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Water soluble Ru(ii)–arene complexes of the antidiabetic drug metformin: DNA and protein binding, molecular docking, cytotoxicity and apoptosis-inducing activity
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Water soluble Ru(II)–arene complexes of the
antidiabetic drug metformin: DNA and protein
binding, molecular docking, cytotoxicity and
apoptosis-inducing activity†
Durairaj Gopalakrishnan,a Mani Ganeshpandian,
Nattamai S. P. Bhuvanesh,c Xavier Janet Sabina
*a Rangasamy Loganathan,b
and J. Karthikeyand
d
Two water soluble Ru(II)–arene complexes of the type [Ru(h6-arene)(met)Cl]Cl 1 and 2, where the arene is
either p-cymene (1) or benzene (2) and met is metformin (antidiabetic drug) have been isolated and
characterized by analytical and spectral methods. The X-ray crystal structure of 1 reveals that the
coordination geometry around Ru(II) is described as the familiar pseudo-octahedral “piano-stool”
structure. Absorption and emission spectral studies reveal that the complexes interact with calf thymus
DNA through hydrophobic and hydrogen bonding interactions of coordinated ligands with the DNA base
pairs. Molecular docking studies show that complex 1 effectively docks in the major groove of DNA. The
decrease in viscosities of CT DNA upon binding to 1 and 2 suggest the covalent mode of DNA binding of
complexes. Further, the covalent mode of binding is validated by the retardation of the mobility of
supercoiled (SC) plasmid DNA by the formation of covalent adducts observed in gel electrophoretic
mobility studies. The protein binding affinity of the complexes depends upon the arene ligand and
follows the order of p-cymene (1) > benzene (2), which is the same as that for DNA binding affinity.
Docking studies with BSA and transferrin show that the complex–protein interaction is stabilized by
hydrophobic as well as hydrogen bonding interactions. The a-amylase inhibition assay of 1 and 2
indicates that they have the potency to exhibit the antidiabetic activity in vitro. A study of cytotoxicity of
1 and 2 against human breast carcinoma (MDA-MB-231), human lung carcinoma (A549), human ovarian
carcinoma (A2780) cell lines and non-tumorigenic human embryonic kidney (HEK293) cells reveals that
they are specifically toxic to cancerous cells and non-toxic to normal cells. Remarkably, complexes 1
and 2 exhibit cytotoxicity with potency more than the metformin suggesting that the incorporation of
Received 11th June 2017
Accepted 18th July 2017
antidiabetic drug with the organometallic Ru-arene frameworks is potential approach to develop
DOI: 10.1039/c7ra06514k
effective anticancer drugs. The morphological changes observed by employing AO/EB and Hoechst
33258 staining methods reveal that the complexes 1 and 2 induce an apoptotic mode of cell death in
rsc.li/rsc-advances
breast cancer cells.
Introduction
Platinum-based anticancer agents such as cisplatin, carboplatin
and oxaliplatin are utilized as the most active metal based drugs
for chemotherapeutic treatment.1,2 However the efficacy of these
Department of Chemistry, SRM University, Kattankulathur, Chennai – 603 203, Tamil
Nadu, India. E-mail: ganeshpandian.m@ktr.srmuniv.ac.in; ganeshpandibdu@gmail.
com
a
b
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA
c
X-ray Diffraction Lab, Department of Chemistry, Texas A&M University, College
Station, TX 77842, USA
Department of Chemistry, Sathyabama University, Chennai – 600119, India
d
† Electronic supplementary information (ESI) available. CCDC 1547296. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c7ra06514k
37706 | RSC Adv., 2017, 7, 37706–37719
drugs is limited by acquired resistance against breast and
prostate cancer cells and they exhibit several side effects such as
myelosuppression, immune-suppression, neuro- and nephrotoxicity.3,4 This provides an impetus for the development of nonplatinum based drugs with a broader spectrum of activity and
lower toxicity for cancer treatment.5 In this context, several
ruthenium (Ru) complexes have shown potent anticancer
activity, especially two Ru(III) complexes which are entered in
the clinical trials. The complex imidazolium trans-[tetrachlorido(dimethylsulfoxide)(1H-imidazole)ruthenate(III)]
(NAMI-A) is active against solid metastatic tumors6 and indazolium
trans-[tetrachloridobis(1H-indazole)ruthenate(III)]
(KP1019) or its sodium salt (KP1339) are active against
a number of primary human tumors.7 Following the encouraging clinical studies of Ru-based anticancer agents, current
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attention is focused on the evaluation of anticancer activity of
organometallic Ru-arene complexes, because, by varying the
arene, substituent ligand and leaving group, the “piano-stool”
framework of the Ru-arene provides a handle for optimizing the
design of anticancer agents with improved pharmacological
properties.8–10 The Ru(II)–arene–PTA complex (RAPTA-C, PTA is
1,3,5-triaza-7-phosphaadamantane) is identied as the lead
structure that has been extensively studied for its antimetastatic
and antiangiogenic properties.11 Sadler and co-workers found
that Ru(II)–arene complexes of the type [Ru(h6-arene)(en)Cl]+,
where h6-arene is biphenyl, dihydroanthracene or tetrahydroanthracene and en is ethylenediamine, are involved in
covalent mode of binding with DNA considerably enhancing the
cytotoxicity of the complexes against different tumor cells.12
Palaniandavar et al. have found that the Ru(II)–arene complexes
with intercalating anthracenyl–methyldiazepane ligand moiety
show higher DNA and protein binding affinity and exhibit
prominent cytotoxicity than those containing simple diazepane
ligand.13
The study of metal complexes bearing a bioactive drug is
gaining more interest now than before because of the synergetic
effect of drugs on coordination with a metal.14–16 In particular,
several organometallic Ru-arene complexes incorporated with
some biocompatible drugs have been reported to exhibit increased
anticancer
activity.
[Ru(h6-benzene)(metronidazole)Cl2],
where
metronidazole
is
1-(b-hydroxyethyl)-2-methyl-5nitroimidazole, is the rst organometallic Ru(II)–arene
complex with the anti-infective agent metronidazole evaluated
for cytotoxic properties.17 Turel et al. have established the antiproliferative activity of Ru(II)–p-cymene complex of the antibacterial drug nalidixic acid18 and its thionated derivative19
against three different cancer cell lines in vitro. [Ru(h6-pcymene)(ox)Cl], where H(ox) is oxicam (nonsteroidal antiinammatory drug), has shown cytotoxicity against human
colon carcinoma with the IC50 value of 80 mM.20 Combining
organometallic Ru(II)–p-cymene fragment with the antiinammatory drug motifs like indomethacin and diclofenac
induces more cytotoxic activity than the original drugs and
also displays higher cytotoxicity and selectivity than cisplatin
towards the human ovarian carcinoma (A2780) cell lines.21
Metformin (N,N0 -dimethylbiguanide), which belongs to the
family of oral hypoglycemic agents, is the most commonly
prescribed antidiabetic drug for the treatment of type II diabetes world wide.22 Interestingly, recent clinical data reveals
that the usage of metformin for diabetic treatment reduces the
risk of cancer23 in such a way that it exhibits signicant growth
inhibitory and proapoptotic activity against several cancer cells
alone or in combination with the well-known chemotherapeutic
drug cisplatin.24,25 The mechanism of antitumor effect of metformin is still unclear; the main effect at the molecular level is
thought to be involved in the activation of AMP (50 -adenosine
monophosphate)-activated protein kinase, which plays a vital
role in cellular sensing of metabolism and oxidative stress.26
By keeping in mind the various established biological
properties of metformin, the incorporation of metformin with
the organometallic Ru(arene) pharmacophore is a promising
approach, which can further enhance the efficacy of the
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RSC Advances
Scheme 1
Structures of the complexes and ligand used.
metformin leading to the development of new anticancer
chemotherapeutics. In this article, we have isolated water
soluble Ru(II)–arene complexes of the type [Ru(h6-arene)(met)Cl]Cl 1 and 2 where arene is p-cymene (1) or benzene (2) and met is
metformin (Scheme 1) and studied their interaction with DNA,
which is considered as the primary pharmacological target of
many anticancer drugs. We have also studied the protein binding
affinity of the complexes using bovine serum albumin (BSA),
since the serum albumins are recognized as major proteins to
manipulate the transportation, distribution, and efficacy of the
many anticancer drugs.27,28 The hydrophobicity of the arene
ligands enhances the ability of complexes to exhibit cytotoxicity
more potent than metformin alone against three different
human cancer cell lines and induces the apoptotic mode of cell
death in MDA-MB-231 human breast carcinoma cells. To the best
of our knowledge, the complexes 1 and 2 are the rst organometallic Ru(II)–arene complexes of antidiabetic drug metformin
reported so far to exhibit apoptotic mode of cell death.
Experimental section
Reagents and materials
The reagents and chemicals were obtained from commercial
sources (Sigma-Aldrich, USA; Himedia, India; Merck, India;
SRL, India). RuCl3$3H2O, a-phellandrene, 1,3-cyclohexadiene,
ethidium bromide (EthBr), distamycin, calf thymus (CT) DNA
(highly polymerized, stored at 20 C) (Aldrich), pUC19 supercoiled DNA, agarose and BSA (SRL) were used as received.
Human breast carcinoma (MDA-MB-231), human lung carcinoma (A549), human ovarian carcinoma (A2780) and human
embryonic kidney (HEK293) cells were obtained from American
Type Culture Collection (ATCC), USA. Roswell Park Memorial
Institute (RPMI) medium, fetal bovine serum (FBS) were
purchased from GIBCO (Grand Island, NY). Glutamine and
penicillin–streptomycin were obtained from Life Technologies,
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USA. Ultrapure MilliQ water was used in all experiments.
Tris(hydroxymethyl)aminomethane$HCl (Tris–HCl) (pH 7.1)
was prepared by the reported procedure.29 Commercial solvents
were distilled and then used for preparation of complexes.
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Preparation of ruthenium(II) precursor complexes
The starting materials [(h6-arene)RuCl2]2, where arene is pcymene or benzene, were prepared by using the procedures
already reported.30,31
Preparation of [Ru(h6-p-cymene)(met)Cl]Cl (1)
This complex was prepared by adding methanolic solution of
metformin (0.26 g, 2 mmol) to a solution of ruthenium dimer
precursor [(h6-p-cymene)RuCl2]2 (0.61 g, 1 mmol) in methanol
and then mixture was stirred at 40 C for 5 h. The orange-brown
solution obtained was ltered and the volume of solvent was
reduced. The product formed as an orange-brown microcrystalline solid on leaving the above reaction mixture to stand at
4 C for 24 h. The product separated out was collected by
suction ltration, washed with small amount of cold methanol
and excess of ether and then dried in vacuum. The orangebrown colored crystals of 1 suitable for X-ray diffraction
studies were obtained by dissolving the complex in aqueous
methanol and allowing it to crystallize. Yield: 84%; mp: 176 C.
Anal. calcd for [Ru(h6-p-cymene)(met)Cl]Cl: C, 38.62; H, 5.79; N,
16.09. Found: C, 38.41; H, 5.36; N, 16.82%. ESI-MS: [Ru(h6-pcymene)(met)Cl]+ displays a peak at m/z 400.0 (calcd 400.08). 1H
NMR (500 MHz, D2O): d 1.11–1.33 (m, CH(CH 3)2), 2.04
(s, C6H4(CH 3)), 2.63–2.76 (m, CH(CH3)2), 2.97 (s, N(CH 3)2), 3.10
(s, NH 2), 5.44–5.45 (d, CH of C6H 4), 5.68–5.70 (d, CH of
C6H 4) ppm. 13C NMR (125 MHz, D2O): d 17.70 (C6H4(CH3)),
21.35–21.49 (CH(CH3)2), 30.32–30.37 (CH(CH3)2), 38.33
(N(CH3)2), 80.98, 83.40, 96.83, 98.57, 99.88 (h6-C6H4) ppm. FT-IR
(KBr, cm1): 3406 (br; n(N–H)), 1674, 1641 (s; n(C]N)). LM
(acetonitrile, 103 M): 64 U1 cm2 mol1.
Preparation of [Ru(h6-benzene)(met)Cl]Cl (2)
This complex was prepared by adopting the procedure used for
the preparation of 1 but using benzene dimer precursor [(h6benzene)RuCl2]2 (0.50 g, 1 mmol) instead of p-cymene dimer.
Yield: 76%; mp: 173 C. Anal. calcd for [Ru(h6-benzene)(met)Cl]
Cl: C, 31.67; H, 4.52; N, 18.47 Found: C, 31.29; H, 4.33; N,
18.72%. ESI-MS: [Ru(h6-benzene)(met)Cl]+ displays a peak at
m/z 344.0 (calcd 344.02). 1H NMR (500 MHz, D2O): d 5.73 (s, h6C6H 6), 3.01 (s, N-(CH 3)2), 3.17 (s, NH 2), ppm. 13C NMR (125
MHz, D2O): d 38.25 (N(CH3)2), 80.5, 82.8 (h6-C6H6) ppm. FT-IR
(KBr, cm1): 3406 (br; n(N–H)), 1678, 1638 (s; n(C]N)). LM
(acetonitrile, 103 M): 62 U1 cm2 mol1.
Experimental methods
Microanalysis (C, H, and N) was carried out with a Vario EL
elemental analyzer. UV-Vis spectroscopy was recorded on
a Specord 210 UV-Vis spectrophotometer using cuvettes of 1 cm
path length. 1H NMR spectra were recorded on a Bruker 500
MHz NMR spectrometer. Mass spectrometry was performed on
37708 | RSC Adv., 2017, 7, 37706–37719
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a Shimadzu LCMS-2020 spectrometer. IR spectra were recorded
from 4000 to 400 cm1 on a Agilent Carry 600 FT-IR spectrophotometer. Electrical conductivity measurements of the
complexes were recorded using SYSTRONICS 304 conductivity
meter at room temperature. Melting point values were recorded
on a PPI-96 Inlab equipment. Emission intensity measurements
were carried out using Perkin Elmer LS-45 spectrouorometer.
Viscosity measurements were carried out using Brookeld
viscometer.
Solutions of calf thymus (CT) DNA in 5 mM Tris HCl/50 mM
NaCl buffer gave a ratio of UV absorbance at 260 and 280 nm,
A260/A280, of 1.9,32 indicating that the DNA was sufficiently free
of protein. Concentrated stock solutions of DNA (20.5 mM) were
prepared in buffer and sonicated for 25 cycles, where each cycle
consisted of 30 s with 1 min intervals. The concentration of DNA
in nucleotide phosphate (NP) was determined by UV absorbance at 260 nm aer 1 : 100 dilutions by taking the extinction
coefficient, 3260 nm, as 6600 M1 cm1. Stock solutions of DNA
were stored at 4 C and used aer no more than 4 days.
Supercoiled plasmid pUC19 DNA was stored at 20 C, and the
concentration of DNA in base pairs was determined by UV
absorbance at 260 nm aer appropriate dilutions taking 3260 nm
as 13 100 M1 cm1.
X-ray crystallography
A BRUKER Venture X-ray diffractometer was employed for
crystal screening, unit cell determination and data collection of
complex 1 (ESI†).33–37 A Leica MZ 75 microscope was used to
identify a suitable brown block with very well dened faces from
a representative sample of crystals of the same habit. The crystal
1 of suitable size selected was mounted on a nylon loop and
then placed in a cold nitrogen stream (Oxford) maintained at
110 K. The goniometer was controlled using the APEX3 soware
suite.33 The sample was optically centered with the aid of a video
camera such that no translations were observed as the crystal
was rotated through all positions. The X-ray radiation employed
was generated from a Cu-ImsX-ray tube (Ka ¼ 1.5418 Å with
a potential of 50 kV and a current of 1.0 mA). 45 data frames
were taken at widths of 1 . These reections were used to
determine the unit cell and the unit cell was veried by examination of the hkl overlays on several frames of data. No supercell or erroneous reections were observed. Aer careful
examination of the unit cell, an extended data collection
procedure (10 sets) was initiated using omega and phi scans.
Molecular docking studies
Molecular docking studies were performed to identify the
possible binding site of complex with DNA, BSA and transferrin.
The 3D crystal structures of B-DNA dodecamer
d(CGCAAATTTCGC)2 (PDB ID: 1BNA), BSA (PDB ID: 3V03) and
transferrin (PDB ID: 1D3K) were downloaded from the RCSB
Protein Data Bank website (http://www.rcsb.org/pdb) and used.
Docking studies were performed with Discovery Studio (Accelrys) by simulation of the complex into the binding site of the
biomolecules. The docking was run using Ligand Fit dock
protocol of Discovery Studio program and the receptors were
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typed with CHARMm force eld. Ligands and water molecules
were removed from the binding sites and default parameters
were used for the docking calculations.
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concentration). Aer 4 h of incubation at 37 C, uorescence of
the highly red uorescent resorun product was quantied at
590 nm emission with 540 nm excitation wavelength in a Versa
max tunable microplate reader.
DNA binding and electrophoretic mobility studies
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DNA binding and electrophoretic mobility studies were carried
out by using the procedures reported already (ESI†).13
Tryptophan uorescence quenching
The tryptophan uorescence quenching studies were carried
out as reported previously (ESI†).13
In vitro antidiabetic activity
a-Amylase inhibitory activity was studied for control and sample
solution based on starch-iodine method.38 a-Amylase enzyme
(1% w/v) solution was added to the sample tube containing 1
mL of different concentration of sample in 0.1 M sodium
acetate/30 mM NaCl buffer (pH 7.2) and the mixture was incubated for 15 min. Aer pre-incubation, aliquots (1 mL) of that
incubate were transferred to sample tubes containing starch
substrate (1% w/v) in acetate buffer and the mixture was reincubated for 15 min. 0.1 mL of the reaction mixture was
withdrawn from each tube aer mixing and discharged into 10
mL of iodine solution. Solutions were thoroughly mixed and the
absorbance was measured immediately at 565 nm. Percentage
inhibition was calculated according to the formula:
Inhibition of a-amylase (%) ¼ (Acontrol Asample)/Acontrol 100
where, Acontrol is the absorbance of the control and Asample is the
absorbance of the test sample. All experiments were performed
in triplicate (n ¼ 3) and results were expressed as mean
standard error mean (SEM).
Cell culture
Human breast carcinoma cells (MDA-MB-231), human lung
carcinoma cells (A549), human ovarian carcinoma cells (A2780)
and human embryonic kidney cells (HEK293) were cultured in
RPMI (Gibco) media with 5% fetal calf serum (FCS, Gibco), 100
U mL1 penicillin and 100 mg mL1 streptomycin at 37 C and
5% CO2.
Cytotoxicity study: resazurin assay
The cytotoxicity of the complexes 1 and 2 were studied in three
different human cancer cell lines, namely, human breast
carcinoma (MDA-MB-231), human lung carcinoma (A549),
human ovarian carcinoma (A2780) cells and in one non-tumor
human embryonic kidney cells (HEK293). The cytotoxicity of
the compounds was measured by a uorimetric cell viability
assay using Resazurin (Sigma, USA). Cells were plated in triplicates in 96-well plates at a density of 5 103 cells per well in
100 mL of media 24 h prior to treatment. Cells were then treated
with increasing concentrations of compounds for 24 h. Aer
24 h in the incubator, the medium was replaced by 100 mL
complete medium containing resazurin (0.2 mg mL1 nal
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Morphological studies
AO/EB staining. For both suspension and adherent cells, 96well plates were centrifuged at 1000 rpm for 5 min using an
Eppendorf 5810 centrifuge with inserts for 96-well plates. An
AO/EB dye mix (8 mL) was added to each well, and cells were
viewed under Nikon Eclipse90i upright microscope equipped
with a Nikon DS-Ri1 12MP color camera. Tests were done in
triplicate, counting a minimum of 100 total cells each.
Hoechst 33258 staining. The cell pathology was detected by
staining the nuclear chromatin of trypsinized cells (4.0
104 per mL) with 1 mL of Hoechst 33258 (1 mg mL1) for 10 min
at 37 C. Staining of suspension cells with Hoechst 33258 was
used to detect apoptosis.39 A drop of cell suspension was placed
on a glass slide, and a coverslip was overlaid to reduce light
diffraction. At random, 300 cells were observed in a uorescent
microscope (Nikon Eclipse90i upright microscope) equipped
with a Nikon DS-Ri1 12MP color camera and observed at 40
magnication, and the percentage of cells reecting pathological changes was calculated. Data were collected for triplicate
and used to calculate the mean and standard deviation.
Results and discussion
Synthesis and characterization of Ruthenium(II) complexes
Two water soluble half-sandwich organometallic complexes of
the type [Ru(h6-arene)(met)Cl]Cl (1, 2), where arene is p-cymene
(1) or benzene (2) and met is metformin, have been isolated by
the reaction of the appropriate dimeric complex [(h6-arene)
RuCl2]2 with a slight excess of equimolar amount of metformin
ligand in methanol at 40 C and the desired products were
isolated as chloride salts in good yield. The formulae of the
complexes [Ru(h6-arene)(met)Cl]Cl, as determined by elemental
analysis and mass spectral studies, are supported by structural
elucidation of 1 by using X-ray crystallography (cf. below). The
complexes are soluble in water, Tris–HCl/NaCl (5 : 50 mM)
buffer (pH 7.1), methanol and acetonitrile. The UV-Vis absorption spectra of complexes 1 and 2 show low energy ligand eld
band due to low-spin d6 conguration of Ru(II) complexes and
exhibit absorbance tail around 350–410 nm, which is assigned
to mixed metal-centered and metal-to-ligand charge transfer
transitions.40 The weak shoulder observed for 1 (240 nm) and 2
(236 nm) is ascribed to the characteristic intra-ligand transitions. (Table S1 and Fig. S1†). The mass spectral data conrm
the formation of complexes and the retention of identity of
complexes even in solution (Fig. S2 and S3†). This is supported
by the values of molar conductivity (LM) in acetonitrile (1, 64; 2,
62 U1 cm2 mol1), falling in the range for 1 : 1 electrolytes.41
The 1H NMR spectra of 1 and 2 in D2O show downeld shis in
positions of the arene protons as compared to the corresponding precursor complexes.42a This may be attributed to the
incorporation of metformin ligand, which modies the electron
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density around metal center. The N-CH3 protons of the metformin are observed as singlet in the range of 2.9–3.0 ppm
(Fig. S4 and S5†). 13C NMR spectra of 1 and 2 were recorded in
D2O to support the formation of the respective complexes
(Fig. S6†). Infra-red spectra of complexes 1 and 2 exhibit characteristic bands due to n(N–H) and n(C]N) stretching vibrations of metformin (Fig. S7†). The coordination of metformin
ligand to metal center is conrmed by the broad band observed
in the region of 3300–3400 cm1 due to n(N–H) stretching
vibrations, which exhibit medium intensity for free
metformin.42b
Description of the crystal structure of [(h6-p-cymene)Ru(met)
Cl]Cl$H2O 1
An ORTEP representation of the coordination environment of
the complex monocation 1 including the atom numbering
scheme is shown in Fig. 1. The crystallographic data and
selected bond lengths and bond angles are listed in Tables 1
and 2 respectively. The asymmetric unit of 1 contains a cationic
complex, a lattice chloride anion and water molecule. The
complex cation adopts the familiar pseudo-octahedral coordination geometry43,44 by coordinating all the six carbon atoms
(C5–C10) of p-cymene ring, both the nitrogen atoms (N1, N3) of
metformin and chlorido ligand (Cl1). The bond angles made by
N1 and N3 atoms of metformin and Cl constitute the three legs
of the ‘piano-stool’ structure [N(1)–Ru(1)–Cl(1) (84.56(8) ) and
N(3)–Ru(1)–Cl(1) (84.97(8) )]. The six Ru–C bonds have almost
similar bond distances (2.155(3)–2.207(3) Å) with an average
bond length of 2.178 Å forming the seat of the piano-stool.45 The
N1 and N3 atoms of metformin are coordinated to the metal
Paper
Table 1
Crystal data and structure refinement for complex 1
1
Formula
Formula weight
Crystal system
Space group
a, Å
b, Å
c, Å
a, deg
b, deg
g, deg
Volume [Å]
Z
D(calc) [mg m3]
q, [min–max deg]
Goodness-of-t on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
C14H27Cl2N5ORu
453.37
Monoclinic
P121/n1
16.876(4)
12.155(3)
19.495(4)
90
111.576(2)
90
3718.6(14)
8
1.620
1.369–27.495
1.050
R1 ¼ 0.0416, wR2 ¼ 0.0699
R1 ¼ 0.0611, wR2 ¼ 0.0767
center with almost equal bond distance (Ru–N(1), 2.078(3); Ru–
N(3), 2.073(3) Å) indicating that the tautomerisation is not
involved in between –N(1)H/–N(3)H and –N(2)H groups, when
the coordination complex is formed. The bite angle of metformin in 1 (N(1)–Ru(1)–N(3): 83.10(10) ) is lower than the bond
angles involving chlorido ligand (N(1)–Ru(1)–Cl(1), 84.56(8) ,
N(3)–Ru(1)–Cl(1) (84.97(8) )), as expected.13 The Ru–Nimine
bonds (Ru–N1, 2.078(3), Ru–N3, 2.073(3) Å) formed by metformin in 1 are shorter than Ru–Namine bonds (Ru–N1, 2.130, Ru–
N2, 2.136 Å) formed by en in [(h6-p-cymene)Ru(en)Cl](PF6)12 on
account of sp3 and sp2 hybridizations of the amine and imine
nitrogen atoms respectively. The bite angle of metformin
(83.10(10) ) in 1 is higher than that of en (78.9(10) ) in [(h6-pcymene)Ru(en)Cl](PF6).12 This is expected because the steric
hindrance formed by six membered chelate ring of metformin
in 1 is less than that of ve membered chelate ring formed by en
ligand in [(h6-p-cymene)Ru(en)Cl](PF6). The hydrogen atom of
the imine nitrogen (N3) in metformin is involved in hydrogen
Selected interatomic distances [Å] and bond angles [deg] for
complex 1
Table 2
1
Fig. 1 ORTEP representation of the crystal structure of [Ru(h6-pcymene)(met)Cl]+ (1) showing atom numbering scheme and
displacement ellipsoid (50% probability level) (water molecule and Cl
anion are omitted for clarity).
37710 | RSC Adv., 2017, 7, 37706–37719
Bond distance [Å]
Ru(1)–N(1)
Ru(1)–N(3)
Ru(1)–Cl(1)
Ru(1)–C(5)
Ru(1)–C(6)
Ru(1)–C(7)
Ru(1)–C(8)
Ru(1)–C(9)
Ru(1)–C(10)
2.078(3)
2.073(3)
2.431(9)
2.199(3)
2.160(3)
2.174(3)
2.207(3)
2.177(3)
2.155(3)
Bond angle [deg]
N(1)–Ru(1)–N(3)
N(1)–Ru(1)–Cl(1)
N(3)–Ru(1)–Cl(1)
83.10(10)
84.56(8)
84.97(8)
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Absorption spectra of [Ru(h6-p-cymene)(met)Cl]+ (0.5 103
M) in 5 mM Tris–HCl buffer at pH 7.1, in the absence (R ¼ 0) and
presence (R ¼ 0–2) of increasing amounts of CT DNA (R ¼ [DNA]/
[complex]).
Fig. 2
bonding with the oxygen atom of the neighbouring water
molecule (N(3)–H/O: 2.163 Å).
DNA binding studies: absorption spectral titration
The less intense absorption spectral band observed for 1
(378 nm) and 2 (367 nm) was used to characterize the interaction of the complexes with CT DNA in 5 mM Tris–HCl/50 mM
NaCl buffer at pH 7.1. Upon incremental addition of solution of
CT DNA to complexes 1 and 2, a less decrease in absorption
intensity (Fig. 2) with negligible shi in band position was
observed suggesting weak electrostatic binding of the
complexes with DNA.46–48 In order to quantitatively compare the
DNA binding affinities, the intrinsic binding constant Kb was
calculated using the equation,49
[DNA]/(3a 3f) ¼ [DNA]/(3b 3f) + 1/Kb(3b 3f)
where [DNA] is the concentration of DNA in base-pairs, 3a is the
apparent extinction coefficient calculated as Aobs/[complex], 3f
corresponds to the extinction coefficient of the complex in its
free form and 3b refers to the extinction coefficient of the
complex in the bound form. Each set of data, when tted into
the above equation, gives a straight line with a slope of 1/(3b 3f)
and an y-intercept of 1/Kb(3b 3f) and Kb is determined from the
ratio of the slope to intercept. The values of Kb vary in the order 1
(1.7 0.08 103 M1) > 2 (0.9 0.04 103 M1), illustrating
that the DNA binding affinity of p-cymene complex is higher
than that of benzene complex (Table 3). Both the enhanced
Table 3
(a) Docking of complex 1 at the major groove of the B-DNA
dodecamer d(CGCAAATTTCGC)2; (b) hydrogen bonding interaction
of complex 1 with phosphate group and adenine17 of B-DNA.
Fig. 3
hydrophobic interaction of methyl and isopropyl groups on
arene ring in 1 and that of N-methyl groups on the metformin
ligand lead to confer a higher DNA binding affinity of p-cymene
complex than that of benzene complex. The Ru(II)–arene
complexes like [Ru(h6-p-cymene)(L)Cl](PF6), where L is N-1(anthracen-10-ylmethyl)-4-methylhomopiperazine (Kb, 7.8
104 M1)13 and [(h6-C6H6)Ru(pmpzdpm)Cl], where pmpzdpm is
5-(2-pyrimidylpiperazine)phenyldipyrromethene (Kb, 9.5
104 M1)50 show higher order of DNA binding obviously because
of intercalation of appended ligand moiety with DNA base pairs.
Thus, the order of DNA binding affinity of the complexes 1 and 2
is expected to be low due to non-intercalative mode of DNA
binding. However, the strong hydrogen bonding propensity of
the donor or acceptor groups of metformin with nucleobases or
phosphate groups present in the edge of DNA, which is identied from molecular docking studies (cf. below), would also
contribute to the DNA binding affinity of complexes. Thus, it is
possible that the ability of the metformin ligand with strong
hydrogen bonding interaction enables the Ru(II) metal center to
be located close to the site of nucleobases for covalent mode of
DNA binding by losing labile chloride ion. Similar observations
have been made by Sadler et al. earlier for the complex [Ru(h6-pcymene)(en)]2+, which contains a non-intercalating en ligand
exhibiting a remarkably high preference for covalent binding to
the N7 position of guanine. Such binding is stabilized by
hydrogen bonding between the C6O of guanine and NH of the
en ligand.51 Thus, the mode and extent of DNA binding of
[Ru(h6-arene)(met)Cl]Cl complexes are contributed by both
Absorption spectral propertiesa and fluorescence spectral propertiesb of ruthenium(II) complexes bound to CT DNA
Complexes
lmax (nm)
R
Change in absorbance
Kb (103 M1)
Kappb (103 M1)
[Ru(h6-p-cymene)(met)Cl]+ 1
[Ru(h6-benzene)(met)Cl]+ 2
378
367
2
2
Hypochromism
Hypochromism
1.7 0.08
0.9 0.04
8.6
7.8
a
b
Measurements were made at R ¼ 2, where R ¼ [DNA]/[complex], concentration of solutions of Ru(II) complexes ¼ 0.5 103 M (1 and 2).
Apparent DNA binding constant from competitive DNA binding studies using 0–60 mM increasing concentration of 1 and 2.
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hydrophobic interaction of arene ligand and hydrogen bonding
propensity of metformin ligand.
DNA docking studies. The mode of DNA binding is further
investigated by performing docking analysis of 1 with B-DNA
dodecamer d(CGCAAATTTCGC)2 (Fig. 3) by using Discovery
Studio molecular graphics program. The calculated binding
energy of the complex with B-DNA (32.773 kcal mol1) indicates that the complex effectively binds with DNA.10 Interestingly, the complex docks in the major groove of DNA, which
supports the mode of DNA binding affinity of complex observed
from absorption spectral titration (cf. above). The preference of
the complex to bind in major groove rather than minor groove is
due to the bulky p-cymene ligand otherwise it would cause
immense strain if it is docked on minor groove. The complex is
situated in major groove of DNA in such a way that the –NH2
and –N(CH3)2 groups of metformin are involved in hydrogen
bonding interaction with the phosphate oxygen acceptor in
guanine16 (1.397 Å) and –NH2 donor in adenine17 (2.455 Å)
respectively. It is important to note that the Discovery Studio
program is capable of identifying the feasible binding site of
DNA by assuming that DNA and complex molecules are rigid
and hence it is not suitable for detecting covalent mode of DNA
binding. Thus, the covalent mode of DNA binding is further
evidenced by viscosity measurements and electrophoretic
mobility shi assay (cf. below).
Competitive DNA binding studies. The Ru(II)–arene
complexes 1 and 2 fail to show steady-state emission in all the
solvents studied and lack emission even in the presence of CT
DNA. When they are added (0–60 mM) to CT-DNA (125 mM)
pretreated with EthBr (12.5 mM) ([DNA]/[EthBr] ¼ 10) in 5 mM
Tris–HCl/50 mM NaCl buffer at pH 7.1, the emission intensity of
DNA-bound EthBr (lex, 450; lem, 595 nm) decreases (Fig. 4).
From the plot of the intensity vs. complex concentration, the
values of apparent DNA binding constant (Kapp) were calculated
using the equation,52
KEthBr[EthBr] ¼ Kapp[complex]
where KEthBr is 4.94 105 M1,53 the concentration of EthBr is
12.5 mM and the concentration of the complex is that used to
obtain a 50% reduction of uorescence intensity of EthBr.
Fig. 4 Effect of addition of complex 2 on the emission intensity of the
CT DNA-bound ethidium bromide (12.5 mM) at different concentrations (0–60 mM) in 5 mM Tris-HCl buffer (pH 7.1).
37712 | RSC Adv., 2017, 7, 37706–37719
Addition of a second DNA binding molecule either replaces the
DNA-bound EthBr (if it binds to DNA more strongly than EthBr)
and/or destabilize the excited state of DNA-bound EthBr by
means of molecular collision,54 which would quench the DNAbound EthBr emission. The observed Kapp values of the
complexes decrease in the order, 1 (8.6 103) > 2 (7.8
103 M1), which is consistent with the order of the Kb values
obtained by UV-Vis absorption spectral titration (Table 3). The
observed low order Kapp values reveal that the complexes are not
able to involve intercalation with DNA to replace the strong
intercalating molecule EthBr. However, the complexes 1 and 2
at higher concentrations would destabilize the DNA-bound
EthBr more efficiently through enhanced hydrophobic interaction of arene ring and metformin with the hydrophobic DNA
surface.
Viscosity measurements. The viscosity measurement of CT
DNA upon treatment with varying concentrations of complexes
reveals the DNA binding mode of the complexes. The values of
relative specic viscosity (h/h0), where h and h0 are the specic
viscosities of DNA in the presence and absence of the complex
respectively, are plotted against 1/R (¼[Ru complex]/[DNA]) ¼ 0–
0.50, along with that of EthBr and distamycin treated (Fig. 5).
The intercalation of EthBr in between the DNA base pairs
increase the overall DNA contour length resulting increase in
viscosity whereas the viscosity of DNA is slightly increased upon
the addition of distamycin, which is known to involve in minor
groove DNA binding. In contrast, decrease in viscosity is
observed for 1 and 2 due to kinking or bending of the DNA
helices upon covalent binding of complexes with DNA base
pairs. A similar decrease in viscosity has been observed for
[Ru(h6-p-cymene)(L)Cl](PF6) (L ¼ N-(2-pyridylmethyl)glycine),55
which is reported as a covalently DNA binding molecule. The
decrease in viscosity is also observed for partially intercalating
complex like [Ru(dmb)2(pdpt)](ClO4)2 (dmb ¼ 4,40 -dimethyl2,20 -bipyridine, pdpt ¼ 3-(pyridine-2-yl)-5,6-diphenyl-astriazine).56 However, the possibility of partial intercalation of
complexes 1 and 2 in between DNA base pairs is excluded
because of absence of extended aromatic ring. Thus, the covalent mode of DNA binding of the present complexes is similar to
other monofunctional Ru(II)–arene-en complexes reported.51
Fig. 5 Effect of addition of complexes 1, 2, EthBr and distamycin on
the viscosity of CT DNA; relative specific viscosity vs. 1/R; [CT DNA] ¼
500 mM.
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Electrophoretic mobility studies
The anticancer activity of most of the complexes is related with
the cellular response of metal induced DNA damage or DNA
distortion, which is caused by formation of inter or intrastrand
cross linking adducts with metal complexes.3,8 The gel electrophoresis technique provides an insight into the mode of interaction of DNA with metal complexes by observing the migration
of plasmid DNA in different conformations. The covalent DNA
binding metal complex like [Ru(h6-p-cymene)(en)Cl]+ involve
local unwinding which leads to retardation of migration of
DNA.57 The intercalating complexes rst unwind negatively
supercoiled DNA and then convert it into positively supercoiled
DNA.55 The major/minor groove DNA binding and partial
intercalating complexes convert the supercoiled DNA into
nicked form, if one strand of DNA is cleaved13 and linear form, if
both strands are cleaved.58 In order to explore the DNA interaction of 1 and 2, supercoiled (SC) pUC19 DNA (40 mM) was
incubated with the complexes (100 mM) in 5 mM Tris–HCl/
50 mM NaCl buffer (pH 7.1) at 37 C for 1 h in the absence of an
activator. The complexes 1 and 2 fail to retard the mobility of SC
DNA with an incubation period of 1 h (Fig. S8†). However, when
the incubation period is increased from 1 h to 3 h, the retention
of SC DNA occurred in the well (Fig. 6) illustrating that these
monocationic complexes involve covalent adduct formation
with DNA. This covalent DNA-complex adduct would signicantly reduce the overall negative charge of the DNA, which
could stop the migration of DNA from the well. When the
concentration of complexes is varied from 50 to 200 mM with an
increasing incubation period of 5 h, the rate of migration of SC
DNA decreases in comparison with the control DNA. Also, the
maximum degradation with undetectable fragments occurs at
200 mM concentration of 1 (Lane 5, Fig. 7) where it occurs at 150
mM concentration for complex 2 (Lane 8, Fig. 7). From the
analysis of the DNA gel electrophoresis data, it is proposed that
the present Ru(II)–arene complexes of the type [Ru(arene)(met)
Cl]+ undergo rapid aquation to form [Ru(arene)(met)(H2O)]2+ in
an aqueous environment.12 Then the aqua product covalently
binds to nitrogen of a DNA heterocyclic base by replacing the
aqua ligand forming DNA–complex adduct. Further, with
a prolonged incubation period, the adduct formed undergoes
multiple fragmentation of DNA. However, detailed investigation
Fig. 6 Retardation of mobility of supercoiled pUC19 DNA (40 mM) by
ruthenium(II) complexes 1 and 2 (100 mM) in absence of an external
agent in 5 mM Tris HCl/50 mM NaCl buffer at 37 C. Lane 1: DNA; Lane
2: DNA + metformin (100 mM); Lane 3: DNA + 1 (100 mM, 3 h incubation); Lane 4: DNA + 2 (100 mM, 3 h incubation); Lane 5: DNA + 1
(100 mM, 1 h incubation); Lane 6: DNA + 2 (100 mM, 1 h incubation).
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Fig. 7 Cleavage of supercoiled pUC19 DNA (40 mM) by ruthenium(II)
complexes 1 and 2 in absence of an external agent in 5 mM Tris HCl/50
mM NaCl buffer at 37 C in 5 h incubation. Lane 1: DNA; Lane 2: DNA +
1 (50 mM); Lane 3: DNA + 1 (100 mM); Lane 4: DNA + 1 (150 mM); Lane
5: DNA + 1 (200 mM); Lane 6: DNA + 2 (50 mM); Lane 7: DNA + 2 (100
mM); Lane 8: DNA + 2 (150 mM).
is required to analyze the fragments formed in DNA cleavage.
Thus, the Ru(II)–arene complexes containing metformin ligand
are able to form covalent adduct with DNA as that of previously
reported Ru(II)–arene-en complexes.57
Protein binding studies
BSA has been taken as model serum albumin59 in order to
determine the protein binding affinity of complexes. The major
intrinsic uorescence of BSA when excited at 295 nm is
provided by tryptophan amino acid residues.60,61 Changes in the
emission spectra of tryptophan residue are common in
response to conformational transitions, subunit associations,
substrate binding or denaturation.62 The tryptophan emission
quenching experiments were carried out by adding the
complexes with increasing concentration (10–50 mM) to BSA
(5 mM) at 298 K and following the decrease in uorescence
intensities. Generally, uorescence quenching can be illustrated by the well-known Stern–Volmer equation,63
F0/F ¼ 1 + KSV [Q]
where F0 and F are the uorescence intensities of BSA in the
absence and presence of the complexes respectively, KSV is the
Stern–Volmer quenching constant and [Q] is the concentration
of the quenching complex. The value of Ksv obtained as slope of
the linear plot of F0/F vs. [Q] follows the order 1 (12.6 0.3
103) > 2 (8.5 0.5 103 M1) (Fig. 8), which is in agreement
with the trend in DNA binding affinities. The p-cymene complex
(1) is capable of quenching the tryptophan uorescence 1.5
times more strongly than benzene complex (2) due to enhanced
hydrophobic interaction of p-cymene ligand moiety with the
tryptophan site of BSA. The mechanism of tryptophan emission
quenching takes place through either dynamic or static mode.
In dynamic quenching, a collision between the complex and the
excited state of uorophore takes place. In contrast, the
complex forms a non-uorescent adduct with the uorophore
in the ground state in case of static quenching.13 UV-Visible
absorption spectra of BSA in the absence and presence of the
complexes (Fig. S9†) show that the absorption intensity of BSA
decreased with no shi in band position. It reveals that there is
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Fig. 8 Fluorescence quenching spectra of BSA (5 mM) at different
concentrations of complex 2 (10–50 mM) at 298 K; excitation wavelength, 295 nm. (Inset) Plots of relative integrated emission intensity
(F0/F) vs. [complex 2].
no static interaction between BSA and the added complex, and
quenching occurs through dynamic mechanism.
Molecular docking studies with BSA and transferrin
In order to get further understanding of the complex–protein
interaction, molecular docking analysis was performed by
docking the p-cymene complex 1 with BSA (PDB ID: 3v03) and
transferrin (PDB ID: 1D3K) (Fig. 9). The globular BSA protein
consists of three homologous domains (I, II, and III), which are
made up of 583 amino acid residues and each primary domain
is further divided into sub-domains A and B along with 17
disulphide linkages.64–66 The p-cymene complex is bound at the
interface between the two sub domains IIA and IIIA, which are
identied as the hydrophobic pocket of interaction of various
drugs. The binding site of the complex is comprised of many
amino acid residues like Asp108, Arg144, His145, Leu189,
Ser192, Ala193, Arg196, Glu424, Ser428, Lys431, Tyr451, Leu454,
Ile455 and Arg458. From the docking simulations, it is observed
that the enhanced hydrophobic interaction of methyl and isopropyl groups on p-cymene and that of N-methyl groups on
metformin lead to confer strong binding affinity with BSA. Also,
the complex–protein binding is further stabilized by hydrogen
bonding interaction, which is evident from the formation of
hydrogen bond between –NH2 group of metformin and oxygen
atom of serine residue (NH/Oserine: 2.676 Å).
Experimental evidences show that the accumulation of
antitumor ruthenium complex KP1019 might be mediated by
iron uptake plasma protein transferrin. Since the neoplastic
cells require high concentration of iron for its metabolism,
ruthenium can bind to other sites on transferrin and ‘piggyback’ into the cell when iron is taken into the cell.67–69 In order
to ascertain the binding mode of complex 1 with transferrin, the
docking simulation was performed. Transferrin is a glycoprotein containing two N and C lobes which are further divided
into two sub-domains coordinating with Fe3+. The crystal
structure of transferrin (1D3K) contains only the N lobes, which
are divided into N1 and N2 sub-domains. The complex is bound
at the interface between N1 and N2 sub-domains in a hydrophobic pocket and the binding site is lined with Trp8, Cys9,
37714 | RSC Adv., 2017, 7, 37706–37719
Molecular docked model of 1 located within the hydrophobic
pocket of BSA (a) and transferrin (b).
Fig. 9
Ala10, Val11, Glu15, Tyr45, Val60, Thr61, Leu62, Leu66, Arg124,
Phe186, Gly187, Tyr188, His249, Asp292, Leu294, Phe295 and
Lys296 amino acid residues (Fig. 9). It is interesting to note that
the hydrogen bonds are formed between –N(CH3)2 group of 1
and –NH2 group of Arg124 (2.286 Å) as well as –NH2 group of 1
and the amide oxygen of Asp292 (2.123 Å). From the calculated
values of binding energy, it can be depicted that the binding
affinity of 1 with BSA (46.5 kcal mol1) is higher than that of
transferrin (26.9 kcal mol1).
In vitro antidiabetic activity
The present Ru(II)–arene complexes incorporated with the wellknown antidiabetic drug metformin are expected to show
enhanced antidiabetic activity. a-Amylase, which is an enzyme
present in the pancreatic juice and saliva is involved in the
process of breakdown of insoluble starch into blood absorbable
sugars. The inhibition of a-amylase activity would delay the rate
of absorption of glucose by blood thereby maintaining the
serum blood glucose in hyperglycemic individuals.70 The aamylase inhibition activity of 1 and 2 for 30 min incubation in
sodium acetate buffer pH 7.2 has been studied in comparison
with the standard drug acarbose, which is a well-known aamylase inhibitor. The inhibitory activity of 1 is comparable to
that of 2, but both the complexes show the inhibitory activity
lower than the standard drug. (IC50: 1, 174.9 1.6; 2, 147.5
1.5; acarbose, 65.8 1.5 mM). Further in vivo studies on animal
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models are required to understand the mechanism of antidiabetic activity, which could be related to the mechanism of
cytotoxicity of complexes (cf. below).71
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Cytotoxicity studies: resazurin assay
The cytotoxic activity of various Ru-arene complexes has been
associated with their DNA binding affinity43,72 and/or signicant
reactivity against emerging protein targets.73 Since the present
Ru(II)–arene complexes involve efficient interaction with DNA
and protein, the cytotoxic activity against human breast carcinoma (MDA-MB-231), human lung carcinoma (A549), human
ovarian carcinoma (A2780) and non-tumorigenic human
embryonic kidney (HEK293) cells has been investigated for 24 h
incubation by adopting resazurin assay. The ability of the
complexes to kill the cancer cells vary as 1 > 2 (Table 4) reects
the ability of interaction of the complexes with DNA and
protein. Interestingly, the complexes 1 and 2 show signicant
cytotoxicity against three different human cancer cells respectively, MDA-MB-231, A549, A2780 and the complexes exhibit
potency to kill the breast cancer cells approximately 2.0 times
more than to kill ovarian and lung cancer cells. Moreover, they
were non-toxic to healthy human kidney cells up to the higher
concentration tested (>100 mM). This clearly indicates that the
cell killing activity of the complexes are more specic towards
cancer cells and are non-toxic to non-cancerous cells. Also, the
cytotoxicity of p-cymene complex 1 (IC50: 16.1 (1.8) mM) and
benzene complex 2 (IC50: 24.4 (2.6) mM) is comparable with that
of [Ru(h6-p-cymene)(en)Cl]+ (IC50, 10.0 (1.1) mM) and [Ru(h6benzene)(en)Cl]+ (IC50, 17.0 (8.3) mM) against human ovarian
carcinoma (A2780) cells respectively.74 Sadler et al. established
that the cytotoxicity of [Ru(h6-arene)(en)Cl]+ complexes is due to
distortion of DNA by forming monofunctional adduct, which
leads to recognition of DNA binding protein and repair mechanism.75 Similar mechanism is expected for the cell killing
activity of the present complexes obviously because of similar
mode of DNA binding and comparable cytotoxicity observed.
Previous studies have shown that the lower cationic charge of
complex and hydrophobicity of ligands confer enhanced lipophilicity, which encourages the permeation of complexes
across cell membrane to exhibit cytotoxicity.43,76 Hence, the
efficient cytotoxicity of complexes may be ascribed, in addition
to their DNA and protein binding affinity, to the monocationic
charge of complex and hydrophobicity of arene and metformin
ligands.
It is interesting to note that the present Ru(II)–p-cymene
complex bearing antidiabetic drug shows cell killing activity
(IC50: 1, 7.9 (0.7) mM) higher than those with the antiinammatory drug derived ligands (IC50: >140–170 mM)
against human breast carcinoma (MDA-MB-231) cells20 and
those of antibacterial quinolone drugs (IC50: >320 mM) against
human ovarian cancer cells,18 irrespective of the methodology
employed. Also, the complexes 1 and 2 are remarkable in displaying cytotoxicity against breast, lung and ovarian cancer cells
more prominent than metformin (IC50, >350–500 mM). This
clearly demonstrates the importance of coordination of antidiabetic drug to Ru-arene framework, which confers versatility of
the complexes leading to improved cytotoxic activity.
Morphological assay: uorescence and phase contrast
microscopic studies
The human breast carcinoma cells (MDA-MB-231) were treated
with IC50 concentration of complexes 1 and 2 for 24 h and 48 h
incubation and the morphological alterations brought out by
the complexes have been evaluated by adopting uorescence
microscopic analysis of AO/EB-stained cancer cells.77 Acridine
orange (AO) permeates the cells and makes the nuclei appear
green. Ethidium bromide (EB) is taken up by cells only when
cytoplasmic membrane integrity is lost, and stains the nucleus
red. The observed cytological changes are classied into four
types depending upon the uorescence emission and morphological features of chromatin condensation in the AO/EB
stained nuclei as reported previously:13,43 (i) Normal and viable
cells have a uniform green uorescing nuclei with the organized
structure (ii) early apoptotic cells have bright green nuclei with
perinuclear chromatin condensation (iii) late apoptotic cells
display orange to red uorescing nuclei with highly condensed
or fragmented chromatin(IV) cells that have died from direct
necrosis have a structurally normal orange to red uorescing
nuclei. These morphological changes were more evident from
the bright eld images of phase contrast microscopy also. All of
these morphological changes observed for 1 and 2, suggest that
the cells are committed to both apoptotic and necrotic mode of
Table 4 In vitro cytotoxicity assay for complexes 1 and 2 against human breast carcinoma (MDA-MB-231), human lung carcinoma (A549),
human ovarian carcinoma (A2780) and non-tumorigenic human embryonic kidney (HEK293) cells for 24 h incubation. Inhibitory concentration
(IC50) values are in mM (data are mean SD of three replicates each)
IC50a, mM
Complexes
MDA-MB-231 human breast
carcinoma cells
A549 human lung
carcinoma cells
A2780 human ovarian
carcinoma cells
HEK293-human embryonic
kidney cells
[Ru(h6-p-cymene)(met)Cl]Cl 1
[Ru(h6-benzene)(met)Cl]Cl 2
[Ru(h6-p-cymene)Ru(en)Cl]PF6
[Ru(h6-benzene)Ru(en)Cl]PF6
Metformin
7.9 0.7
21.0 1.3
—
—
>350
15.5 2.8
29.2 1.7
—
—
>500
16.1 1.8
24.4 2.6
10.0 + 1.1b
17.0 + 8.3b
>500
>100
>100
—
—
>800
a
IC50 ¼ concentration of drug required to inhibit growth of 50% of the cancer cells (in mM). b Growth inhibition assay (ref. 74).
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cell death in a time-dependent manner (Fig. 10). In particular,
the observed morphological alterations like membrane blebbings, chromatin condensation or fragmentation and cytoplasm vacuolization indicate that the induction of apoptosis is
the major mode of cell death. The potency of the complexes to
induce apoptotic cell death varies as 1 > 2 revealing that the
higher apoptosis-inducing ability of p-cymene complex is facilitated by its higher hydrophobicity than benzene complex. The
enhanced hydrophobicity and hence lipophilicity of arene and
metformin ligands facilitate the transport of the complexes
Paper
across the cell membrane and their eventual release at various
cellular components leading to apoptosis.
Hoechst staining studies. The molecular mechanism of cell
death has been further conrmed by treating the human breast
carcinoma cells (MDA-MB-231) with IC50 concentration of 1 and
2 for 24 h and then cytological changes are observed by adopting Hoechst 33258 staining. The representative morphological
changes observed for 1 and 2 such as chromatin fragmentation,
bi- and/or multinucleation, cytoplasmic vacuolization and late
apoptosis indication of dot-like chromatin condensation are
(A) AO/EB-staining (a–c) and phase contrast microscopic images (d) of human breast carcinoma cells (MDA-MB-231) at 24 and 48 h
incubation by the treatment of 1 (L: Live cells, EA: Early Apoptosis and LA: Late Apoptosis). (B) Graph shows manual count of percentage of
normal, apoptotic and necrotic cells at 24 and 48 h treatment with compound 1 and 2 (data are mean% SD% of each triplicate).
Fig. 10
37716 | RSC Adv., 2017, 7, 37706–37719
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Conflict of interest
There are no conicts of interest to declare.
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Acknowledgements
Fig. 11 Hoechst 33258 staining of human breast carcinoma cells
(MDA-MB-231) were treated with IC50 concentrations of 1 and 2. (a)
Untreated MDA-MB-231 cells (control), (b and c) cells were treated
with 1 and 2 after 24 h incubation; apoptotic body formation is indicated by arrows.
shown in Fig. 11. This clearly provides further evidence for
induction of apoptosis by the complexes and the complexes
could be developed as efficient anticancer drugs as the
apoptosis-inducing ability is critical in determining the efficacy
of an anticancer drug. However, additional biochemical experiments like cellular uptake and cell cycle analysis are to be
performed to further investigate the mode of cell death
observed for the complexes.
Conclusions
In this study, two water soluble half-sandwich Ru(II)–arene
complexes of the type [Ru(h6-arene)(metformin)Cl]Cl have been
isolated and characterized. The coordination geometry around
Ru(II) in the p-cymene complex is described as familiar pseudooctahedral structure. The p-cymene complex, in addition to the
donor functionalities of metformin, is involved in hydrophobic
interaction with DNA through the methyl and isopropyl groups
of arene ligand, which is responsible for its stronger DNA
binding affinity than benzene complex. DNA docking studies
reveal that p-cymene complex binds in the DNA major groove,
which is stabilized by hydrogen bonding interactions. The
viscosity measurements imply that the complexes are involved
in the covalent mode of DNA binding, which is further supported by the retardation of migration of DNA observed from gel
electrophoretic mobility studies. Remarkably, the p-cymene
complex exhibits higher affinity to bind to BSA than benzene
complex in the hydrophobic region and it effectively docks on
BSA and transferrin. It is noteworthy that it shows lower antidiabetic activity in comparison with the standard drug in vitro.
The same complex is unique in displaying more cytotoxic
activity than its analogue against human breast, lung and
ovarian cancer cells and is found to be non-toxic to normal
kidney cells. The facile transport of the complex cation across
the cell membrane on account of efficient hydrophobicity of
both the arene and metformin also induces the prominent
apoptotic activity of the complex. Thus, a well-known antidiabetic drug such as metformin along with coordinated p-cymene
ligand may be incorporated into Ru-based organometallic
anticancer agents.
This journal is © The Royal Society of Chemistry 2017
We thank Science and Engineering Research Board (SERB), New
Delhi for a Start-up research grant (YSS/2015/000403/CS) to M.
G. and Junior Research Fellowship to D. G. M. G. sincerely
thanks SRM University for providing various instrumentation
facilities. We thank Dr B. Neppolian and Dr M. Mariappan, SRM
University for providing the UV-Vis absorption spectral facilities. Mr K. Manikandan, SRM College of pharmacy is grateful
for help to obtain the ligand. DST-FIST is greatly acknowledged
for creating facilities to the Department of chemistry, SRM
University, Kattankulathur, Chennai.
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