Download Synthesis and characterization of glycoconjugate tin(IV) complexes

Journal of Organometallic Chemistry 696 (2011) 1600e1608
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Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis and characterization of glycoconjugate tin(IV) complexes:
In vitro DNA binding studies, cytotoxicity, and cell death
Sartaj Tabassum a, *, Rais Ahmad Khan a, Farukh Arjmand a, Subrata Sen b, Jyoti Kayal b,
Aarti S. Juvekar b, Surekha M. Zingde b
a
b
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India
Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, Mumbai, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 November 2010
Received in revised form
24 December 2010
Accepted 11 January 2011
New glycosyl derived ligand and its complexes, with SnCl4$5H2O (1) and (CH3)2SnCl2 (2) were synthesized and characterized by spectroscopic (IR, 1H, 13C, and 119Sn NMR, UVevis, ESI-MS) and analytical
methods. Interaction studies of 1 and 2 with CT DNA were studied by using various biophysical techniques, which showed high binding affinity of 2 with CT DNA. In vitro cytotoxicity of complexes 1 and 2
were evaluated against different human cancer cell lines of different histological origins by employing
SRB Assay. The organotin(IV) complex 2 exhibited remarkable activity against DWD (oral cancer) cell
lines with GI50 values <10 mg/ml. Complex 2 induced apoptosis of DWD cell line at a very low concentration of 1e4 mg/mL.
Ó 2011 Elsevier B.V. All rights reserved.
Keywords:
GlycoconjugateeSn(IV) complexes
In vitro DNA binding studies
Cytotoxicity
Nuclear morphology
1. Introduction
One of the most rapidly developing areas of pharmaceutical
research is the discovery of robust drugs for treating cancers.
Cisplatin (cis-diamminedichloroplatinum(II)) [1,2] is an archetypical metal-based drug widely used for treating solid tumors viz;
testicular, ovarian, bladder and head/neck cancers, however, it
possesses serious limitations because of inherent or acquired
resistance in tumor cells and severe side effects [3]. Although,
cisplatin and its other second generation cisplatin drugs remain the
top selling drugs (exceeding $1.4 billion in the US in 2008), still the
toxicity issues pertaining to these drugs remain major impediments in its use. Therefore, there is an exigency to identify effective
metal-based therapeutics particularly, those that overcome
inherent and acquired resistance to drug therapy and show
improved therapeutic properties, stimulating the ongoing investigations of alternative molecular targeted metal-based drugs [4].
Anticancer drugs targeting biomolecules represent a rational
advancement in the modern drug discovery. DNA and enzymes
involved in replication and transcription represent the most targeted bioreceptors for small molecules and a target for the control
* Corresponding author. Tel.: þ91 05712703893.
E-mail address: [email protected] (S. Tabassum).
0022-328X/$ e see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2011.01.012
of gene expression [5e7]. Most anticancer drugs bind to DNA and
proteins either in a reversible or irreversible manner suggesting
a direct relationship between their interactions with macromolecules, hence, leading to their therapeutic effect [8,9].
There is considerable promise in enhancing the targeting and
efficiency of metal-chelators through ligand design. Ligands can
significantly alter the biological properties by modifying reactivity
or substitution related inertness. Carbohydrates are of primary
importance as energy sources for living organisms [4,10,11]. Due to
the properties inherent to this class of molecules, carbohydrates
have been utilized to prepare bioactive materials [12], and bettertargeted drugs [13,14]. Appending a carbohydrate moiety to drug
candidates to form new drugs and/or pro-drugs, offers great
potential in increasing the solubility of the molecule and minimizing the toxicity, and enhancing the stability [15].
Present investigation focuses on the design and synthesis of new
saccharide derivative chelator by the introduction of salicylaldehyde as anchoring group which can provide well defined binding
environment as well as increases the stability of the resultant metal
complexes. D-glucosamine (an amino monosaccharide) which is
widely taken as dietary supplement to relieve discomfort of osteoarthritis related joint pains, has been reported as an inhibitor of
cancer cells in vitro and in vivo [16e19]. A potential benefit of
utilizing this approach is that the carbohydrate can remain pendant,
thereby being freely available to interact with carbohydrate transport and metabolic pathways in the body. Tin(IV) and organotin(IV)
S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608
compounds exhibit wide spectrum biological activity [20e24] and
are notably important in the discovery of new metal-based anticancer chemotherapeutic agents [3,25e27]. Tin(IV) complexes
prefer to bind to the oxygen atom of the phosphate of the polyanionic structure of DNA. Tin(IV) ions possess a hard Lewis acid
nature, neutralize the negative charge of the phosphate moiety of
the DNA backbone and thus brings conformational changes in DNA
[28,29]. This class of compounds has gained tremendous impetus
because their in vitro antitumor activity is greater than in vivo
activity of cisplatin, especially used for treating testicular, ovarian,
bladder and head/neck cancers [3]. Besides this, tin compounds are
known to act as strong apoptotic directors, activate apoptosis
directly via p53 tumor suppressor pathway.
In continuation of our previous work [21,22,29e34], herein we
report the synthesis and characterization of N-glycoside derived
ligand and its complexes with SnCl4$5H2O (1) and (CH3)2SnCl2
(2), respectively. The interaction of complexes 1 and 2 with CT
DNA was carried out by employing various biophysical techniques. The in vitro cytotoxicities of 1 and 2 were tested against
the 14 human carcinoma cell lines of different histological origins.
The changes in nuclear morphology of the DWD (oral cancer cell
line) upon addition of 2 at 1e3 mg/mL concentrations were
photographed.
2. Experimental section
2.1. Materials
Glucosamine hydrochloride, dimethyl tin(IV) dichloride, ethylenediamine (SigmaeAldrich), tin(IV) tetrachloride, tris(hydroxymethyl)aminomethane (E. Merck), imidazole (Fluka), Disodium
salt of calf thymus DNA (highly polymerized stored at 4 C), agarose
(SigmaeAldrich) and pBR322 supercoiled plasmid DNA (Genei)
were used as received.
2.2. Methods and instrumentation
Microanalysis (CHN) was carried out with a Carlo Erba Analyzer
Model 1108. Molar conductances were measured at room temperature on a Eutech conductivity bridge. Interspec 2020 FTIR spectrometer was used for recording IR spectra of KBr pellets in the
range of 4000e400 cm1. Electronic spectra were recorded on
UV-1700 PharmaSpec UVevis spectrophotometer (Shimadzu).
Emission spectra were determined with a Hitachi F-2500 fluorescence spectrophotometer. 1H, 13C and 119Sn NMR were recorded on
Bruker Avance II 400 NMR spectrometer at 25 C. Electrospray mass
spectra were recorded on Micromass Quattro II triple quadrupol
mass spectrometer. Cleavage experiments were performed with
the help of Axygen electrophoresis supported by Genei power
supply with a potential range of 50e500 V, visualized and photographed by Vilber-INFINITY Gel documentation system.
DNA binding experiments that include absorption spectral
studies, fluorescence studies are confirmed to the standard
methods [35e38] and practices previously adopted by our laboratory [30e32]. Standard error limits were estimated using all data
points.
2.3. Cleavage activity
The cleavage experiments of supercoiled pBR322 DNA (300 ng)
by complexes 1 and 2 (10e40 mM) in (5 mM TriseHCl, 50 mM NaCl)
buffer at pH 7.2 were carried and observed using agarose gel
electrophoresis. The samples were incubated for 1 h at 37 C.
A loading buffer containing 25% bromophenol blue, 0.25% xylene
cyanol, 30% glycerol was added and electrophoresis was carried out
1601
at 60 V for 1 h in TriseHCl buffer using 1% agarose gel containing
1.0 mg/ml ethidium bromide. Similarly in photocleavage studies,
reaction mixture was carried out under illuminated conditions at
365 nm (12 W) monochromatic light source. The samples were
incubated for 0.5 h at 37 C and analyzed for photocleaved products
using gel electrophoresis as discussed above. The standard protocols were followed for these experiments.
2.4. In vitro antitumor activity
The human tumor cell lines used for in vitro screening for
antitumor activity were Hop62 and A549 (lung), PC3 and DU145
(prostate), A498 (kidney), DWD (oral), Colo205, HT29 HCT15 and
SW620 (colon) T24 (bladder) MIA-PA-CA-2 (pancreas), MCF7 and
ZR-75-1 (breast), SiHa, ME180 and HeLa (cervix) U373MG (astrocytomaeglyoblastoma), A2780 (ovary), K562 (erythroleukemia).
The human malignant cell lines were procured and grown in
RPMI-1640 medium supplemented with 10% Fetal Bovine Serum
(FBS) and antibiotics to study growth pattern of these cells. The
inhibition of the cell proliferation upon treatment with the
synthesized compounds was determined using the Sulforhodamine-B (SRB) semi-automated assay. Cells were seeded in 96-well
plates at an appropriate cell density to give optical density in the
linear range (from 0.5 to 1.8) and were incubated at 37 C in CO2
incubator for 24 h. Stock solutions of the complexes were prepared
as 100 mg/ml in DMSO and four dilutions, i.e. 10 mg/ml, 20 mg/ml,
40 mg/ml, 80 mg/ml, in triplicates were tested, each well receiving
90 mL of cell suspension and 10 mL of the drug solution. Appropriate
positive control (Adriamycin) and vehicle controls were also run.
The plates with cells were incubated in CO2 incubator with 5% CO2
for 24 h followed by drug addition. The plates were incubated
further for 48 h. Termination of experiment was done by gently
layering the cells with 5 mL of chilled 30% TCA (in case of adherent
cells) and 50% TCA (in case of suspension cell lines) for cell fixation
and kept at 4 C for 1 h. Plates were washed under running tap
water, air-dried and stained with 50 mL of 0.4% SRB for 20 min. The
excess dye was removed by washing with 1% acetic acid. The
bound SRB was eluted by adding 100 mL 10 mM Tris (pH 10.5) to
each of the wells. The absorbance was read at 540 nM with 690 nM
as reference wavelength. All experiments were repeated three
times.
2.5. Effect of compound 2 on DWD cells
A modified procedure for detection of apoptosis in 96-well
plates was used. DWD cells (2.0 104/well) were seeded in
quadruplicate wells in a 96-well plate for each treatment and kept
overnight. Next day the cells were treated with the complex 2 at
1, 2, 3 and 4 mg/ml and the cells were maintained for 24 h at 37 C
in 5% CO2 incubator. The plates containing the control and treated
cells were centrifuged at 1000 rpm for 5 min using a plate
centrifuge (model R-23, Remi, India). The pelleted cells were
stained with acridine orangeeethidium bromide (EB/AO) dye
mixture (1:1), (100 mg/ml in PBS, 4 ml/well). Cells were viewed
under the Zeiss Axiovert 200 M inverted microscope fitted with
FITC (408/490) and rhodamine (550/573) filters. The images of
DWD cells treated with complex 2 were captured using Axiocam
MRM camera. For each treatment at least 500 cells were counted.
Percent live, dead and apoptotic (early þ late) cells were calculated by formula:
n=total no: of cells counted 100;
where ‘n’ represents number of live/dead or apoptotic cells counted
per treatment group.
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S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608
2.6. Synthesis
was stirred for 4 h, which yielded pale yellow coloured product. It
was filtered, washed with cold MeOH and Et2O, dried under
vacuum over P2O5. Yield ¼ 56%. Anal. Calcd for C15H22N3O5SnCl3
(1): C 32.79, H 4.04, N 7.65. Found: C 32.82, H 4.05, N 7.66. LM
(1 103 M, DMSO): 17.0 U1 cm2 mol1 (non-electrolyte). IR (KBr)
(nmax/cm1): 3287 (eOH sugar, broad), 1642 (HC]N), 1474, 1451
(dOCH, dCH2, dCCH), 643 (SneO), 432 (SneN), 293 (SneCl). 1H NMR
(400 MHz, DMSO, d): 10.19 (s, NH, 1H), 8.69 (s, N]CH, aldimine,
1H); 7.52 (m, AreCH, 2H), 6.93 (m, AreCH, 2H), 6.26 (s, br, NH2,
GLcN, 2H), 4.20 (t, CH sugar, 2H), 3.31e3.08 (m, OH, CH, sugar, 9H),
2.56 (s, CH2 of en, 4H). 13C NMR (100 MHz, DMSO, d): 165.24
(carbon of characteristic aldimine), 145.22, 136.18, 135.98, 122.46,
118.40, 117.51 (six aromatic carbons), 78.51, 78.18, 77.85, 58.24,
55.84, 51.32 (six carbons of GlcN), 40.12, 39.91 (two carbons of en,
CH2). 119Sn NMR (149.19 MHz, DMSO, d): 570.23. ESI-MS (m/z):
549 [C15H22N3O5SnCl3]þ.
2.6.1. Synthesis of ligand (L)
To a methanolic solution (20 mL) of N-glycoside synthesized by
a reaction between D-glucosamine hydrochloride (0.215 g, 1 mmol)
and ethane-1,2-diamine (0.06 ml, 1 mmol) in MeOH at 80 C for 1 h,
[39] was added methanolic solution (10 mL) of salicylaldehyde
(0.122 ml,1 mmol) dropwise, and allowed to reflux for 1 h. During the
course of reflux, the mixture turned yellow. The reaction mixture was
then allowed to cool at room temperature. The solvent was removed
by rotavapor and dried under vacuum over P2O5. The product was
obtained as a yellow liquid. Yield ¼ 48%. IR (KBr) (nmax/cm1): 3436
(AreOH), 3289 (eOH sugar), 1646 (HC]N); 1472, 1450 (dOCH, dCH2,
dCCH). 1H NMR (400 MHz, DMSO, d): 13.27 (s, AreOH, 1H), 10.20 (s,
NH, 1H), 8.77 (s, N]CH, aldimine, 1H), 7.50 (m, AreH, 2H), 6.94 (m,
AreH, 2H), 5.32 (s, br, NH2 sugar, 2H), 4.19 (t, CH sugar, 2H), 3.65e3.05
(m, OH, CH, sugar, 9H), 2.53 (s, CH2 of en, 4H). 13C NMR (100 MHz,
DMSO, d): 172.40 (carbon of characteristic aldimine), 146.48, 136.67,
136.53, 122.60, 122.26, 118.85 (six aromatic carbons), 73.84, 71.39,
71.31, 63.58, 51.31, 51.02 (six carbons of GlcN), 40.33, 40.06 (two
carbons of en, CH2). ESI-MS (m/z): 326.4 [C15H23N3O5 þ 1H]þ.
2.6.3. Synthesis of C17H28N3O5SnCl (2)
This complex was prepared in a manner analogous to that of
complex 1, using (CH3)2SnCl2 (0.219 g, 1 mmol) in place of
SnCl4$5H2O. On cooling a yellow coloured complex was obtained,
which is filtered, washed with cold MeOH and Et2O, dried under
vacuum over P2O5. Yield ¼ 52%. Anal. Calcd for C18H34N3O7SnCl
(2): C 38.70; H 6.13; N 7.52. Found: C 38.73, H 6.14, N 7.54.
LM (1 103 M, DMSO): 14.0 U1 cm2 mol1 (non-electrolyte). IR
2.6.2. Synthesis of C15H24N3O6SnCl3 (1)
To a methanolic solution (20 mL) of SnCl4$5H2O (0.352 g,
1 mmol) was added the ligand (L) (0.325 g, 1 mmol). The solution
HO
O OH
OH
+
OH
NH2CH2CH2NH2
HO
MeOH
70oC, 1h
OH
OH
NH3+ Cl-
Glucosamine
hydrochloride
HO
OH
OH
NH2
HN
O
HO
+
NH3+
HO
CHO
in situ
OH
OH
-
Cl
O
NH2
HN
O
NH3+ Cl-
N
NH
HO
NH3+ ClLigand (L)
L
+
SnCl4. 5H2O
MeOH
Reflux
HO
O
OH
HN
Cl
N
H2
OH
N
O
Sn
Cl
Cl
Complex 1
L
+
(CH3)2SnCl2
MeOH
Reflux
HO
O
OH
OH
N
HN
H3C
Sn
N
H2
Cl
Complex 2
Scheme 1.
O
CH3
S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608
(KBr) (nmax/cm1): 3292 (OeH sugar, broad), 1639 (C]N), 1473,
1450 (dOCH, dCH2, dCCH), 1411 (CeN), 635 (SneO), 429 (SneN),
289, 281, 276 (SneCl). 1H NMR (400 MHz, DMSO, d): 10.17 (s, NH,
1H), 8.54 (s, N]CH, aldimine, 1H), 7.37 (d, AreCH, 1H), 7.21
(t, AreCH, 1H), 6.90 (m, CH, aromatic, 2H), 6.49 (s, br, NH2, GLcN,
2H), 3.94 (t, CH sugar, 2H), 3.31e3.05 (m, OH, CH, sugar, 9H), 2.56
(CH2 of en, 4H), 1.26 (s, CH3, bonded to Sn, 6H). 13C NMR
(100 MHz, DMSO, d): 166.55 (C]N, characteristic aldimine);
144.42, 131.96, 131.37, 118.32, 118.23, 116.24 (six aromatic carbons),
78.94, 78.61, 78.28, 58.84, 56.44, 51.34 (six carbons of GlcN);
45.54, 40.12 (two carbons of en, CH2), 8.94, 8.51 (two carbons of
methyl group). 119Sn NMR (149.19 MHz, DMSO, d): 603.01. ESIMS (m/z): 508.4 [C17H28N3O5SnCl]þ.
3. Results and discussion
3.1. Synthesis and characterization
The glucosamine-based ligand was synthesized by the reaction
of N-glycoside formed through condensation of 2-amino-2-deoxyD-glucose hydrochloride with ethylenediamine, by salicylaldehyde
in methanol. The reaction of Schiff base ligand with SnCl4$5H2O
and (CH3)2SnCl2 in 1:1 molar ratio, respectively, led to the
formation yellow coloured complexes 1 and 2 (Scheme 1). Both the
complexes are soluble in H2O and DMSO. Elemental analyses
obtained are in good agreement with the suggested chemical
formulae of the compounds. Structural proposals are based on IR,
1
H, 13C, 119Sn NMR, ESI-MS. The results obtained through these
techniques are in agreement with the proposed 1:1 stoichiometry
between the Sn(IV) moieties and ligand. The molar conductance
values of 103 M solutions of the synthesized complexes 1 and 2
are in the range of non-electrolytes. The in vitro binding studies of
1 and 2 with CT DNA were carried out by using absorption,
emission spectroscopic titrations, and gel electrophoresis. In vitro
cytoxicity of the complexes on human cell lines was evaluated by
SRB assay.
1603
eOH and the eNH of the linker amine. However, on complexation, the phenolic eOH peak disappears which suggest the
deprotonation of eOH and bonding to central tin(IV) ion. The 1H
NMR spectra of the ligand and its complexes 1 and 2, displayed
the characteristic aldimine peak at 8.77e8.54 ppm. The aromatic
proton signatures were observed in the range of 7.55e6.86 ppm.
A broad peak for the NH2 of the glucosamine appeared at
around 5.32 ppm in the ligand, which on complexation
undergoes a significant chemical shift suggestive of coordination
with the central tin(IV) ion. Furthermore, the characteristic signal
for the sugar moiety appeared in the range of 4.20e3.05 ppm,
due to the typical-saccharide coupled system; however, the
individual assignments of the peak were not possible. The
protons of CH2 of ethylenediamine appeared at around w2.5 ppm
[44,45]. In case of complex 2, the signal of the methyl group
bound to tin appeared at 1.26 ppm. The absence of phenolic OH
peak in the complexes suggested the deprotonation and confirms
the SneO bonding.
13
C NMR spectrum of the ligand exhibited the strong characteristic signal of aldimine carbon at 172 ppm. A large shift to
165e166 ppm was observed in the spectra of the complexes
1 and 2 which ascertained the coordination of tin(IV) to nitrogen
atom. The six aromatic carbon peaks appeared in the range
146e116 ppm. The six distinct carbon signals of the D-glucosamine were exhibited in the range of 78.94e51.32 ppm [46,47].
The two carbons of the ethylenediamine moiety appeared in the
range 45.54e39.91 ppm. The additional peak in complex 2, for
the two methyl groups bonded to tin was observed at 8.94 and
8.51 ppm [23].
The validation of the geometry of tin(IV) metal ion in 1 and 2
was done by 119Sn NMR spectroscopy. It is known that 119Sn
chemical shift d (119Sn) is sensitive towards the coordination sphere
around the tin atom. 119Sn NMR showed a single peak
at 570.23 ppm and 603.01 ppm for complexes 1 and 2, respectively, confirming the octahedral geometry around the tin atoms in
both the complexes which was in good agreement with the
previous literature reports [22,30].
3.2. IR spectroscopy
4. Binding studies
In the IR spectrum of the free D-glucosamine, the sharp bands
of n(OeH) were observed in the range of 3200e3500 cm1. The
characteristic envelop at around 3400 cm1 merged and broadened due to the complexation [40]. The IR spectrum of ligand
shows a very sharp and strong band at 1646 cm1 assignable to
characteristic n(HC]N) [24]. The IR spectra of the complexes 1
and 2 exhibited significant shift in n(HC]N) to lower wave
numbers viz; 1642 and 1639 cm1, respectively, indicating the
coordination of Sn(IV) moieties to aldimine nitrogen. The bands
appearing between 1475 and 1449 cm1 were attributed to d
(OCH, CH2, CCH) for the sugar moiety [41]. The above modes of
coordination of ligand are further supported by the appearance of
two new bands at 446 cm1 and 432 cm1 in the spectra of tin
(IV) complexes, which are assigned to n(SneN) [42,43], respectively. Additional, absorption bands at 647e643 cm1 for n(SneO)
were observed which correspond to deprotonation of phenolic
OH due to coordination with central tin(IV) atom [30]. In the IR
spectrum of the complex 2, a medium intensity band at 572 cm1
corresponds to n(SneC). The n(SneCl) was also revealed by the
presence of the bands at w290 to 270 cm1, in the far IR region
[22].
3.3. NMR spectroscopy
The 1H NMR spectrum of the ligand exhibited signals
at 13.27 and 10.20 ppm, which were attributed to the phenolic
4.1. Absorption studies
Titration with UV absorption spectroscopy is an effective
method to examine the binding mode of DNA with metal
complexes [48]. The interaction of complexes with DNA is
expected to perturb the ligand based transitions of the complexes.
It is known that the metal complexes can bind to DNA via both
covalent and/or non-covalent interactions [49,50]. The absorption
spectra of 1 and 2 in the absence and presence of CT DNA (at
a constant concentration of complexes, 0.16 104 M) are shown
in Fig. 1. In the UV region complexes 1 and 2 exhibited bands at
252 nm and 254 nm, respectively, which are assigned to the pep*
transitions, due to long living triplet excited state of the aromatic
moiety. The other bands at around 372 nm are associated with
LMCT bands and in the region of MLCT bands at w435 nm. Upon
increasing concentration of CT DNA (0e0.4 104 M), the UV
region exhibited an increase in absorption intensity ‘hyperchromic’ effect with a red shift of 2e4 nm in pep* region.
However, spectral changes in the LMCT transition and MLCT
region also showed hyperchromism but to a lower extent
compared with the pep* region. The strong hyperchromic effect
with a significant red shift is suggestive of higher binding
propensity to CT DNA, possibly by electrostatic mode, and stabilization of the complex-DNA adduct. The Sn(IV) ions (non-transition metal ion) of the complexes exhibit preferential selectivity
1604
S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608
absorbance of the pep* bands with increasing concentration of
CT DNA:
½DNA=3a 3f ¼ ½DNA=3b 3f þ 1=Kb 3b 3f (1)
where [DNA] represents the concentration of DNA, 3a, 3f and 3b are
the apparent extinction coefficient Aobs/[M], the extinction coefficient for free metal complex and the extinction coefficient for metal
complex in the fully bound form, respectively. In the plots of [DNA]/
3a 3f versus [DNA], Kb is given by the ratio of the slope to the
intercept. The intrinsic binding constants of 1 and 2 were found to
be 1.2 104 M1 and 9.4 104 M1, respectively. The Kb values
revealed that 2 exhibits greater propensity towards CT DNA,
thereby shows stronger binding affinity.
4.2. Fluorescence interaction studies
4.2.1. Luminescence studies
The interaction of complexes 1 and 2 were also investigated by
monitoring its fluorescence intensities both in the presence and
Fig. 1. Variation of UVevis absorption (a) for complex 1 and (b) complex 2, respectively, with increase in the concentration of CT DNA in buffer 5 mM TriseHCl/50 mM
NaCl, pH ¼ 7.2 at 25 C. Inset: plots of [DNA]/3a 3f versus [DNA].
towards the phosphate group of the DNA backbone [30]. Sn(IV)
complex interacts with the phosphate group of DNA, and cause the
contraction and conformational change of DNA helix, due to the
fact that phosphate group can provide the suitable anchors for
coordination in Sn(IV) complexes and consequently results in
breakage of the secondary structure of the DNA. Furthermore, the
Schiff base ligand would encourage minor groove binding by
engaging in hydrogen-bonding between coordinated eNHe and
eOH with the functional groups positioned on the edge of DNA
bases [22,30]. However, the presence of carbohydrate in a drug is
important in molecular recognition at the specific site of DNA and
has profound effect on biological activity. This effect was first
studied in family of anthracycline antibiotics [51], which showed
that carbohydrate makes the van der Waal’s contacts with the
minor groove of the DNA. Aminosugars that interact with the
nucleic acid backbone mainly through electrostatic interactions
between the positive charged amino groups and the negative
charged phosphate of the furanose moiety. In addition to electrostatic interactions, other kinds of forces involving non-polar
interactions between carbohydrates cannot be excluded [52].
Therefore, the above results indicate that complex 2 may first
bind with the phosphate group of DNA, neutralize the negative
charge of DNA phosphate group, and cause the contraction and
conformational change of DNA.
To evaluate quantitatively, the binding strengths of 1 and 2
with CT DNA, the intrinsic binding constants Kb of the complexes
were determined with Eq. (1) by monitoring the changes in
Fig. 2. Emission spectra of (a) complex 1, and (b) complex 2, in the absence and
presence of CT DNA in buffer 5 mM TriseHCl/50 mM NaCl, pH ¼ 7.2 at 25 C.
S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608
1605
absence of CT DNA [22,30,53]. The complexes 1 and 2 exhibited
emission bands at 450 nm when excited at 594 nm. In the absence
of CT DNA, 1 and 2 emit weak luminescence in 5 mM TriseHCl,
50 mM NaCl buffer at ambient temperature. The enhancements in
the emission intensity of 1 and 2 with increasing CT DNA
concentration are depicted in Fig. 2a and b. Since it is found that
complexes with increased hydrophobicity show greater increase in
emission intensities upon binding to polyelectrolytes viz; DNA,
thus, we conclude that there is some interaction between the CT
DNA and complexes that occurs due to the hydrophobicity of both
the molecules. The CT DNA binding abilities of the complexes
were obtained from Scatchard equation and follow the order,
2 (K ¼ 7.6 104 M1) > 1 (K ¼ 1.4 104 M1). These results are
consistent with the findings obtained from UVevis spectral
studies.
the adjacent DNA base pairs. Addition of second molecule, which
binds to DNA more strongly than EthBr, would quench the DNAinduced EthBr. The extent of quenching of the fluorescence of
EthBr bound to DNA would reflect propensity of DNA binding of 1
and 2.
On addition of complexes 1 and 2, to CT DNA pretreated with
EthBr ([DNA]/[EthBr] ¼ 1) the decrease in emission intensity was
observed. The emission intensity in the absence and presence of 1
and 2 with EthBreDNA are depicted in Fig. 3. As there is incomplete
quenching of the EthBr-induced emission intensity, thus the
intercalative mode for 1 and 2 was ruled out. Furthermore quantitatively the quenching extents Ksr were evaluated for 1 and 2,
were found to be 2.1 104 M1 and 8.9 104 M1, respectively. The
higher value of Ksr for 2 was attributed to increased hydrophobicity
of the complex.
4.2.2. Ethidium bromide displacement assay
To further investigate the mode of binding of the complexes 1
and 2, the ethidium bromide displacement assay was carried out
[22]. The molecular fluorophore EthBr emits intense fluorescence
in the presence of CT DNA due to its strong intercalation between
4.2.3. Effect of phosphate group on the binding of complexes with
DNA
To further validate the interaction of the complexes 1 and 2 with
the DNA, the fluorescence titrations were performed in the
Fig. 3. Quenching spectra of CT DNA bound ethidium bromide in the presence of (a)
complex 1 and (b) complex 2, in buffer 5 mM TriseHCl/50 mM NaCl, pH ¼ 7.2 at 25 C.
Fig. 4. Emission spectra of (a) complex 1, and (b) complex 2, with increasing
concentration of K2HPO4, in the absence and presence of CT DNA in TriseHCl buffer at
pH ¼ 7.2 at 25 C.
1606
S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608
presence of K2HPO4 at 25 C. With increasing amount of K2HPO4,
fluorescence intensity of complexes 1 and 2 increase appreciably
demonstrating a competitive binding behavior between the phosphate group of K2HPO4 and the phosphate component of DNA
backbone [54]. This competitive interaction of the phosphate
groups weakens the interaction of the complexes with DNA [55]
which provide supportive evidence for electrostatic interaction of
the Sn(IV) complexes with the phosphate backbone of DNA (Fig. 4
(a) and (b)). However, the enhancement in the intensity of
complex 2 is higher in comparison to complex 1, due to hydrophobicity of the complexes. These observation provides supportive
evidence for electrostatic interaction of the Sn(IV) complexes which
exhibit selective binding to the phosphate group of DNA double
helix.
5. Cleavage experiments
5.1. Chemical nuclease activity
a
Form II
Form I
10
20
The DNA cleavage activity of the complex 2 at 10e40 mM
concentration was studied using pBR322 DNA (w80% Supercoiled
form) in 5 mM TriseHCl/50 mM NaCl buffer at pH 7.2 and upon
irradiation with UV light of 365 nm [58,59]. On increasing the
concentration, complex 2 produces conversion of SC DNA (Form I)
to NC form (Form III) completely. The ligand moiety could generate
the photoexcited (nep*) and/ or (pep*) states, causing the cleavage
of DNA as observed. The photocleavage experiment results suggested, the possible involvement of a metal assisted photoexcitation process in the visible light-induced DNA cleavage activity of
the complex 2. Thus as evident from the photoinduced electrophoretic pattern of 2, the photonuclease activity observed for 2 at
micro molar concentration upon irradiation of 365 nm UV light for
a shorter time of 10 min exhibited significantly promising activity
(Fig. 6).
6. In vitro anticancer activity
There has been considerable interest in DNA endonucleolytic
cleavage reactions that are activated by metal complexes [56,57].
Thus, DNA mobility shift assays were carried out to investigate the
ability of Sn(IV) complexes to interact with plasmid DNA. The initial
amount of pBR322 DNA (300 ng) was constant and incubated with
increasing concentrations of the complexes 1 and 2. The complex 1
(10e40 mM) cleaves double stranded supercoiled plasmid DNA (SC
form: Form I) (300 ng) on incubation in 5 mmol TriseHCl/50 mmol
NaCl buffer into nicked circular form (NC form: Form II) at physiological pH 7.2 and temperature 37 C as depicted in Fig. 5(a). Upon
gel electrophoresis of the reaction mixture, with varied concentration of complex 2 (10e40 mM) and keeping the DNA concentration (300 ng) constant efficient cleavage was observed (Fig. 5b).
The DNA results suggested that the efficient nuclease activity
was exhibited by the complex 2 at the concentration of 40 mM. In
the concentration-dependent electrophoretic pattern exhibited
conversion of SC form (Form I) to NC form (Form II) with increase in
the concentration of 2. The distribution of supercoiled and nicked
forms of DNA in the agarose gel electrophoresis provides a measure
of the extent of hydrolysis of the phosphodiester bond which
suggested the more binding propensity of complex 2 as compared
to 1.
CT
5.2. Photonuclease activity
30
40 µM
b
In vitro anticancer activity of complexes 1 and 2 was screened
against 20 different human carcinoma cell lines of different histological origin. The Sulforhodamine-B (SRB) assay was used to assess
cellular proliferation [60]. The result showed good potential of the
complexes 1 and 2 as drug candidate. Interestingly, complex 2
showed specificity and exhibited good cytotoxic activity only
against DWD (human oral carcinoma cell line). The results in terms
of GI50 values are given in Table 1. The reason for its selectivity
needs further investigations.
7. Cell death induced by complex 2
To ascertain whether the cell death induced by complex 2
involves changes in nuclear characteristics, EB/AO dual staining
was performed (Fig. 7). To identify the possible involvement of
apoptosis, DWD cells treated with complex 2 (1e4 mg/ml for 24 h)
were stained with EB/AO dual stain. Changes in the nuclear
morphology such as extensive chromatin aggregation or nuclear
condensation were observed in the treated cells. This can be
explained by the fact that, in dual staining with EB/AO, AO
permeates the cells and stains the nucleus green. However, EB is
taken up only by the cells when cell membrane integrity is lost and
stains nucleus orange [61,62]. Thus with dual staining, the live cells
show green coloured intact nucleus while dead or necrotic cells
show structurally normal nucleus with even orange staining. The
early apoptotic cells, where membrane integrity is not lost show
green coloured condensed or fragmented nucleus while late
apoptotic cells display fragmented and condensed chromatin
which is stained orange. The cells treated with complex 2 exhibited
fragmented orange coloured chromatin, a hall mark of late
apoptotic cells even at a concentration as low as 1 mg/ml A five to
nine fold increase in percent apoptotic cells was observed in treated
groups (at 1e4 mg/ml concentrations) as compared to control
group.
Form II
Form II
Form III
Form I
Form I
CT
10
20
30
40 µM
Fig. 5. Agarose gel electrophoresis patterns of pBR322 plasmid DNA (300 ng) cleaved
by (a) complex 1 (10e40 mM), and (b) complex 2 (10e40 mM), after 1 h incubation
time in buffer (5 mM TriseHCl/ 50 mM NaCl, pH ¼ 7.2 at 25 C) (concentration
dependent).
CT
10
20
30
40 µM
Fig. 6. Photoinduced electrophoretic separations showing the cleavage of pBR322
plasmid DNA (300 ng) by induced 2 after 0.5 h incubation time, in buffer (5 mM
TriseHCl/50 mM NaCl, pH ¼ 7.2 at 25 C).
S. Tabassum et al. / Journal of Organometallic Chemistry 696 (2011) 1600e1608
1607
Table 1
Summary of the screening data of 1 and 2, for the in vitro anti tumor activity GI50 (in mg/ml).a
Compound
Hop62
A549
PC3
DU145
A498
DWD
Colo205
HT29
HCT15
SW620
2
1
ADRb
16
79
<10
50
>80
<10
36
76
<10
33
65
<10
61
57
<10
<10c
<10c
<10
26
50
<10
>80
>80
<10
12
76
<10
49
>80
<10
Compound
2
1
ADR
T24
29
49
<10
MIA-PA-CA2
23
>80
<10
MCF7
22
44
<10
ZR-75-1
47
71
<10
SiHa
33
60
<10
ME180
53
>80
<10
HeLa
58
>80
<10
U373MG
45
>80
<10
A2780
25.6
47.5
<10
K562
55
>80
<10
a
b
c
GI50 ¼ growth inhibition of 50% (GI50) calculated from [(Ti Tz)/(C Tz)] 100 ¼ 50, drug concentration results in a 50% reduction in the net protein increase.
ADR ¼ adriamycin (positive control compound).
GI50 value <10 mg/ml is considered to demonstrate activity.
phosphate backbone of DNA double helix. Furthermore, 2 exhibited
an efficient DNA photocleavage activity. Complex 2 showed
significantly good cytotoxic activity against DWD cell lines (human
oral carcinoma cell line). Indeed, complex 2 is an illustration of an
antitumor drug candidate which could exhibit fewer side effects,
low toxicity, specificity (due to glycosylated unit) and therefore,
warrants further in vivo investigations.
Acknowledgements
We express our gratitude to Department of Biotechnology, New
Delhi, for generous financial support (Scheme No. BT/PR6345/Med/
14/784/2005). Thanks to RSIC, Panjab University, Chandigarh for
providing NMR facility, RSIC, CDRI Lucknow for providing CHN
analysis data, ESI-MS.
References
Fig. 7. Effect of complex 2 on nuclear morphology of DWD cells: (A) control cells without
any treatment. Pink arrows showing normal green coloured nuclei and white arrow
showing occasional orange coloured necrotic or dead cells. (B) Cells treated with complex
2, at 1 mg/mL showing fragmented, condensed orange coloured chromatin (yellow arrows)
indicating late apoptotic cells while blue arrow indicating early apoptotic cells with green
condensed and fragmented chromatin. (C) (LefteRight) showing a magnified view of
necrotic, live, early and late apoptotic cells, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article).
8. Conclusions
We have described herein, the synthesis and characterization of
new water soluble glycoconjugate derived Sn(IV) complexes 1 and
2, as antitumor drug candidates. The in vitro DNA binding studies of
complexes 1 and 2 revealed that Sn(IV) ions interact through the
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