 45°C in the dark for 6–10 days. The resulting silver
chloride was removed by filtration (by Millipore filter
(0.22 μm)). The filtrate was concentrated under a reduced
pressure to ∼5 ml in volume. The white precipitate formed
was filtered off, washed with small portions of water and
dried in vacuo, as presented in Scheme 2.
Yield 20%. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 13.47
(s, 2H, 2× NH), 7.84 (m, 2H, 2× ArH), 7.49 (m, 2H, 2×
ArH), 7.28 (m, 4H, 2× ArH), 6.23 (broad s, 2H, 2× OH),
5.00 (s, 4H, 2× -CH2-), 4.28 (m, 1H, -CH-), 1.16 (m, 3H,
CH3). IR (ATR): υ (cm−1) 3395-2981 (NH, OH, = CH,
-CH), 1643 (C = O), 1397 (C-O), 553 (Pt-O). Anal. Calcd.
for C20H20N4O6Pt.2H2O: C, 37.33; H, 3.76; N, 8.71. Found:
C, 37.76; H, 3.51; N, 8.12.
[PtL2(DL-malato)] (6) and [PtL2(D-malato)] (7)
[Ethylmalonato-di(2-
hydroxymethylbenzimidazole)platinum(II)]. H2O
[PtL2 (ethylmalonato)] .H2O (5)
A suspension of [PtL2I2] (1 mmol) and disilver DL or
D-malate (Ag2C4H4O5) (1 mmol) in water/acetone (4/1)
(20 ml) were stirred at 45°C in the dark for 14–16 days. The
resulting silver chloride was removed by filtration (by
Millipore filter (0.22 μm)). The filtrate was concentrated
under a reduced pressure to ∼5 ml in volume. The white
precipitate formed was filtered off, washed with small por-
tions of water and dried in vacuo, as presented in Scheme 2.
Yield 33%. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 13.47
(s, 2H, 2× NH), 7.50 (m, 8H, 2× ArH), 6.22 (s, 2H, 2× OH),
5.00 (s, 4H, 2× -CH2-), 3.74 (s, 1H, -CH-), 1.99 (m, 2H,
CH2CH3), 0.93 (m, 3H, CH2CH3). IR (ATR): υ (cm−1) 3393-
2981 (NH, OH, = CH, -CH), 1610 (C = O), 1381 (C-O),
553(Pt-O). Anal. Calcd. for C21H22N4O6Pt. H2O: C, 39.44; H,
3.78; N, 8.76. Found: C, 39.19; H, 3.59; N, 8.81.
[Monosulfat-monoaqua-bis(2-
hydroxymethylbenzimidazole)platin(II)]. 2H2O (1)
[DL-malato-di(2-hydroxymethylbenzimidazole)
Yield 22%. 1H NMR (400 MHz, DMSO-d6): δ (ppm) δ 8.31
(m, 2H, 2× ArH), 8.01 (m, 2H, 2× ArH), 7.37 (m, 4H, 2×
ArH), 5.15 (m, 4H, 2× -CH2-). IR (ATR): υ (cm−1) 3286
(NH, OH, = CH, -CH), 584 (Pt-O). Anal. Calcd. for
C16H19N4O7PtS. 2 H2O: C, 29.91; H, 3.61; N, 8.72; S,4.99.
Found: C, 29.53; H, 3.30; N, 9.02; S,3.87.
platinum(II)]. 2H2O [PtL2(DL-malato)]. 2H2O (6)
Yield 45%. 1H NMR (DMSO-d6): δ (ppm) 13.10 (s, 2H, 2×
NH), 7.47-7.09 (m, 8H, 2× ArH), 6.15 (broad s, 1H,
-CHOH), 5.97 (broad s, 2H, 2× –CH2OH ), 5.10-5.00
(m, 4H, 2× CH2OH), 4.30-4.28 (m, 1H, -CH-), 3.85-3.84
(d, J = 3.2 Hz, 1H, -CH2-), 3.82-3.81 (d, J = 3.2 Hz, 1H,
-CH2-). IR (ATR): υ (cm−1) 3390-2981 (NH, OH, = CH,
-CH), 1659 (C = O), 1376 (C-O), 551 (Pt-O). Anal. Calcd.
for C20H20N4O7Pt. 2H2O: C, 36.42; H, 3.67; N, 8.50. Found:
C, 36.34; H, 3.68; N, 8.97.
[Oxalato-di(2-hydroxymethylbenzimidazole)
platinum(II)]. 1.5 H2O [PtL2(oxalato)] 1.5 H2O (2)
Yield 23%. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 13.56
(s, 2H, 2× NH), 7.74 (d, J = 7.2 Hz, 2H, 2× ArH), 7.50 (m,
2H, 2× ArH), 7.28 (m, 4H, 2× ArH), 6.24 (s, 2H, 2× O-H ),
4.95(s, 4H, 2× -CH2-). IR (ATR): υ (cm−1) 3395-2981 (N-H,
O-H, = CH, -CH), 1692 (C = O), 1403 (C-O), 567 (Pt-O).
Anal. Calcd. for C18H16N4O6Pt 1.5 H2O: C, 35.35; H, 3.15; N,
9.23. Found: C, 35.29; H, 2.76; N, 9.20.
[D-malato-di(2-hydroxymethylbenzimidazole)
platinum(II)] [PtL2(D-malato)]. 0.5 H2O (7)
Yield 46%. 1H NMR (DMSO-d6): δ (ppm) 13.10 (s, 2H, 2×
NH), 7.46-7.10 (m, 8H, 2× ArH), 5.14-4.97 (m, 4H, 2×
CH2OH), 4.31-4.28 (m, 1H, -CH-), 3.89-3.88 (d, J = 4 Hz,
1H, -CH2-), 3.86-3.85 (d, J = 3.6 Hz, 1H, -CH2-). IR (ATR):
υ (cm−1) 3390-2981 (NH, OH, = CH, -CH), 1663 (C = O),
1381 (C-O), 553 (Pt-O). Anal. Calcd. for C20H20N4O7Pt. 0.5
H2O: C, 36.93; H, 3.56; N, 8.61. Found: C, 37.03; H, 3.70; N,
8.50.
[Malonato-di(2-hydroxymethylbenzimidazole)
platinum(II)]. H2O [PtL2(malonato)]. H2O (3)
Yield 45%. 1H NMR (400 MHz, DMSO-d6): δ (ppm)
13.56 (s, 2H, 2× NH), 7.74 (d, J = 7.2 Hz, 2H, 2× ArH),
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40 V. At the end of the electrophoresis, the gel without
ethidium bromide was stained in the same buffer contain-
ing ethidium bromide (0.5 μg/ml). The gel was then viewed
with a transilluminator, and the image was captured with a
video camera (GelDoc-It Imaging System, UVP; Upland,
CA, USA) as a TIFF file. The experiments were repeated
three times.
In-vitro cytotoxicity studies
Cell lines and growth conditions
Cisplatin (Cas No: 15663-27-1) and carboplatin (Cas No:
41575-94-4), used as reference compounds, were obtained
from Sigma. The human HeLa cervix (ER−), MCF-7 (ER+)
and MDA-MB 231 (ER−) breast cancer cell lines used in
this study were obtained from the Cell Culture Collection
(HUKUK numbers 90061901, 00092502 and 02031201,
respectively) of the Institute for Foot and Mouth Disease,
Turkey. The cells were grown as monolayer cultures in T75
flaks, subcultured three times at 37°C in an atmosphere of
5% CO2 in air and 100% of relatively humidity, and main-
tained in third passage.
Those three cell suspensions were prepared at the con-
centration of 8.7 × 103 cell/ml and dispensed onto 96-well
cell culture plates (100 μl per well). The multi-well plates
were incubated at 37°C and 5% CO2 in air condition for
24 h. After 24 h, the culture medium was removed from the
wells, and equal volumes (100 μl) of serial dilutions (80, 40,
20, 10 and 5 μm) of the test complexes 1–7 were added to
each well. After 72-h incubation period, cytotoxicity was
evaluated colorimetrically by MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay.[28] Three
experiments were carried out for each complex.
BamHI and HindIII restriction enzyme digestion
In this series experiments, the drug-DNA mixtures were first
incubated for 24 h and then subjected to enzyme BamHI or
HindIII digestion for 1 h at 37°C. To each 8 μl of incubated
drug-DNA mixtures were added 1 μl of 10× digestion buffer
and then 0.1 μl of BamHI or HindIII (1 unit). The mixtures
were left in shaking water bath at for 1 h at 37°C. The diges-
tion was terminated by rapid cooling. The restricted DNA was
run in 1% agarose gel electrophoresis for 3 h at 40 V in TAE
buffer. The gel was stained with ethidium bromide, and the
gels were then viewed with a transilluminator, and the image
was captured by a video camera as a TIFF file. These studies
were repeated three times.
Statistical analysis
Cytotoxic activity values were performed in triplicates, and
data are expressed as mean standard deviation (n = 3).
Results were analysed using one-way analysis (Table 1)
and two-way analysis (Table 2) of variance, followed by
Dunnett’s post-hoc test for multiple comparisons with
controls. A value of P < 0.05 was considered statistically
significant.
In-vitro cytotoxic activity of complex 2, which was found
to be the most active among the tested complexes, was also
investigated in combination with estrogen (0.1 μm), or pro-
gesterone (0.1 μm) or estrogen (0.1 μm) and progesterone
(0.1 μm) in MCF-7 and MDA-MB 231 cancer cell lines
using MTT assay in comparison with cisplatin.
Results and Discussion
Studies of interaction with pBR322 plasmid DNA
Chemistry
The cisplatin and carboplatin used as reference compounds,
plasmid DNA pBR322, ethidium bromide, agarose and
enzyme BamHI and HindIII, were purchased from Sigma.
The interaction of platinum(II) complexes 1–4 and 7,
carboplatin and cisplatin with pBR322 plasmid DNA was
studied by agarose gel electrophoresis.[29] Stock solutions to
the tested complexes in dimethylformamide (DMF) were
prepared and used within 1 h. The final amount of DMF
never exceeded 0.1%. In brief, 40-μl aliquots of increasing
concentrations of the complexes, ranging from 0 to 160 μm,
were added to 1 μl of plasmid DNA (concentration of
0.5 μg/ml) in a buffer solution containing TE (10 mM Tris-
HCl, 0.1 mM EDTA, pH = 7.4). The samples were incubated
at 37°C for 24 h in the dark, and then 10-μl aliquots of
drug-DNA mixtures were mixed with loading buffer (0.1%
bromophenol blue, 0.1% xylene cyanol) and loaded into 1%
agarose gel with or without ethidium bromide. Electropho-
resis was carried out under TAE buffer (0.05 M Tris base,
0.05 M glacial acetic acid, 1 mM EDTA, pH = 8.0) for 5 h at
The carrier ligand L was prepared according to the Phillips
method,[24] as shown in Scheme 1, and its melting point was
in accordance with the literature.[25]
The procedures of the synthesis of [PtL2Cl2] and [PtL2I2]
platinum(II) complexes, which were synthesised in this
study, were also reported in our previous article.[19]
Briefly, dichloro-platinum(II) complex was synthesised
by the reaction of L and K2PtCl4 in ethanol/water solution.
IR and 1H NMR spectral data of the [PtL2Cl2] complex were
in accordance with the data reported previously.[19] Reaction
of K2PtCl4 with an excess amount of KI produced K2PtI4 in
solution. K2PtI4 was then reacted with two equivalents of L
to obtain diiodo-platinum(II) complex.
[PtL2I2] complex was treated with AgSO4 to obtain
monosulfato-monoaqua-2-hydroxymethylbenzimidazole
platinum(II) complex 1. The other platinum(II) complexes
[PtL2(oxalato)] 2, [PtL2(malonato)] 3, [PtL2(methyl
malonato)] 4 and [PtL2(ethylmalonato)] 5 were prepared by
1598
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Semra Utku et al.
Cytotoxic activity of platinum(II) complexes
Table 1 Cytotoxicity of benzimidazole-platinum(II) complexes 1–7, cisplatin and carboplatin on the HeLa, MCF-7 and MDA-MB-231 cell lines
Inh.% (μM SD)
Comp.
1
C (μM)
HeLa
9.90 0.47*,†
MCF-7
MDA-MB-231
80
40
20
10
5
10.12 1.77*,†
8.68 2.03*,†
6.39 1.32*,†
5.09 0.70*,†
3.10 0.57*
29.36 1.19†
25.79 0.56†
24.18 1.29*,†
22.00 1.42*,†
13.54 0.90†
15.62 1.64*,†
12.88 1.35*
10.47 2.06*
7.62 1.04*
12.54 1.07*,†
10.01 0.77*,†
7.35 1.15*
7.40 0.28*,†
5.78 0.36*,†
3.75 0.52*,†
1.88 0.96*,†
25.92 0.75*,†
19.63 1.01*,†
15.10 1.15*
9.86 0.53*,†
5.29 0.69*,†
18.95 1.43*
16.51 1.16*
13.87 0.78*
11.42 0.77*
6.65 1.02*,†
13.85 1.79*,†
9.03 0.78*,†
8.79 0.63*,†
6.51 1.07*,†
5.54 0.51*,†
11.37 0.65*,†
9.98 1.50*,†
7.65 1.01*,†
5.83 1.46*,†
3.65 1.79*,†
8.12 0.67*,†
5.04 1.03*,†
2.69 1.21*,†
0.86 0.72*,†
0.25 0.53*,†
16.31 0.50*,†
9.82 0.70*,†
6.26 0.26*,†
4.62 0.39*,†
2.72 1.03*,†
41.13 0.30*,†
37.41 0.41*,†
32.95 0.53*,†
24.45 1.16*,†
18.63 0.58*,†
18.56 1.26*,†
16.57 1.03*,†
14.31 0.93*,†
12.98 0.30*,†
9.63 1.14*,†
5.17 0.78*
3.88 0.85*
2
80
40
20
10
5
29.17 1.16*,†
24.11 1.69†
14.60 1.19*,†
7.51 0.60*,†
4.60 1.15*,†
15.27 0.53*
11.72 1.30*,†
8.35 0.53*
3
80
40
20
10
5
6.27 0.39*
4.62 1.29*
5.46 1.13*,†
11.77 0.59*,†
8.43 0.33*,†
6.07 0.48*,†
3.86 0.15*,†
2.46 0.72*
4
80
40
20
10
5
12.81 0.76*,†
10.96 1.35*,†
8.43 0.65*,†
5.95 0.60*,†
9.35 2.44*,†
13.88 0.26*,†
12.79 0.27*
11.73 0.19*
9.24 0.44*
5
80
40
20
10
5
9.13 0.97*,†
7.70 0.79*,†
5.46 0.96*,†
3.02 0.72*,†
1.52 0.65*
6.40 0.63*
6
80
40
20
10
5
6.23 0.15*,†
2.91 0.68*,†
2.60 0.12*,†
0.44 0.71*,†
0.15 0.19*,†
8.34 0.50*,†
6.76 0.52*,†
4.41 0.45*,†
1.92 0.52*,†
1.01 0.45*,†
28.09 0.73*,†
24.75 0.79*,†
18.75 1.17*,†
16.00 0.63*,†
13.87 1.02*,†
18.38 1.01*,†
15.07 0.56*,†
12.20 0.94*,†
9.76 1.48*,†
4.61 0.69*,†
6.89 0.43*,†
4.13 0.34*,†
2.19 0.63*,†
0.86 0.12*,†
0.46 1.10*,†
13.83 1.91*,†
9.77 0.83*,†
6.63 1.09*
7
80
40
20
10
5
3.86 0.31*,†
0.67 0.60*
Cisplatin
Carboplatin
80
40
20
10
5
26.56 0.86*,†
24.93 1.04*,†
19.70 0.91*,†
14.69 0.92*,†
11.36 0.71*,†
16.49 0.50*,†
14.07 0.70*,†
9.13 1.86*,†
5.34 0.77*,†
2.16 0.65*,†
80
40
20
10
5
Results are expressed as mean standard deviation (SD) from three independent experiments. One-way analysis of variance followed by Dunnett’s
post-hoc test was used for statistical analysis. *Denotes P < 0.001 compared with cisplatin, and †denotes P < 0.05 compared with carboplatin on
using cell lines.
reaction of the [PtL2Cl2] complex with the disilver salt of
dicarboxylic acid (oxalic, malonic, methylmalonic or
ethylmalonic acid) derivatives, respectively, as shown in
Scheme 2.
We have synthesised the other series of neutral
malatoplatinum(II) complexes of [PtL2(DL-malato)] (6)
and [PtL2(D-malato)] (7) by the reaction of [PtL2I2] with
disilver salt of DL or D-malic acid.
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1593–1605
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Cytotoxic activity of platinum(II) complexes
Semra Utku et al.
Table 2 Cytotoxicity of complex 2, cisplatin and carboplatin in combination with estrogen, progesterone and estrogen + progesterone in MCF-7
and MDA-MB 231 cancer cell lines
Cell line
Comp.
2
MCF-7 Inh.% (μM SD)
Complex + estrogen Complex + progesterone
C (μM)
Complex
Complex + estrogen + progesterone
100
50
42.36 1.07*
30.71 1.72*
31.01 0.38*
21.68 2.13*
13.88 0.62*
11.93 0.81*
42.18 1.01*
32.63 0.81*
30.34 0.84*
22.09 0.79*
14.21 0.04*
11.29 0.37*
39.78 1.90*
34.53 0.62*
30.73 0.24*
23.08 0.50*
14.39 0.20*
13.81 0.46*
33.77 0.23*
36.03 1.29*
31.45 0.41*
23.90 0.54*
15.62 0.78*
13.62 0.34*
Cisplatin
100
50
Carboplatin
100
50
Cell line
Comp.
2
MDA-MB 231 Inh.% (μM SD)
C (μM)
Complex
Complex + estrogen
Complex + progesterone
Complex + estrogen + progesterone
100
50
40.27 0.66
35.04 0.54
39.91 0.19*
35.19 0.74*
15.72 0.54*
13.67 0.36*
39.00 0.87
33.40 1.46*
39.18 0.71*
36.03 0.90*
15.18 0.98*
13.77 0.57*
38.09 0.93
33.85 0.30
39.12 0.76*
33.90 0.67*
14.75 0.47*
13.53 0.37*
34.38 0.52*
34.33 0.87*
39.62 0.37*
28.12 0.70*
19.21 1.04*
15.89 0.71*
Cisplatin
100
50
Carboplatin
100
50
Results are expressed as mean standard deviation (SD) from three independent experiments. Two-way analysis of variance followed by Dunnett’s
post-hoc test was used for statistical analysis. *Denotes P < 0.001, significantly different from cisplatin and carboplatin.
The structures of [PtL2Cl2], [PtL2I2] and complexes 1–7
The large downfield shifts in the benzimidazole NH
signal in the spectra of the complexes with respect to the
ligand are a result of an increase in the NH acid character
after platinum binding.[32] Their NH chemical shifts vary
between 13.56 and 13.10 ppm. Therefore, the signals of
–CH2 or –CH groups of the dicarboxylate derivatives were
often weaker (or not seen) than expected for these proton/
protons exchanged quite rapidly with the D atoms of the
solvent DMSO-d6. Therefore, the spectrum showed a low
intensity multiplet for the –CH2- or –CH- (or -CDH-,
-CD2- or -CD-), which is consistent with the literature.[33,34]
1HNMR spectra of 6 DL-maloto complexes showed
broad singlets at 6.15 ppm for OH protons but did not
appear for D-maloto complex 7.
are shown in Schemes 1 and 2, respectively. All complexes
1
were characterised by elemental analysis, IR and H NMR
spectroscopic techniques. The elemental analyses data for
each complex were in good agreement with the empirical
formula proposed.
In the IR spectrum L, a very broad band was seen in the
region of 3400–2500 cm−1 due to the imidazole NH.
Platinum(II) complexes 1–7 exhibited υ (NH) stretching
bands centred at 3230 cm−1 sharper than that of free ligands
due to breaking of tautomerism, indicated that the N-H
group was not involved in the coordination.[30,31]
The carboxylate group of the complexes 2–7 exhibited
a band in the range 1610–1692 cm−1 corresponding to
the asymmetric carboxylate υa (COO−) band and 1376–
1403 cm−1 symmetric carboxylate υs (COO−) stretching
band. Trends in the positions and separation between these
bands are the most useful tools in assigning structures from
IR spectra. The structure of the complexes was confirmed
by the appearance of the peak of υ Pt-O band in the range
of 548–584 cm−1.
Cytotoxicity
As an extension of our previous studies on benzimidazole
Pt(II) complexes, this study evaluates the in-vitro cytotoxic
activity of Pt(II) complexes with 2-hydroxymethyl
benzimidazole carrier ligand and oxalate, malonate,
methylmalonate, ethylmalonate, DL-malate or D-malate
leaving ligands on the human HeLa, MCF-7 and MDA-MB-
231 cell lines.
The growth inhibitory effects of platinum(II) complexes
1–7, the reference compounds cisplatin and carboplatin
against the cell lines used were measured by the MTT assay.
The cytotoxic activity values of platinum complexes 1–7
and the reference compounds, expressed as inhibition
(inh.)%, are presented in Tables 1 and 2.
The insolubility of the complexes in the other organic
1
solvents made it necessary to record H NMR spectra in
DMSO-d6. All 1H NMR measurements were recorded
immediately to avoid the ligand exchange reaction between
the platinum complexes synthesised and DMSO-d6. The 1H
NMR spectral data of the ligand and complexes are pre-
sented in the experimental section. Almost all signals were
shifted upon complexation as the result of the electric field
effect caused by complexation.
1600
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1593–1605

Semra Utku et al.
Cytotoxic activity of platinum(II) complexes
In the test on the cell lines used, the cytotoxicity of
complex 2 bearing oxalate leaving ligand, which was found to
be the most active complex among the benzimidazole-
platinum complexes tested, was approximately two or five-
fold greater than those of the other benzimidazole-platinum
complexes tested. Complex 2 was found to be cytotoxic equal
to cisplatin against the MCF-7 and MDA-MB-231 cell lines.
It may be concluded that the potency of the platinum(II)
complexes tested may also be leaving group dependent.
The complex 3 bearing non-substituted malonate leaving
ligand was found to be more active than the other two com-
plexes 4, 5, bearing methyl or ethyl substituted malonate
ligands, respectively, for all cell lines used.
In general, complexes 2–5 showed higher activity against
the MCF-7 (ER+) cell line than did to the MDA-MB-231
(ER−) cell lines.
It is interesting to note that complex 7 having D-malate
leaving ligand was found to be more active than the
complex 6 having DL-malate against all cell lines used. It is
also interesting to note that the two complexes, particularly
complex 7 (D-malate), were found to be more active against
the (ER−-) cell lines HeLa and MDA-MB 231 than the
(ER+) cell line MCF-7.
To determine whether estrogen enhances platinum sensi-
tivity, in-vitro cytotoxic activity of complex 2, which was
found to be the most active among the tested complexes,
was investigated in combination with estrogen, progester-
one and estrogen + progesterone in MCF-7 and MDA-MB
231 cancer cell lines.
In MCF-7 cells, estrogen or progesterone co-treatment
slightly increased the cytotoxicity of complex 2, cisplatin
and carboplatin compared with the complex tested alone in
50 μm concentration. Particularly, the effect of combination
of estrogen–progesterone and complex 2 was more evident
in MCF-7 cells. Because estrogen has been implicated in the
aetiology of breast cancer owing to its proliferative proper-
ties, progesterone may be preferred for patients with breast
cancer.
In general, inh.% of the platinum complexes tested
in combination with estrogen or/and progesterone
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
4
5
6
7
8
9 10 11 12
P
1 2 3 4 5 6 7 8 9 10 11 12
1
2
3
1
2
3
4
5
6
7
8
9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
4
7
1
2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10111213141516
Cisplatin
Carboplatin
Figure 1 Modification of gel electrophoretic mobility of pBR322 plasmid DNA when incubated with various concentrations of complexes 1–4, 7,
cisplatin and carboplatin. Concentrations (in μM) are as follows: (lines 1 and 12) untreated pBR322 plasmid DNA; For complexes 1–4 and 7 and
carboplatin: (line 2) 0.312; (line 3) 0.625; (line 4) 1.25; (line 5) 2.5; (line 6) 5; (line 7) 10; (line 8) 20; (line 9) 40; (line 10) 80; (line 11) 160. For
cisplatin: (lines 1 and 16) untreated pBR322 plasmid DNA; (line 2) 0.019; (line 3) 0.039; (line 4) 0.078; (line 5) 0.156; (line 6) 0.312; (line 7) 0.625;
(line 8) 1.25; (line 9) 2.5; (line 10) 5; (line 11) 10; (line 12) 20; (line 13) 40; (line 14) 80; (line 15) 160. The top and the bottom bands correspond to
form II (open circular) and form I (covalently closed circular) plasmids, respectively.
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1593–1605
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Cytotoxic activity of platinum(II) complexes
Semra Utku et al.
were lower than that of the complexes alone in
MDA-MB 231 cell, except for carboplatin in combination of
estrogen–progesterone.
pBR322 plasmid DNA was used as a control. No significant
pBR322 plasmid DNA binding affinity was observed for
complexes 5 and 6 (data not shown).
In this work, using in-vitro assays, it was shown that
estrogens, combined with cisplatin, were able to enhance
cytotoxicity slightly in the MCF-7 cell line.
Nuclear DNA is an important molecular target for plati-
num complexes, which bind purine bases at the N7 posi-
tion. The resulting Pt-DNA damage triggers downstream
effects, including inhibition of replication and transcrip-
tion. If the cell cannot remove the damage, then it dies by
one of several pathways.[35]
It is widely documented that platinum-based complexes
can untwist or bend plasmid-DNA, depending on the kind
of specific adducts formed on the DNA.[36] Monofunctional
or intercalating adducts may untwist dsDNA, whereas
bifunctional adducts and intra- and interstrand cross-links,
in addition, bend DNA.
A decrease in the rate of migration of Form I bands is the
result of untwisting the DNA, as this reduces the number of
supercoils. It can be seen in Figure 1 that compound 2–4
and cisplatin caused a significant decrease in the rate of
migration of the Form I band of the pBR322 plasmid DNA.
In contrast, complexes 1 and 7 and carboplatin did not
untwist the DNA significantly.
Interaction with pBR322 plasmid DNA
To detect whether complexes 1–4 and
7 induced
conformational changes on the DNA helix, and whether
there was a relationship between the plasmid DNA binding
affinity and the cytotoxicity of the complexes, we investi-
gated their capacity to remove and reverse the supercoiling
of closed circular pBR322 plasmid DNA, as assessed by
electrophoretic mobility measurements on agarose gels.
Figure 1 shows the electrophoretic mobility pattern of the
covalently closed circular Form I and open circular Form II
bands of pBR322 plasmid DNA after incubation with
compounds in a range of concentrations from 0 to 160 μm
at 37°C for 24 h. In the electrophoretograms, the untreated
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
1
2
3
4
5
6
7
8
9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
4
7
1 2 3 4 5 6 7 8 9 10111213141516
1
2
3
4
5
6
7
8
9 10 11 12
Cisplatin
Carboplatin
Figure 2 Electrophoretograms for the BamHI digested mixtures of pBR322 plasmid DNA after their treatment with various concentrations of com-
plexes 1–4, 7, cisplatin and carboplatin. Concentrations (in μM) are as follows: (lines 1 and 2) untreated pBR322 plasmid DNA and pBR322 DNA
linearised by BamHI, respectively. For complexes 1–4 and 7 and carboplatin: (line 3) 0.312; (line 4) 0.625; (line 5) 1.25; (line 6) 2.5; (line 7) 5; (line 8)
10; (line 9) 20; (line 10) 40; (line 11) 80; (line 12) 160. For cisplatin, (line 3) 0.019; (line 4) 0.039; (line 5) 0.078; (line 6) 0.156; (line 7) 0.312; (line 8)
0.625; (line 9) 1.25; (line 10) 2.5; (line 11) 5; (hat 12) 10; (line 13) 20; (line 14) 40; (line 15) 80; (line 16) 160. The top, the bottom and the middle
bands indicate form I (covalently closed circular) and form II (open circular) and form III (linear) plasmids, respectively.
1602
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Semra Utku et al.
Cytotoxic activity of platinum(II) complexes
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
1
2 3 4 5 6 7 8 9 10 11 12
1
2 3 4 5 6 7 8 9 10 11 12
4
7
1
2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10111213141516
Cisplatin
Carboplatin
Figure 3 Electrophoretograms for the HindIII digested mixtures of pBR322 plasmid DNA after their treatment with various concentrations of com-
plexes 1–4, 7, cisplatin and carboplatin. Concentrations (in μM) are as follows: (lines 1 and 2) untreated pBR322 plasmid DNA and pBR322 DNA
linearised by HindIII, respectively. For complexes 1–4 and 7, and carboplatin: (line 3) 0.312; (line 4) 0.625; (line 5) 1.25; (line 6) 2.5; (line 7) 5; (line 8)
10; (line 9) 20; (line 10) 40; (line 11) 80; (line 12) 160. For cisplatin, (line 3) 0.019; (line 4) 0.039; (line 5) 0.078; (line 6) 0.156; (line 7) 0.312; (line 8)
0.625; (line 9) 1.25; (line 10) 2.5; (line 11) 5; (hat 12) 10; (line 13) 20; (line 14) 40; (line 15) 80; (line 16) 160.
The concentrations required to achieve total unwinding
of the pBR322 plasmid DNA for complexes 2, 3, 4 and
cisplatin were 160, 20, 160 and 10 μm, respectively. The
presence of a coalescence point indicates a strong unwind-
ing of the supercoiled DNA.
When pBR322 plasmid DNA interacted with complexes
1–4, 7, cisplatin and carboplatin, followed by BamHI or
HindIII, it was observed that complexes 2–4 and cisplatin
prevent enzyme digestion at high concentrations indicated
by binding the complexes A/A and A/G site of DNA
(Figures 2 and 3).
BamHI and HindIII restriction enzymes activity. of pBR322
plasmid DNA modified and then the inhibition of BamHI
and HindIII restriction enzymes.
The growth inhibitory effect results showed that the
cytotoxicity of complex 2 bearing oxalate leaving ligand was
found to be the most active complex among the synthesised
complexes (1–7) on the cell line used. The complex 2 was
found to be cytotoxic equal to cisplatin and higher than
carboplatin against the MCF-7 and MDA-MB-231 cell lines.
Furthermore, to determine whether estrogen enhances
platinum sensitivity in MCF-7 and MDA-MB 231 cells, the
estrogen or progesterone co-treatment slightly increased the
cytotoxicity of complex 2, cisplatin and carboplatin com-
pared with the complex 2 tested alone in 50-μm concentra-
tion. Especially, combination of estrogen–progesterone with
complex 2 was more obvious in MCF-7 cells.
The results of the plasmid DNA interaction and the
restriction studies suggest that complexes 1–4,7 modified
the tertiary structure of pBR322 plasmid DNA, and com-
plexes 2–4 prevent enzyme digestion at high concentrations
indicated by binding the complexes A/A and A/G site of
DNA.
Conclusion
The aim of this study was to evaluate the effect of the con-
version of leaving ligand moieties of dichloro Pt(II)
complex into bidentate dicarboxylato groups moieties
(complex 2–7), in-vitro cytotoxic activity against the
human HeLa (ER−), MCF-7 (ER+) and MDA-MB 231
(ER−) cell lines. It also aims to determine whether the com-
plexes 1–4, 7 modified the tertiary structure of pBR322
plasmid DNA interaction, and then the inhibition of
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1593–1605
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Cytotoxic activity of platinum(II) complexes
Semra Utku et al.
It can be also concluded that the in-vitro cytotoxic activ-
ity of complex 2 against the MCF-7 and MDA-MB 231 cell
lines is noteworthy and must be taken into consideration in
future studies.
Acknowledgements
This work was supported by Research Foundation of Gazi
University (EF 02/2007-24). The authors are grateful to Dr
Bahar Tas¸delen (Mersin University, Faculty of Medicine
Department of Biostatistics and Medical Informatics) for
helpful statistical analysis support.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
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mination by accelerator mass spec-
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