Improving of Anticancer Activity and Solubility of Cisplatin by Methylglycine and Methyl Amine Ligands Against Human Breast Adenocarcinoma Cell Line

Article abstract of DOI:10.1007/s12010-018-2715-5

Methylglycine, also known sarcosine, is dramatically used in drug molecules and its metal complexes can interact to DNA and also do cleavage. Hence, to study the influence of methylglycine ligand on biological behavior of metal complexes, two water-solubl

Full text of DOI:10.1007/s12010-018-2715-5

Appl Biochem Biotechnol
https://doi.org/10.1007/s12010-018-2715-5
Improving of Anticancer Activity and Solubility
of Cisplatin by Methylglycine and Methyl Amine Ligands
Against Human Breast Adenocarcinoma Cell Line
Fatemeh Safa Shams Abyaneh1
&
Mahboube Eslami Moghadam2 & Adeleh Divsalar3
Davood Ajloo4 & Moyaed Hosaini Sadr1
&
Received: 15 November 2017 /Accepted: 6 February 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract Methylglycine, also known sarcosine, is dramatically used in drug molecules and its
metal complexes can interact to DNA and also do cleavage. Hence, to study the influence of
methylglycine ligand on biological behavior of metal complexes, two water-soluble platinum
(II) complexes with the formula cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-
gly)]NO3 (where CH3-gly is methylglycine) have been synthesized and characterized by
spectroscopic methods, molar conductivity measurements, and elemental analyzes. The anti-
cancer activity of synthesized complexes was tested against human breast adenocarcinoma cell
line of MCF7 using MTT assay and results showed excellent anticancer activity with Cc50
values of 126 and 292 μM after 24 h incubation time, for both complexes of
cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3gly)]NO3, respectively. Also, the
interaction between Pt(II) complexes with calf thymus DNAwas extensively studied by means
of absorption spectroscopy, fluorescence titration spectra displacement with ethidium bromide
(EtBr), and circular dichroism studied in Tris-buffer. The obtained spectroscopic results
revealed that two complexes can bind to highly polymerized calf thymus DNA cooperatively
and denature at micromolar concentrations. The fluorescence data indicate that quenching
effect for cis-[Pt(NH3)2(CH3gly)]NO3 (Ksv = 9.48 mM−1) was higher than that of cis-[Pt(NH2-
CH3)2(CH3gly)]NO3 (Ksv = 1.98 mM−1). These results were also confirmed by circular
dichrosim spectra. Consequently, docking data showed that cis-[Pt(NH3)2(CH3gly)]NO3 with
* Mahboube Eslami Moghadam
eslami_moghadam@ccerci.ac.ir; eslami_moghadam@yahoo.com
1
Department of Chemistry, Science and Research branch, Islamic Azad University, Tehran, Iran
2
Chemistry & Chemical Engineering Research Center of Iran, Tehran, Iran
3
Department of Cell & Molecular Sciences, Faculty of Biological Sciences, Kharazmi University,
Tehran, Iran
4
School of Chemistry, Damghan University, Damghan, Iran

Appl Biochem Biotechnol
more interaction energy binds on DNA via groove binding which is more compatible with
experimental results.
.
.
.
.
.
Keywords Cisplatinanalog Anticancerdrug Cytotoxicity Methylglycine DNAinteraction
.
Thermodynamic parameters Molecular docking
Abbreviations
CT-DNA
Calf thymus DNA
Methylgly Methyl glycine
CD
EB
Circular dichroism
Ethidium bromide
Introduction
Cisplatin, Cis-[Pt(NH3)2Cl2] has been widely used as anticancer drugs for treatment of
testicular and solid malignancies like bone, neck, and bladder cancer [1]. Due to the dose
limiting toxic side effects of cisplatin, much current research work is aimed at the discovery
and design of new platinum complexes which are not cross-resistant with cisplatin and have
also fewer toxic side effects and natural clinical activity against a broad range of types of
cancer [2, 3]. However, an effective oral formulation of cisplatin is not always achievable
because of its poor water solubility and low level of bioavailability of attached ligands [4]. For
overcoming the serve side effects and improve clinical effectiveness, the Pt(II) compounds
should be tailored to meet the various requirements such as water solubility, chemical stability,
biodegradability, and tumor targeting [5]. So, with choosing an appropriate attached ligand
sphere, activity of the drugs should be increased, where as it is acceptable to decreased their
general toxicity [6].
Hence, amino acids are the fundamental material of life and the basis of metabolism [7].
Introducing amino acid into an antitumor drug can improve selectivity to tumor cells, enhance
their liposolubility, and remit their toxicity to normal cells [8]. Indeed, certain tumors and
cancer cells are unable to produce all the synthesized amino acids by the normal cells.
Therefore, these cells require an external supply of such essential amino acids to pass on to
the cancer cells by the blood stream [9]. Furthermore, the amino acids especially the first of
them, glycine are very important compounds for the transfer inside the cell of biologically
active alkylating agents [10]. In the recent past, numerous of studies have widely used the
glycine ligand in the synthesis of Pt(II) complexes and their structure has highlighted in
various significant applications [11–13].
In the present work, design, synthesis, and characterization of two new cationic cisplatin
analog (see Scheme 1) were bearing with glycine amino acid derivative as chelating ligands
are reported [14]. The biological activity of two synthesized Pt complexes were tested against
the human breast adenocarcinoma cancer model cell line of MCF7 and showed excellent
cytotoxicity effects at micromolar concentration. The binding mechanism of these water-
soluble Pt(II) complexes with calf thymus DNA under simulated physiological conditions
(pH 7.4) has also been investigated by various spectroscopic techniques including UV-Vis
spectroscopy, fluorescence measurements and circular dichroism. Moreover, to quantitatively
analyze the interaction of Pt complex DNA, the relative thermodynamic characteristics were
investigated. Theoretical methods such as computer simulations and docking could be

Appl Biochem Biotechnol
Scheme 1 Proposed structure of
a cis-[Pt(NH3)2(CH3-gly)]NO3
and b cis-[Pt(NH2-CH3)2
(CH3-gly)]NO3
CH3
a
b
H3N
H3N
NH CH2
NO3
Pt
O
O
CH3
H2
H3C
H3C
N
NH CH2
NO3
Pt
O
O
N
H2
examined the interactions and described important features for DNA complex recognition. So,
binding affinities of the new complexes interactions were also estimated by docking calcula-
tions [15].
Experimental Procedures
Highly polymerized calf thymus DNA sodium salt and Tris-HCl (tris (hydroxymethyl) amino
methane hydrochloride were purchased from Aldrich Co. (England). Acetone, ethanol, and
anhydrous ether, methyl amine and potassium tetrachloride palatinate, barium chloride, sodium
chloride, sodium bicarbonate, sodium hydroxide, hydrochloric acid, sulfuric acid, and silver
nitrate were purchased from Merck Co. (Germany) used for synthesis complexes. Moreover,
methylglycine ligand was made according to the procedure previously described [14].
Spectra of 1H NMR were recorded using a Bruker Avance spectrometer at 300 MHz, using
DMSO-d6 as the solvent and TMS as the internal reference. Infrared spectra of these
complexes were determined on a Perkin Elmer-10.20.00 plus FT-IR spectrophotometer in
the range of 400–4000 cm−1 in KBr pellets. UV-Vis spectra were recorded on SPEKOL 2000
spectrophotometer. Conductivity of the synthesized complexes was measured on a Metrohm-
712 conduct meter, using water as the solvent. Elemental analysis of carbon, hydrogen, and
nitrogen was carried out on an elemental analysis of CHN Rapid Heraus. Circular dichroism
measurements were recorded on an Aviv-215 spectrophotometer and fluorescence measure-
ments were carried out with a JASCO FP- 6500 spectrophotometer.
Synthesis of Pt(II) Complexes
Preparation of two new platinum complexes was described as follow (Scheme 2):
Â
Ã
Cis− PtðNH3Þ2ðCH3−glyÞ NO3 : ðM:W ¼ 381:9 g=molÞ

Appl Biochem Biotechnol
H2
N
H2
N
R
R
OH2
OH2
R
R
I
2AgNO3
NO3
Pt
2AgI
+
2
Pt
H2O/Aceton
N
I
N
H2
H2
+
CH3
H2
O
CH3
R
N
NH CH2
N
H
NO3
Pt
O
O
O
R
N
H2
R=H or methyl
Scheme 2 Schematic reaction of synthesis of two new Pt complexes: cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-
[Pt(NH2-CH3)2(CH3-gly)]NO3
This complex was synthesized by [Pt(NH3)2I2] made according to the reported method
[16]. One hundred milligrams [Pt(NH3)2I2] (0.2 mmol) and 67 mg AgNO3 (0.4 mmol) were
dissolved and refluxed in 120 mL of double distilled water/acetone (1/3, v/v). The mixture
reaction was refluxed at 50 °C for 24 h and string continued for 12 h at room temperature
under darkness. After filtering the suspension and removing the AgI precipitate, aqua solution
was mixed with 25 mg methylglycine (0.2 mmol) and 33 mg NaHCO3 (0.4 mmol) dissolved
in 10 mL distilled water at 40 °C. Then, the solution was evaporated and concentrated to 5 mL
at 35 °C. The brown precipitate was formed and washed with distilled water and acetone and
dried in a vacuum oven at room temperature. The yield of reaction was 51%. Analytical
calculated for C3H11N4O5Pt compound (378.08) is C, 9.52; H, 2. 9; N, 14.81%. Analytical
found: C, 9.83; H, 2.57; N, 14.59%. UV Band maxima in nanometer ( M in liter mol−1 cm−1
×
−1
10−4): 200 (29.1). Molar conductance, M (10−4 M, H2O): 162.4 Ω cm2 mol−1. IR (cm−1,
1
KBr disk): 3419 (s, N-H), 2968 (w, C-H), 1630 (s, C=O), 1381 (s, (NO3)−), H NMR
(300 MHz, DMSO-d6, δ in ppm): 2.6345 (s, 2H), 1.64 (m, 4H), 1.21(m, 4H), 2.9(m, 7H NH).
Â
Ã
Cis− PtðNH2−CH3Þ2I2 : ðM:W: ¼ 510:19 g=molÞ
41.5 mg (0.1 mmol) K2PtCl4 was dissolved in 200 μl double distilled water and then
100 mL solution of 0.1 g KI (0.6 mmol) was added to dark red of K2PtCl4 solution, the
mixture was stirred continuously at 35 °C for 1 h until dark brownish red appeared. Thirty
microliters (0.2 mmol) methyl amine was added drop wise to the following solution. The light
yellow precipitates were filtered and washed with a small volume of ice water and acetone,
respectively. The formed cis-[Pt(NH2-CH3)2I2]was dried at vacuum oven for 1 day. The yield
of synthesis reaction was 73%. IR (cm−1, KBr disk): 3213 (s, N-H), 2955 (s, C-H).
Â
Ã
Cis− PtðNH2−CH3Þ2ðCH3−glyÞ NO3 : ðM:W: ¼ 406:08 g=molÞ
One hundred milligrams cis-[Pt(NH2-CH3)2I2] (0.195 mmol) was suspended in 120 mL
of double distilled water/acetone (1/3, v/v). The solution of 66 mg of AgNO3 (0.39 mmol)
in 10 mL water was added slowly to this suspension with continuous stirring. The reaction
mixture was refluxed at 50 °C for 24 h and cooled at the room temperature for 12 h under

Appl Biochem Biotechnol
darkness. Then, the mixture was filtered to remove gray AgI precipitate by centrifugation.
This filtrated solution was mixed with 24 mg synthesized methylglycine hydrochloride
(0.1 mmol) and 32 mg NaHCO3 (0.39 mmol) dissolved in 5 mL distilled water. The
reaction mixture was further stirred at 45 °C for 3 h. The reaction mixture was concen-
trated to 1 mL until the withe precipitate was formed. The product obtained was filtered
and washed with small amounts of chilly distilled water and acetone and dried in a
desiccator. The reaction yield of complex was 51%.
Analytical calculated for C5H15N4O5Pt compound (406.08) is C, 14.77; H, 3.69; N, 13.79%.
Analytical found: C, 14.65; H, 3.77; N, 13.59%. UV Band maxima in nanometer ( M in liter
mol−1 cm−1 × 10−4): 205 (29.8). Molar conductance, M (10−4 M, H2O): 178.5 Ω −1 cm2 mol−1.
IR (cm−1, KBr disk): 3204 (s, N-H), 3121(s, C-H), 1606 (s, C=O), 1360 (s, (NO3)−), 1H NMR
(300 MHz, DMSO-d6, δ in ppm): 2.21(m, 9H), 2.24 (m, 2H), 5.3729(m, 5H NH).
In Vitro Cytotoxicity Studies
Cell Culture
Human breast cancer cell line of MCF7 was purchased from the National Cell Bank of
Iran (NCBI), Pasteur Institute of Iran. The cells were grown on the DMEM medium
(Sigma) supplemented with L-glutamine (2 mM), streptomycin, penicillin (5 μg/ml), and
10% heat-inactivated fetal calf serum at 37 °C under a 5% CO2/95% air atmosphere.
Cell Proliferation Assay
The anticancer and antiproliferative activity of the designed Pt(II) complexes was measured
by MTT assay. The cleavage and conversion of the soluble yellowish MTT to the insoluble
purple formazan by active mitochondrial dehydrogenase of living cells have been used to
develop an assay system alternativeto other assays for measurement of cell proliferation. The
harvested cells were seeded into a 96-well plate (1 × 104 cell/ml) and were left to adhere
overnight. Prior to the experiments, the cells were twice washed with phosphate-buffered
saline (PBS). Then, the MCF7 cancer cells were incubated with different concentrations of
sterilized both of the Pt(II) complexes (0–350 μM) and incubated for 24 h. Four hours to the
end of the incubation, 25 μL of the MTT solution (5 mg/ml in PBS) was added to each well
containing fresh and cultured media. At the end, the insoluble formazan produced was
dissolved in a solution containing 10% SDS and 50% DMF (left for 2 h at 37 °C in darkness)
and optical density (OD) was read against reagent blank with multi-well scanning spectro-
photometer (ELISA reader, Model Expert 96, Asys Hitech, Austria) at the wavelength of
570 nm. The OD value of the study groups was divided by the OD value of the untreated
control and presented as the percentage of control (as 100%).
Metal Complexes Interaction with Calf Thymus DNA
Absorption spectroscopy is one of the most commonly useful techniques to study interaction
of any metallodrug to CT-DNA [17]. The application of UVabsorption is based on monitoring
the difference absorption changes upon addition of platinum (II) complexes. The stock
solution of DNA was prepared by dissolving 4 mg of DNA in 20 mLTris-buffer as accordance

Appl Biochem Biotechnol
to the reports. Here, due to no absorption for Pt complexes in UV-Vis range, the ΔΑs for each
titration of metal complex was recorded at λmax (258 nm) of DNA [18].
UV-Vis Spectroscopic Experiment of DNA Denaturation
In this experiment, the reference cell was filled with 1.8 mL Tris-buffer and sample cell was
filled with 1.8 mL DNA (0.08 mM). Both cells were settled at constant temperature of 300 and
310 K which were separately titrated with various concentrations of each platinum (II)
complex. After 1 min, the absorption was recorded at 258 nm for DNA and at 640 nm to
eliminate the interference of turbidity. The injection of metal complex to both cells was
continued until no further changes in the absorption readings were observed; these absorption
readings of DNA solution were plotted separately versus different concentrations of each metal
complex. Thermodynamic parameters like molar Gibbs free energy (ΔG°), molar enthalpy
(ΔH°), and molar entropy of DNA binding with complex (ΔS°) were obtained by using the
Pace method. The application of UV absorption method to the study of interaction of DNA
with Pt(II) complex is similar to that reported earlier [19].
Thermal Denaturation Studies
Sequence differences between two different DNA sequences can also be detected by
using DNA denaturation. DNA is heated and denatured into single-stranded state, and the
mixture is cooled to permit strands to rehybridize. This experiment was contested in to
Tris-buffer using continues heating from 25 to 100 °C. The growth in the temperature
was ~2 °C/min. The change of absorbance difference was recorded at 258 nm. Since
examined, the profile of absorbance agent temperature is almost linear in the melting
temperature [20].
Fluorescence Quenching Studies
A quantitative analysis affinity of the Pt(II) complexes was bound to DNA was carried out by
luminescence titration method with JASCO spectrofluorimeter (FP6200). A fixed solution of
DNA (60 μmol L−1) and ethidium bromide (2 μmol L−1) was titrated by various concentra-
tions of each Pt complex (50, 100, 150, 200, 250, 300, 350, and 400 μmol L−1). The emission
spectra for both Pt(II) complexes were recorded between 540 and 700 nm with excitation
wavelength at 471 nm. The fluorescence intensities of Pt(II) complexes at 471 nm excitation
wavelength have been checked which was small and negligible. This experiment, fluorescence
emission spectra of intercalated ethidium bromide with increasing of each Pt(II) complexes,
was observed [21, 22].
Circular Dichroism Studies
Positive band of DNA due to base stacking (273 nm) and negative one due to right-handed
helicity (246 nm) are quite sensitive to interaction mode of DNA small molecules [23]. So,
CD spectroscopy is a worthwhile tool to diagnose change in DNA morphology during
drug-DNA interactions. CD measurements were conducted on JASCO (J-810) spectropo-
larimeter by keeping the concentration of DNA constant (5 × 10−5 M) while varying the
complex concentration from 0.48 to 1.09 mM at room temperature [24].

Appl Biochem Biotechnol
Docking Studies
AutoDock 4.2 program was used to obtain the energetic and binding site for the interaction of
the titled complexes with DNA. Docking simulation was done in a box with dimension of
52 × 100 × 80 points and the spacing of 0.375 (maximum number of generations and energy
evaluation were set to 27,000 and 2.5 × 105, respectively. For each run, 250 docking energies
were reported with the initial population of 250 individuals [25].
Result and Discussion
Determination of the Binding Parameters
Electronic absorption spectroscopy is one of the most important techniques for DNA-binding
studies of platinum complexes. The final result of interaction of CT-DNA with
cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 are shown in Fig. 1 and
summarized in Table 1. DNA denaturation experiment was done by looking at the systematic
changes in the UV absorption spectrum of DNA solution at 258 nm upon addition of two
platinum complexes. Addition of metal complex and the absorption monitoring was continued
until no further changes in the absorption reading were observed at two different temperatures
of 300 and 310 K [26]. Some thermodynamic parameters were discussed by using DNA
denaturation plots (Fig. 1) and the Pace method. The value of K unfolding equilibrium constant
and ΔGo(H2O), unfolding of free energy of DNA in the presence of cis-[Pt(NH3)2(CH3-
gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 have been calculated by Eqs. (1) and (2)
[27], where Aobs, AN, and AD are the absorbance readings in the transition region, the natural
and denatured conformations of DNA, respectively.
An−Aobs
Aobs−An
K ¼
ð1Þ
ΔG° ¼ −RTLnK
ð2Þ
The straight line is determined when ΔG° are plotted against concentration of
cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 in transition region at 300
and 310 K. These plots are shown in Fig. 2. The value of m, that is that is slop of each group in the
same plots (a measure of platinum complex ability denature DNA) and intercept of each plot is,
ΔG° (conformational stability of DNA in the absence of platinum complexes) [28]. The m value of
cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 system is higher than those of other Pt(II) complex in room
temperature which indicate the much higher ability of cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 to
denature in 300 K temperature. As we know, the higher value of ΔG°H2O indicate a large
systematic conformational stability of DNA. However, the value of ΔGo decreases by increasing
the temperature for cis-[Pt(NH2-CH3)2(CH3gly)]NO3 because most of the macromolecules are less
stable at higher temperature. Another important thermodynamics parameter is the molar enthalpy
of DNA denaturation in the absence and presence of cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-
CH3)2(CH3gly)]NO3 complexes. ΔHoconformational or ΔHodenaturation is calculated in the range of 300

Appl Biochem Biotechnol
a
1.6
1.4
1.2
1
300 K
310 K
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
[L]t (mM)
b
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
300 K
310 K
0
0.2
[L]t (mM)
0.4
0.6
Fig. 1 The changes of absorbance of DNA at λmax = 258 nm due to increasing total concentration of cis-
[Pt(NH3)2(CH3-gly)]NO3 insert with cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex in (ο) 300 and (•) 310 K
to 310 K temperature by using the Gibbs-Helmholtz equation as follows [29]:
ΔH° ¼ ðΔG°T =T1−ΔG°T =T2Þ=ð1=T1−1=T2Þ
ð3Þ
1
2
On plotting the value of these enthalpies against the concentration of each Pt(II) complex,
straight lines will be obtained which are shown in Fig. 3 for cis-[Pt(NH3)2(CH3-gly)]NO3 and
insert for cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex. Interpolation of these lines (interception

Appl Biochem Biotechnol
Table 1 Thermodynamic parameters of CT-DNA denaturation by cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-
[Pt(NH2-CH3)2(CH3-gly)]NO3
Compound
Temperature am (kJ/mol)
(K)
bΔG°(H2O) cΔH°(H2O) dΔS°(H2O)
(mmol/L)−1
(kJ/mol)
(kJ/mol)
(kJ/molK)
cis-[Pt(NH3)2(CH3-gly)]NO3
300
310
44.3
63.1
52.3
63.1
10.3
28.5
25.9
20.4
561.5
673.7
1.83
1.71
2.15
2.11
cis[Pt(NH2CH3)2(CH3gly)]NO3 300
310
a Ability of platinum complex to denature DNA(kJ/mol) (mmol/L)−1
b Conformational stability of DNA in the absence of platinum complex (kJ/mol)
c The heat needed for DNA denaturation in the absence of platinum complex(kJ/mol)
d The entropy of DNA denaturation by platinum complex(kJ/molK)
ordinate in absence of each metal complex) gives the value of ΔH°(H2O) (Table 1). These plots show
that in the range of 300 to 310 K changes in enthalpies in the presence of cis-[Pt(NH3)2(CH3-
gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 are descending.
The observation indicates that an increase in the concentration of Pt(II) complexes de-
creases the stability of DNA. In addition, it can be concluding that the interaction both
cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex with DNA are
spontaneous and exothermic. Moreover, the entropy of DNA unfolding by Pt(II) complex,
ΔS°(H2O), has been calculated using equation, ΔG° = ΔH° − TΔS°, and the data are given in
Table 1. These data show that Pt(II)-DNA complex is more disordered that the native DNA,
because the entropy changes are positive for both cis-[Pt(NH3)2(CH3-gly)]NO3 and
cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex in the denaturation process of CT-DNA [30].
Thermal Denaturation (Tm) Studies
Due to raising temperature of DNA solution, the double-stranded DNA gradually dissociated
in to single strand. Tm is considered as the temperature where half of the total base pairs of
DNA are pairs [31]. Thermal behavior of DNA in the presence of complex can give deep
insight in to their basic conformational changes when temperature is raised and offer infor-
mation about interaction strength of complexes with DNA. The stabilization of CT-DNA
through the groove bonding and electrostatic interactions of the noncovalent complex was
carefully assessed by measuring the melting temperature [32].
This experiment was carried out for CT-DNA (0.13 mM) in the absence and presence of
0.108 mM of cis-[Pt(NH3)2(CH3-gly)]NO3 or cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex. The
melting plot of DNAwas monitored by plotting the UV maximum absorption of DNA at 258 nm vs
various temperature. Tm of CT-DNA in the absence of complex has been found to be 78 °C.
Increasing of DNA melting temperature by 2° for cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-
CH3)2(CH3gly)]NO3 were observed, separately (see Fig. 4). The values show clearly that Pt(II)
complexes are able to stabilize DNA helix through non-intercalation mode [33].
Fluorescence Spectroscopic Studies
Cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 are non-fluorescent
on excitation invisible region. So, completive binding studies using ethidium bromide

Appl Biochem Biotechnol
a
4
3
y = -63.102x + 28.534
R² = 0.9961
2
1
0
0
0.2
0.4
0.6
-1
-2
-3
-4
-5
y = -44.321x + 10.342
R² = 0.9982
[Complex] (mM)
b
10
5
y = -52.321x + 25.921
R² = 0.9837
0
0.25
0.3
0.35
0.4
y = -63.051x + 20.42
R² = 0.9955
-5
[Complex] (mM)
Fig. 2 The molar Gibbs free energy plot of unfolding (ΔGo vs. [L]t) of the CT-DNA in the presence of a cis-
[Pt(NH3)2(CH3-gly)]NO3 and b cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex
(EtBr) bound to CT-DNA were carried out for two systems. Ethidium bromide, a fantastic
cationic dye, is used widely as a probe for native DNA [34]. The fluorescence intensity of
free EtBr is very week but it is strongly enhanced when EtBr intercalated with DNA base
pairs. The quenching extent of fluorescence of EtBr bound to CT-DNA is usually used to
determine the mode of binding between the second molecule and CT-DNA. The emission
spectra of EtBr-DNA in the absence and the presence of cis-[Pt(NH3)2(CH3-gly)]NO3 and
cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 are given in Fig. 5. Gradual reduction emission inten-
sity can be seen by adding different concentrations of each complex when ethidium is
removed from duplexes of DNA and released to buffer medium by the action of Pt(II)
complex [35].
It has been reported that there are two quenching mechanism of binding quencher-macro-
molecules: statistic and dynamic quenching. Statistic generally offer formation of a non-

Appl Biochem Biotechnol
a
800
600
400
y = -520.43x + 561.54
R² = 1
200
0
0
0.5
1
1.5
2
2.5
3
-200
-400
-600
-800
-1000
[Complex] (mM)
b
800
600
400
200
0
y = -1953.8x + 673.77
R² = 1
0
0.1
0.2
0.3
0.4
0.5
[Complex] (mM)
-200
Fig. 3 Plots of molar enthalpies of CT-DNA denaturation (ΔHoconnformation) in the interaction of a cis-
[Pt(NH3)2(CH3-gly)]NO3 and b cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex in the range of 300 to 310 K
fluorescent fluorophore quencher complex and the dynamic quenching lead to diffuse the
fluorophore during the lifetime of the exited state and the fluorophore returns to ground state
without emission of a photon.
To determine the quenching mechanism, the Stern-Volmer is often applied:
(4)
where Fo and F are the fluorescence intensity of CT-DNA in the absence and presence of
quencher (here cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3gly)]NO3, respectively)
and kq is the EtBr-DNA-Pt(II) complex quenching rate constant; o is the average lifetime of the
fluorophore in the absence of Pt(II) complex as a quencher and the value of fluorescence lifetime
of the biopolymer is between10−7 and 10−9 s−1. The plot of F0/F versus [Q] for Pt(II) complexes
yeilded a linear graph (Fig. 6). [Q] is the total concentration of the quencher. Ksv is the linear Stern-
Volmer quenching constant [15, 36]. Results are summarized in Table 2.

Appl Biochem Biotechnol
a
0.6
0.55
0.5
DNA
DNA+COM
0.45
0.4
0.35
0.3
0
20
40
60
80
100
120
Temperature (°C)
b
0.65
0.6
0.55
0.5
DNA
DNA+ COM
0.45
0.4
0.35
0
20
40
60
Temperature(°C)
80
100
120
Fig. 4 Melting curves of CT-DNA (1 mM) in the absence and presence of a cis-[Pt(NH3)2(CH3-gly)]NO3 and b
cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complex
Circular Dichrosim Spectral Studies
CD technique gives us useful information on how the conformation of DNA is influenced by
binding of complex to DNA. The CD spectrum of CT-DNA exhibits appositive band effect at
275 nm due to base stacking and a negative band effect at 245 nm due to the helicity of B-
DNA form [23].
The CD spectrum of CT-DNA shows conformational changes by both
cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 complexes titrations
(Fig. 7). In CD spectra for both DNA-Pt(II) complex systems, intensity shift of the
negative band to the positive level at 245 nm was observed, while the positive band at
275 nm is increased to higher value. This suggest that DNA binding with Pt(II) complexes
induce certain conformational changes, such as the conversion from a model B-like to A-
like structure within the DNA molecules. DNA is not a rigid molecule and water mole-
cules are playing an important role on the conformation of DNA. The major and minor
grooves are coated by a molecular layer of water molecules with exposed -C=O, -N and -
NH functions of glycine in Pt(II) complex structure. The interaction of Pt(II) complexes

Appl Biochem Biotechnol
a
900
EtBr
800
700
600
500
400
300
200
100
0
EtBr+DNA
[com]= 0.0182 mM
[com]= 0.0357 mM
[com]= 0.0523 mM
[com]= 0.068 mM
[com]= 0.083 mM
[com]= 0.097 mM
[com]= 0.111 mM
[com]= 0.125 mM
500
550
600
650
700
750
Wavelenght (nm)
b 1000
EtBr
EtBr+DNA
[com]=0.0182 mM
[com]=0.0357 mM
[com]=0.0523 mM
[com]=0.068 mM
[com]=0.083 mM
[com]= 0.097 mM
[com]=0.111 mM
[com]=0.125 mM
800
600
400
200
0
500
550
600
650
700
750
Wavelenght (nm)
Fig. 5 Fluorescence emission spectra of interacted EtBr-DNA in the absence and presence of different
concentration of a cis-[Pt (NH3)2(CH3gly)] NO3and b cis-[Pt(NH2-CH3)2(CH3gly)]NO3
with minor groove of DNA were occurred with competition between water molecules that
solvated the DNA. So, water content in the microsurrounding around the DNA may be
decreased and the conformational change may be due to stronger hydrogen binding from
B-like to A-like and at A-like in platinum binding [17, 37].
Docking Results
Autodock software moves a molecule or drug around the receptor such as protein, enzyme,
DNA, or the other macromolecules randomly based on genetic and Lamarkian algorithm. The
basic theory of docking software is molecular mechanic and their force fields are same as
molecular dynamics software. So, the molecules translate in the box around the macromole-
cules. The box is divided by several smaller boxes as grid. Whenever number of grid becomes

Appl Biochem Biotechnol
a
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
y = 1.5277x + 1.4644
R² = 0.9829
0
-0.5
-1
-1.5
-2
Log [COM] (mM)
-1.5
-1.2
-0.9
-0.6
-0.3
0
b
y = 0.8579x + 0.1882
R² = 0.9937
0
-0.5
-1
-1.5
-2
Log [COM] (mM)
Fig. 6 Plots of log(F°-F)/F against log [complex] for a cis-[Pt(NH3)2(CH3gly)]NO3 and b cis-[Pt(NH2-
CH3)2(CH3gly)]NO3 quenching effect on EtBr-DNA fluorescence at room temperature
higher and grid size becomes smaller, then results will be more precise, but spend more
computer time. All docking parameters were determined by the above-mentioned meth-
od. In this study, 500 docking sites were obtained and sorted based on their energies
(Table 3). Some of docking sites are located near each other and so form a cluster. The
cluster ranks are listed in Table 3. First column is related to cluster rank and the first
cluster is the most negative rank. Second column is related to the most negative docking
energies and column 3 is related to mean dock energy, and last column is the number of
conformation with approximately similar energies in a cluster. The cluster rank 1 has the
most population in a cluster and most probable docking site [13, 38]. In comparison of
(a) cis-[Pt(NH3)2(CH3gly)]NO3 as Pt1 and (b) cis-[Pt(NH2-CH3)2(CH3gly)]NO3 com-
plexes as Pt2, it is observed that the docking energy of Pt2 is more negative than that
of Pt1, while it is compatible with experimental results. In order to find the molecular
basis of this difference, docking sites of both Pt complexes were analyzed.
Figure 8 shows DNA structure which is taken from protein data bank 453D (www.
rcsb.org). The 12 cluster ranks also are observed in Fig. 8a as yellow for Pt1 and blue for
Pt2. Figure 8b shows the docking site of first cluster rank that is located in minor groove.

Appl Biochem Biotechnol
Table 2 Binding parameters of CT-DNA interaction with cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-
CH3)2(CH3gly)]NO3 complex obtained from the Stern-Volmer equation
Compound
aKSV × 10−3(M−1
)
bkq × 1013(Ms)−1 R2
cKb × 104(M−1
)
n
R2
cis-[Pt(NH3)2(CH3gly)]NO3
cis-[Pt(NH2CH3)2(CH3gly)]NO3 1.98
9.48
9.48
1.98
0.97 29.13
1.52 0.99
0.85 0.98
0.99
1.54
a KSV is the Stern-Volmer quenching constant
b kq is the quenching constant
c Kf is the binding constant
Both complexes occupied the same site and approximately overlap on each other.
Figure 8c confirms our claim that two Pt complexes are located in one position. Label
of nearest nucleotide and number of hydrogen bond (green line color) were also shown.
As seen from this hydrogen bond for both complexes shown in Fig. 8d, e, there are four
hydrogen bonds. In order to know the molecular level difference between two
complexes, the thermodynamic parameters were compared in Table 4. The results
show that the van der Waals, hydrogen bond, and desolvation energy are more
effective than that of electrostatic interactions [39].
a
22
17
12
DNA
r=25
r=50
r=75
7
2
Ø
220
240
260
280
300
320
340
-3
-8
-13
-18
wavelenght (nm)
20
15
10
5
b
DNA
r=20
r=40
r=80
0
220
240
260
280
300
320
340
-5
-10
-15
-20
Wavelenght (nm)
Fig. 7 Circular dichroism Spectra of DNA (1 × 10−3 M) in the absence and presence of increasing amount of (a)
cis-[Pt(NH3)2(CH3gly)]NO3 and (b) cis-[Pt(NH2-CH3)2(CH3gly)]NO3 with ratio of r = [com]/[DNA]

Appl Biochem Biotechnol
Table 3 Binding energies (in
kcal/mol) obtained by Autodock 4.2
Cluster
rank
Lowest binding
energy
Mean binding
energy
Number of
conformations
in the cluster
Pt1
0
2
3
4
5
6
7
8
− 7.07
− 6.94
− 6.87
− 6.87
− 6.80
− 6.77
− 6.73
− 6.72
− 6.64
− 6.63
− 6.46
− 6.45
− 6.99
− 6.90
− 6.79
− 6.83
− 6.80
− 6.75
− 6.65
− 6.65
− 6.63
− 6.63
− 6.46
− 6.44
334
9
19
26
1
39
25
38
4
9
10
11
12
Pt2
1
2
3
4
5
6
7
8
9
10
11
12
1
1
3
− 7.33
− 7.22
− 7.02
− 6.98
− 6.96
− 6.94
− 6.84
− 6.82
− 6.82
− 6.79
− 6.75
− 6.71
− 7.17
− 7.20
− 6.78
− 6.90
− 6.90
− 6.93
− 6.70
− 6.80
− 6.80
− 6.76
− 6.65
− 6.71
402
35
13
10
11
3
5
9
2
2
7
1
Cytotoxicity Studies of Pt(II) Complexes
The anticancer and growth inhibitory activity of the two new synthesized complexes of
cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3gly)]NO3 were investigated
against human model breast cancer cell line of MCF7 using MTT assay. Human breast
adenocarcinoma cancer model cell line of MCF7 was incubated with various concentra-
tions of both complexes ranging from 0 to 350 μM (Fig. 9). According to the results
(Fig. 9), the 50% cytotoxic concentrations (Cc50) of the complexes were calculated 126
and 292 μM for complexes of cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-
CH3)2(CH3gly)]NO3, respectively, after 24 h incubation time.
The effects of synthesized Pt(II) complexes on the morphological features of human
cancer cell line of MCF7 (Fig. 10 from left to right, incubated in the absence of complex
or control, and in the presence of cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-
CH3)2(CH3-gly)]NO3) were studied using phase-contrast microscopy. Phase-contrast
microscopy results have shown a decreasing in cell density in the presence of complexes
relative control image. Also, the results indicated cell rounding and shrinking in the
presence of complexes. Also, cytotoxicity results show that the presence of the methyl
groups and increasing of lipophilicity of cisplatin analog have reverse influence on the
growth suppression activity of the complex on MCF7 cells and cause more Cc50 value of
cis-[Pt(NH2-CH3)2(CH3gly)]NO3 complex.

Appl Biochem Biotechnol
Fig. 8 (a) Structure of DNA taken from protein data bank (453D) (b) binding sites of two platinum complexes
(c) binding site of 12 cluster ranks (d) binding site of the most negative cluster rank, first cluster rank in Table 3
(e) zoom structure and nearest nucleotide around the complex

Appl Biochem Biotechnol
Table 4 Thermodynamic parameters for the most negative cluster rank (rank 1 in Table 3) for two platinum
complexes
Thermodynamic parameters (kcal/mol)
cis-[Pt(NH3)2
(CH3-gly)]NO3
cis-[Pt(NH2CH3)2
(CH3gly)]NO3
Estimated free energy of binding
Estimated inhibition constant, Ki
Final intermolecular energy
vdW + Hbond + desolv energy
Electrostatic energy
Final total internal energy
Torsional free energy
Unbound system’s energy
− 7.07
6.59 uM
− 7.62
− 3.29
− 4.33
+ 0.84
+ 0.55
+ 0.84
− 7.17
5.59 uM
− 7.71
− 3.75
− 3.97
+ 0.58
+ 0.50
+ 0.58
Conclusion
In the present study, two new created and water-soluble compounds, cis-[Pt(NH3)2(CH3gly)]NO3
and cis-[Pt(NH2-CH3)2(CH3gly)]NO3, were synthesized and characterized by spectroscopic
techniques as IR, 1H NMR, UV-Vis, and elemental analysis. Cytotoxic data show that the Cc50
value of cis-[Pt(NH3)2(CH3gly)]NO3 is lower than that of cis-[Pt(NH2-CH3)2(CH3gly)]NO3
complex which indicate more cytotoxicity and anticancer activity for this compound. According
to spectroscopic results, cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3gly)]NO3 com-
plexes can bind to CT-DNA by groove binding. Different instrumental methods were used to the
investigation of the interaction modes. Determinations of several binding and thermodynamic
100
90
80
70
60
50
40
30
20
10
0
0
100
200
300
400
[complex] ( M)
Fig. 9 The growth inhibitory activity of the complexes of cis-[Pt(NH3)2(CH3gly)]NO3 Ο) and cis-[Pt(NH2-
CH3)2(CH3gly)]NO3 (•) on breast cancer line of MCF7; incubated with varying concentrations of the complexes
for 24 h using MTT assay

Appl Biochem Biotechnol
Fig. 10 Phase-contrast microscopy of the effects of synthesized Pt(II) complexes on the morphological features
of human cancer cell line of MCF7 (from left to right, incubated in the absence of complex or control, in the
presence of cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3)
parameters have also been attempted. Overall, experimental results indicate that the interaction
affinity of cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 is more than that of cis-[Pt(NH3)2(CH3-gly)]NO3
complex analog. It is hoped that current study will open the new approaches for the development
of efficient pharmaceutical systems in cancer treatment with low cytotoxic effects.
Acknowledgments The Research Council of Chemistry & Chemical Engineering Research Center of Iran is
gratefully acknowledged for supporting this work.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
References
1. Weaver, A., Flemming, S., Kish, J., Vandenberg, H., Jacob, J., Crissman, J., & Al-Sarraf, M. (1982). Cis-
platinum and 5-fluorouracil as induction therapy for advanced head and neck cancer. The American Journal
of Surgery, 144(4), 445–448.
2. Storr, T., Thompson, K. H., & Orvig, C. (2006). Design of targeting ligands in medicinal inorganic
chemistry. Chemical Society Reviews, 35(6), 534–544.
3. Farrell, N. (2015). Multi-platinum anti-cancer agents. Substitution-inert compounds for tumor selectivity
and new targets. Chemical Society Reviews, 44(24), 8773–8785.
4. Deubel, D. V. (2004). Factors governing the kinetic competition of nitrogen and sulfur ligands in cisplatin
binding to biological targets. Journal of the American Chemical Society, 126(19), 5999–6004.
5. Yousefi, R., Aghevlian, S., Mokhtari, F., Samouei, H., Rashidi, M., Nabavizadeh, S. M., Tavaf, Z.,
Pouryasin, Z., Niazi, A., Faghihi, R., & Papari, M. M. (2012). The anticancer activity and HSA binding
properties of the structurally related platinum (II) complexes. Applied Biochemistry and Biotechnology,
167(4), 861–872.
6. Barabas, K., Milner, R., Lurie, D., & Adin, C. (2008). Cisplatin: a review of toxicities and therapeutic
applications. Veterinary and Comparative Oncology, 6(1), 1–18.
7. Wu, G. (2009). Amino acids: metabolism, functions, and nutrition. Amino Acids, 37(1), 1–17.
8. Gentilucci, L., De Marco, R., & Cerisoli, L. (2010). Chemical modifications designed to improve peptide
stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. Current
Pharmaceutical Design, 16(28), 3185–3203.
9. Zhao, M., Yang, M., Li, X.-M., Jiang, P., Baranov, E., Li, S., & Hoffman, R. M. (2005). Tumor-targeting
bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proceedings of
the National Academy of Sciences of the United States of America, 102(3), 755–760.
10. Gorre, M. E., Mohammed, M., Ellwood, K., Hsu, N., Paquette, R., Rao, P. N., & Sawyers, C. L. (2001).
Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science,
293(5531), 876–880.

Appl Biochem Biotechnol
11. Eslami Moghadam, M., Saidifar, M., Divsalar, A., Mansouri-Torshizi, H., Saboury, A. A., Farhangian, H., &
Ghadamgahi, M. (2016). Rich spectroscopic and molecular dynamic studies on the interaction of cytotoxic
Pt (II) and Pd (II) complexes of glycine derivatives with calf thymus DNA. Journal of Biomolecular
Structure and Dynamics, 34(1), 206–222.
12. Safa Shams Abyaneh, F., Eslami Moghadam, M., Hoseini Sadr, M., & Divsalar, A. (2017). Effect of
lipophilicity of amylamine and amylglycine ligands on biological activity of new anticancer cisplatin
analog. Journal of Biomolecular Structure and Dynamics, 1–13. https://doi.org/10.1080
/07391102.2017.1301273.
13. Ajloo, D., Moghadam, M. E., Ghadimi, K., Ghadamgahi, M., Saboury, A. A., Divsalar, A., & Yousefi, K.
(2015). Synthesis, characterization, spectroscopy, cytotoxic activity and molecular dynamic study on the
interaction of three palladium complexes of phenanthroline and glycine derivatives with calf thymus DNA.
Inorganica Chimica Acta, 430, 144–160.
14. Kantoury, M., Eslami Moghadam, M., Tarlani, A. A., & Divsalar, A. (2016). Structure effect of some new
anticancer Pt (II) complexes of amino acid derivatives with small branched or linear hydrocarbon chains on
their DNA interaction. Chemical Biology & Drug Design, 88(1), 76–87.
15. Heydari, M., Moghadam, M. E., Tarlani, A., & Farhangian, H. (2017). DNA as a target for anticancer phen-
imidazole Pd (II) complexes. Applied Biochemistry and Biotechnology, 182(1), 110–127.
16. Lippert, B. (1999). Cisplatin: chemistry and biochemistry of a leading anticancer drug. Zürich: John Wiley
& Sons, Verlag Helvetica Chimica Acta.
17. Hadian Rasanani, S., Eslami Moghadam, M., Soleimani, E., Divsalar, A., & Tarlani, A. (2017). Improving
activity of anticancer oxalipalladium analog by the modification of oxalate group with isopentylglycine.
Journal of Coordination Chemistry, 70(22), 3769–3789.
18. Eslami Moghadam, M., Divsalar, A., Abolhosseini Shahrnoy, A., & Saboury, A. A. (2016). Synthesis,
cytotoxicity assessment, and interaction and docking of novel palladium (II) complexes of imidazole derivatives
with human serum albumin. Journal of Biomolecular Structure and Dynamics, 34(8), 1751–1762.
19. Mansouri-Torshizi, H., Shahraki, S., Nezami, Z. S., Ghahghaei, A., Najmedini, S., Divsalar, A., Ghaemi, H., &
Saboury, A.-A. (2014). Platinum (II)/palladium (II) complexes with n-propyldithiocarbamate and 2, 2′-bipyridine:
synthesis, characterization, biological activity and interaction with calf thymus DNA. Complex Metals, 1(1), 23–31.
20. Yakovchuk, P., Protozanova, E., & Frank-Kamenetskii, M. D. (2006). Base-stacking and base-pairing
contributions into thermal stability of the DNA double helix. Nucleic Acids Research, 34(2), 564–574.
21. Mansouri-Torshizi, H., Saeidifar, M., Divsalar, A., & Saboury, A. A. (2010). Interaction studies between a 1,
10-phenanthroline adduct of palladium (II) dithiocarbamate anti-tumor complex and calf thymus DNA. A
synthesis spectral and in-vitro study. Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, 77(1), 312–318.
22. Shahabadi, N., Kashanian, S., & Darabi, F. (2009). In vitro study of DNA interaction with a water-soluble
dinitrogen Schiff base. DNA and Cell Biology, 28(11), 589–596.
23. Macquet, J. P., & Butour, J. L. (1978). A circular dichroism study of DNA· platinum complexes. The FEBS
Journal, 83(2), 375–387.
24. Sullivan, E. (1979). Circular dichroism of palladium (II) and platinum (II) diamine complexes. Canadian
Journal of Chemistry, 57(1), 67–70.
25. Ajloo, D., Yoonesi, B., & Soleymanpour, A. (2010). Solvent effect on the reduction potential of anthra-
quinones derivatives. The experimental and computational studies. International Journal of Electrochemical
Science, 5, 459–477.
26. Saeidifar, M., Mansouri-Torshizi, H., Palizdar, Y., Divsalar, A., & Saboury, A. A. (2013). Synthesis,
characterization, and cytotoxicity studies of a novel palladium (II) complex and evaluation of DNA-
binding aspects. Nucleosides, Nucleotides and Nucleic Acids, 32(7), 366–388.
27. Shahraki, S., Mansouri-Torshizi, H., Heydari, A., Ghahghaei, A., Divsalar, A., Saboury, A., et al. (2015).
Platinum (II) and palladium (II) complexes with 1, 10-phenanthroline and pyrrolidinedithiocarbamato
ligands: synthesis, DNA-binding and anti-tumor activity in leukemia K562 cell lines. Iranian Journal of
Science and Technology, 39(A2), 187.
28. Aminzadeh, M., Mansouri-Torshizi, H., & Modarresi-Alam, A. R. (2016). 2, 2′-bipyridine coplanar with
coordination square of Pd (II) nonyldithiocarbamato antitumor complex interacting with DNA in two
distinct steps. Journal of Biomolecular Structure and Dynamics, 35, 1–13.
29. Saeidifar, M., Mansouri-Torshizi, H., Palizdar, Y., Eslami-Moghaddam, M., Divsalar, A., & Saboury, A. A.
(2014). Synthesis, characterization, cytotoxicity and DNA binding studies of a novel anionic
organopalladium (II) complex. Acta Chimica Slovenica, 61(1), 126–136.
30. Mansouri-Torshizi, H., Saeidifar, M., Khosravi, F., Divsalar, A., Saboury, A. A., & Hassani, F. (2011). DNA
binding and antitumor activity of α-diimineplatinum (II) and palladium (II) dithiocarbamate complexes.
Bioinorganic Chemistry and Applications, 2011, 1–11.

Appl Biochem Biotechnol
31. Marmur, J., & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its
thermal denaturation temperature. Journal of Molecular Biology, 5(1), 109–118.
32. Mansouri-Torshizi, H., Saeidifar, M., Divsalar, A., & Saboury, A. (2011). Study on interaction of DNA from
calf thymus with 1, 10-phenanthrolinehexyldithiocarbamatopalladium (II) nitrate as potential antitumor
agent. Journal of Biomolecular Structure and Dynamics, 28(5), 805–814.
33. Shahabadi, N., Kashanian, S., & Purfoulad, M. (2009). DNA interaction studies of a platinum (II) complex,
PtCl 2 (NN)(NN= 4, 7-dimethyl-1, 10-phenanthroline), using different instrumental methods.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 72(4), 757–761.
34. Ware, W. R. (1962). Oxygen quenching of fluorescence in solution: an experimental study of the diffusion
process. The Journal of Physical Chemistry, 66(3), 455–458.
35. Shahabadi, N., Maghsudi, M., Mahdavi, M., & Pourfoulad, M. (2012). Interaction of calf thymus DNAwith
the antiviral drug lamivudine. DNA and Cell Biology, 31(1), 122–127.
36. Ghalandari, B., Divsalar, A., Eslami-Moghadam, M., Saboury, A. A., Haertlé, T., Amanlou, M., & Parivar,
K. (2015). Probing of the interaction between β-lactoglobulin and the anticancer drug oxaliplatin. Applied
Biochemistry and Biotechnology, 175(2), 974–987.
37. Mostafavinia, S. E., & Hoshyar, R. (2016). Spectroscopic studies on the interaction of anticancer rosemary
with ctDNA. Gene, Cell and Tissue, 3(2), e35638.
38. Lang, P. T., Brozell, S. R., Mukherjee, S., Pettersen, E. F., Meng, E. C., Thomas, V., Rizzo, R. C., Case, D.
A., James, T. L., & Kuntz, I. D. (2009). DOCK 6: combining techniques to model RNA–small molecule
complexes. RNA, 15(6), 1219–1230.
39. Arjmand, F., Parveen, S., Afzal, M., & Shahid, M. (2012). Synthesis, characterization, biological studies
(DNA binding, cleavage, antibacterial and topoisomerase I) and molecular docking of copper (II) benz-
imidazole complexes. Journal of Photochemistry and Photobiology B: Biology, 114, 15–26.