A Co(III) Complex of 1-Amino-4-hydroxy-9,10-anthraquinone Exhibits Apoptotic Action against MCF-7 Human Breast Cancer Cells

Abstract

A Co(III) complex of 1-amino-4-hydroxy-9,10-anthraquinone (QH) (Scheme-1) having the molecular formula CoQ3 (Scheme-2) was prepared and characterized by elemental analysis, FTIR spectroscopy, UV–vis spectroscopy, fluorescence spectroscopy, and mass spectrometry. In the absence of a single crystal, the energy-optimized molecular structure of CoQ3 was determined by employing computational methods that was validated using spectroscopic evidences, elemental analysis, and mass spectrometry data. The electrochemical properties of the complex were analyzed using cyclic voltammetry and indicate a substantial modification of the electrochemical properties of the parent amino-hydroxy-9,10-anthraquinone. CoQ3 was thereafter tested on MCF-7 human breast cancer cells. The IC50 value for a 24 h incubation was found to be (95 ± 0.05) μg/mL. The study showed that such cancer cells underwent both early and late apoptosis following the interaction with CoQ3.

Full text

A Co(III) Complex of 1‑Amino-4-hydroxy-9,10-anthraquinone
Exhibits Apoptotic Action against MCF‑7 Human Breast Cancer Cells
Somenath Banerjee, Sanjay Roy, Dhanasekaran Dharumadurai, Balaji Perumalsamy,
Ramasamy Thirumurugan, Saurabh Das,*Asoke Prasun Chattopadhyay,*and Partha Sarathi Guin*
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ABSTRACT: A Co(III) complex of 1-amino-4-hydroxy-9,10-anthraquinone (QH) (Scheme-1) having the molecular formula CoQ3
(Scheme-2) was prepared and characterized by elemental analysis, FTIR spectroscopy, UV−vis spectroscopy, fluorescence
spectroscopy, and mass spectrometry. In the absence of a single crystal, the energy-optimized molecular structure of CoQ3was
determined by employing computational methods that was validated using spectroscopic evidences, elemental analysis, and mass
spectrometry data. The electrochemical properties of the complex were analyzed using cyclic voltammetry and indicate a substantial
modification of the electrochemical properties of the parent amino-hydroxy-9,10-anthraquinone. CoQ3was thereafter tested on
MCF-7 human breast cancer cells. The IC50 value for a 24 h incubation was found to be (95 ±0.05) μg/mL. The study showed that
such cancer cells underwent both early and late apoptosis following the interaction with CoQ3.
1. INTRODUCTION
Anthracycline drugs are anticancer agents used in treating
different forms of human carcinoma.
1−4
Although they enjoy
wide acceptance in chemotherapy, their use is often questioned
for the associated cardiotoxicity and high cost involved,
particularly for people from economically weaker sections of
the society. Hence, there is an effort worldwide
5−19
to find
alternative cheaper analogues that are less cardiotoxic.
5−10
These are either derivatives of anthracyclines that are less
costly or their simpler analogues.
11−20
The limitation due to acute and chronic toxicity,
21−25
of
which cardiotoxicity receives the maximum attention, is the
most disturbing regarding the use of anthracyclines or their
derivatives and analogues as anticancer agents.
26−31
Participat-
ing in reactions of the respiratory chain, they produce
semiquinone radical anions and related intermediates by one-
electron reduction of the quinone. Although a pre-requisite for
chemotherapeutic efficacy, such generation is also responsible
for cardiotoxicity.
26−30
Semiquinone upon reaction with O2
generates superoxide radical anion (O2
•−) that in turn produce
H2O2/OH•.
20,30−33
These species participate in a wide range
of redox reactions as in oxidative phosphorylation, complex
formation with phospholipid, and in lipid peroxidation.
30−32
Previous research on the subject suggests that complex
formation of these drugs with different metal ions leads to
decreased toxicity, the magnitude of which depends on the
metal ion. Those metal ions having a stable lower oxidation
state were found to cause maximum decrease in O2
•−formation
in an assay where NADH was the electron donor and
cytochrome c was the electron acceptor. Hence, studies related
to metal complexes gained a lot of importance regarding this
matter.
7,12,14,31,32
Metal complexes stabilize the semiquinone
radical anion formed. Hence, superoxide formation due to a
reaction between a semiquinone radical anion and molecular
oxygen is either inhibited or decreased drastically. It is
Received: November 1, 2021
Accepted: December 22, 2021
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therefore imperative to study such metal complexes,
particularly with regard to their electrochemical behavior
under different experimental conditions.
It is worth mentioning that although several metal
complexes of adriamycin, daunorubicin, mitoxantrone, and
their analogues with Fe(III), Al(III), Cu(II), Ni(II), Pd(II),
and Tb(III) were prepared and characterized,
7,10−14,33−46
comprehensive knowledge on structures of these metal
complexes is lacking due to inherent difficulties in obtaining
single crystals for X-ray diffraction studies. Single-crystal X-ray
diffraction structures of only a few hydroxy-9,10-anthraquinone
complexes are reported.
44,47,48
In this study also, different
methods were employed to obtain single crystals of CoQ3
taking different compositions of solvents. However, all efforts
in getting an appropriate single crystal for CoQ3failed. The
planarity of the anthraquinone unit in these complexes could
possibly be a hindrance in getting single crystals.
7
For this
reason, we made an effort to characterize CoQ3theoretically
using density functional theory (DFT) based on experimental
data we obtained such as elemental analysis, IR spectroscopy,
mass spectrometry, powder X-ray diffraction, molecular
spectroscopy, and electrochemistry. DFT is helpful in
generating the energy optimized structure, and various
essential parameters of the complex may also be obtained
from this study. The thus prepared complex was tested on
MCF-7 human breast cancer cells to see whether it initiates
apoptosis and thus could be considered as a less costly
alternative to anthracyclines already in use.
2. RESULTS AND DISCUSSION
2.1. Analysis of the Mass Spectra of CoQ3.Assuming
that the formula of the complex is CoQ3(Scheme 2), an
analysis of its mass spectrum (Figure S1, SI) was attempted.
The molecular ion peak or that of the protonated molecular
ion expected at m/z = 773.62 was not found. However, a clear
signal at m/z = 689.46 corresponds to a fragment remaining of
the complex following loss of a carbon-bound −NH2group
from each ligand (a loss of 28 mass units from each ligand, i.e.,
78 mass units from the complex) to result in a peak
theoretically expected at m/z = 689.62. From this peak, loss
of two quinone oxygens would result in a peak theoretically
expected at m/z = 661.62. The experimental value is 661.45,
which tallies with the expected value. Loss of four quinone
oxygens from the first fragment results in a peak theoretically
expected at m/z = 633.62 and experimentally found at 633.42.
Here also, the agreement is close. Similarly, loss of six quinone
oxygens from all the three ligands of the first fragment should
result in a theoretical peak at m/z = 605.62. This was
experimentally observed at 605.39, again pointing to a close
agreement. At this stage of fragment formation in mass
analysis, the metal center is bound to three ligands via the
three phenolic −OH groups on each of them. The peaks
identified above therefore categorically indicate the formation
of a 1:3 complex. Subsequent to the fragmentations mentioned
above, further loss of two carbon atoms and a few hydrogens at
a time explains peaks at an m/zvalue of 577.35 and also the
cluster of peaks at m/z values of 533.99, 532.99, and 531.98,
respectively. Peaks at lower m/z values correspond to smaller
fragments. Therefore, from an analysis of the mass spectrum of
the cobalt complex, it may be concluded that the complex has
the formula CoQ3as shown in Scheme 2.
2.2. Analysis of the IR Spectra of CoQ3.The FTIR
spectrum for QH (Figure S2, SI) shows a peak at 3431 cm−1,
which is due to N−H bond stretching, while that at 3300 cm−1
is due to stretching of O−H bonds.
6
The O−H stretching is
modified significantly in the complex (Figure S3,SI),
indicating an involvement of the −OH group during complex
formation. Since there is deprotonation of −OH during
complex formation, the molecule ceases to show intra-
molecular hydrogen bonding identified in QH. Peaks in this
region do not disappear completely in the complex when
compared with QH, indicating the presence of free −NH2on
each ligand (just as that observed or the IR spectrum of QH).
In the IR spectrum of CoQ3(Figure S3, SI), peaks at 1625,
1586, and 1537 cm−1are attributed to stretching due to free
carbonyl and CC or a combination of both, respectively. In
an earlier study,
6
we showed that peaks obtained in the region
1464−1031 cm−1in the IR spectrum of the ligand (QH) may
be attributed to combinations of O−H, N−H, and C−H
bending modes. Natures of the peaks in this region are
somewhat different in the complex. More specifically, the peak
at 1121 cm−1is reduced significantly, probably due to binding
of oxygen of the −OH group to Co(III) following its
deprotonation.
2.3. Powder X-ray Diffraction of CoQ3.The powder X-
ray diffraction (PXRD) pattern of CoQ3is shown in Figure 1.
All peaks can be indexed with the space group R32(155), and
Cu Kα= 1.5406 Å using the WINPLOTR program. Refined
cell parameters were found to be a= 7.45 Å, b= 6.52 Å, and c
= 27.8 Å. The unit cell volume was 1352 Å3;α= 33.43°,β=
90°, and γ=90°. Thus, PXRD analysis provides information
about the dimension of the unit cell of a crystalline CoQ3.
2.4. Structure of CoQ3from Density Functional
Theory Methods. The energy-optimized molecular structure
of CoQ3is shown in Figure 2, and structural parameters are
summarized in Table S1 (SI). Figure 2 shows three QH
molecules coordinated to Co(III) through phenolic O−and
quinone oxygen, forming CoQ3.
Scheme 1. 1-Amino-4-hydroxy-9,10-anthraquinone (QH)
Scheme 2. Structure of CoQ3
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The energy level diagrams of QH and CoQ3are shown in
Figure S3 (SI). The HOMO (H) and LUMO (L) are
indicated in each case (Figure 3). Red lines indicate the π
orbitals, black lines indicate σ, and blue lines represent mixed
metal−ligand (M-L) orbitals. Some M-L type MOs may have
mixed σand πcharacters as the three ligands are arranged in
such a manner that σof one may mix with πof another. Metal
orbitals are mainly dπ, with some pπmixed. Co(III) orbitals
are much lower in energy to be shown in the above diagram. It
should also be noted that the HOMO and LUMO are M-L
type orbitals.
2.5. UV−vis Spectroscopy of CoQ3.The absorption
spectrum of QH (Figure 4a) in 30% ethanol
6,7
shows four
absorption bands (at 250, 290, 530, and 565 nm) due to π−π*
and n−π*transitions of its various tautomeric forms in rapid
equilibrium in aqueous solution.
6,7,49
From the UV−vis
spectrum of CoQ3(Figure 4b), it is clear that the absorption
peaks at 250, 290, 530, and 565 nm remain almost unaltered,
which indicate that the electronic absorption spectrum of
CoQ3depends weakly on the nature of the metal and is
primarily defined by the ligand (QH).
49
However, the
appearance of a new peak at 600 nm is characteristic of the
complex (CoQ3). It is important to mention here that
tautomeric structures found for free QH
49
in aqueous media
are not possible for CoQ3since phenolic −OH groups in the
QH molecule are deprotonated owing to coordination of
Co(III) by phenolic oxygens.
2.6. Fluorescence Spectroscopy of CoQ3.Fluorescence
spectra of QH and CoQ3are shown in Figure S4 (SI) recorded
following an excitation at 530 nm. The emission spectrum
exhibits a maximum at 590 nm for QH and 594 nm for CoQ3.
The difference in the emission peak of CoQ3compared to QH
is due to the metal ligand bond.
2.7. Electrochemical Reduction of CoQ3in Organic
Polar Solvents. Electrochemical behavior of CoQ3was
studied in anhydrous DMSO and DMF in the presence of
TBAB as the supporting electrolyte using cyclic voltammetry.
In anhydrous DMSO, CoQ3undergoes successive three one-
electron reductions having peak potentials (Epc)at−0.795,
−1.010, and −1.295 V, respectively, vs Ag/AgCl/saturated
KCl (Figure 5 and Table 1). In this case, the first reduction is
reversible, while the other two are quasi-reversible at different
scan rates. These three one-electron reduction steps are due to
the reduction of the three free quinone centers of the three Q−
bound to Co(III) inCoQ3(Scheme 3). For these reductions,
the formal potentials (E) of the respective reduction steps were
found at −0.750, −0.987, and −1.255 V, respectively. It is
noted that although there are three equivalent free quinone
sites in CoQ3(Scheme 3), there exists a difference in their
formal potential values, which is quite appreciable. Thus, after
reduction at the first free quinone in CoQ3, reduction of the
second and third quinone sites is significantly delayed. In other
words, the reduced species (semiquinone radical anion) that
formed due to the first or second reduction is stabilized in the
metal ion environment due to delocalization of the negative
charge. This is important with regard to the compound’s
biochemical action since a stabilized semiquinone would delay
the reaction between semiquinone and molecular oxygen
30−34
within cells where it would be employed.
In anhydrous DMF, under similar experimental conditions,
CoQ3undergoes three-one electron reductions having peak
potentials (Epc)at−1.025, −1.225, and −1.475 V, respectively,
with the corresponding formal potentials (E) being −0.950,
−1.195, and −1.405 V, respectively (Figure 6 and Table 1).
Considering the fact that the polarity of DMF is less than that
of DMSO
51
and comparing the three reduction potentials of
CoQ3in the two solvents, it can be said that with the
increasing polarity of the medium, reduction potentials move
in a positive direction and that reductions become more
feasible as the polarity of solvent increases. This means stability
of the formed semiquinone species is increased with an
increase in the polarity of the medium. Stabilization of the
Figure 1. Powder X-ray diffraction patterns of CoQ3.
Figure 2. Energy optimized structure of CoQ3.
Figure 3. Different HOMOs (H) and LUMOs (L) of CoQ3.
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semiquinone is also reflected in the formal reduction potential
data. This aspect is important with respect to its chemo-
therapeutic efficiency.
30−34
Owing to stabilization of the
semiquinone radical anion, the probability for reaction of a
semiquinone radical anion with molecular oxygen would be
delayed and that may reduce cardiotoxicity if the molecule
were to be employed as an anticancer agent.
30−34
Under similar experimental conditions, a cyclic voltammo-
gram of QH shows two reversible waves at −0.816 and −1.355
V in anhydrous DMSO and at −0.832 and −1.309 V in
anhydrous DMF vs Ag/AgCl, with saturated KCl forming a
semiquinone radical anion and quinone dianion, respec-
tively.
7,8
Formal potentials for such reductions were evaluated
as −0.770 and −1.308 V in anhydrous DMSO and −0.785 and
−1.258 V in anhydrous DMF.
8
Comparing electrochemical
parameters and cyclic voltammograms (Figures 5 and 6)of
CoQ3with those of QH in anhydrous DMSO and anhydrous
DMF,
8
one can say that the electrochemical behavior of QH
bound to a metal ion as Q−(as in CoQ3) is significantly altered.
It is seen that the reduction peak currents (Ipc) for three
successive reductions of CoQ3in both DMSO and DMF have
a linear relationship with the square root of the scan rate and
that it passes through the origin (Figures 5 and 6). This
suggests that such reductions are fully diffusion controlled and
that there is no adsorption on the electrode surface. The
diffusion coefficient (DO) of CoQ3was determined by the
relation shown in eq 1
50
and found as 3.04 ×10−5and 6.31 ×
10−5cm2s−1in DMSO and DMF, respectively (summarized in
Table 1).
I
nD ACv(2.69 10 )
pc 53/2
O1/2 1/2
=× (1)
where Ipc = cathodic peak current (A), n= number of electron
involved in the reduction, A= area of the electrode (cm2), C=
concentration (mol·cm−3), and v= scan rate (V·s−1).
From values of diffusion coefficients of CoQ3in two
different solvents (Table 1), it is evident that DOincreases as
the polarity of the solvent decreases, clearly indicating greater
solvation of CoQ3in a more polar solvent that causes lower
diffusion onto the surface of the electrode. Thus, CoQ3is more
solvated in DMSO due to hydrogen bonding and other
electrostatic interactions.
8
Intermolecular hydrogen bonding
between one of the two hydrogen of aromatic amino group
(−NH2) of QH and negatively charged oxygen of the solvent
(DMSO) is very strong.
8
This type of hydrogen bonding
would be weak in the case of DMF since for this solvent,
oxygen has a less partial negative charge than that on oxygen in
DMSO.
8
Figure 4. UV−vis spectrum of (a) QH and (b) CoQ3in aqueous ethanol.
Figure 5. (a) Cyclic voltammogram of CoQ3in anhydrous DMSO media. Scan rate: 0.10 Vs−1. [CoQ3]=1×10−3M, [TBAB] = 0.1 M, T=
298.15 K. (b) Plot of cathodic peak current vs square root of scan rate for first (solid circles), second (open squares), and third reduction (solid
squares) of CoQ3in anhydrous DMSO.
Table 1. Electrochemical Parameters of CoQ3
a
media Epc-1 (V) Epc-2 (V) Epc-3 (V) E-1 (V) E-2 (V) E-3 (V) D0(cm2s−1)
DMSO −0.795 −1.010 −1.295 −0.750 −0.987 −1.255 3.04 ×10−5
DMF −1.025 −1.225 −1.475 −0.950 −1.195 −1.405 6.31 ×10−5
a
Potentials were measured with respect to vs Ag/AgCl/saturated KCl.
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2.8. Effect of CoQ3on Viability of MCF-7 Human
Breast Cancer Cells by the MTT Assay. Using the MTT
assay, the cytotoxic activity of CoQ3was analyzed against
MCF-7 human breast cancer cells (Figure 7). It was estimated
according to dose values of exposure of CoQ3required to
reduce the survival to 50% (IC50) in comparison to that of
untreated cells. The IC50 value for 24 h was found to be (95 ±
0.05) μg/mL. This indicates that CoQ3is cytotoxic against
MCF-7 breast cancer cells.
2.9. AO/EB Staining. Apoptosis is the hallmark of cell
death and can be characterized by cellular morphological
Scheme 3. Three Step One-Electron Reductions of CoQ3in Organic Polar Solvents Like DMSO and DMF
Figure 6. (a) Cyclic voltammogram of CoQ3in anhydrous DMF
media. Scan rate: 0.10 Vs−1. [CoQ3]=1×10−3M, [TBAB] = 0.1 M,
T= 298.15 K. (b) Plot of cathodic peak current vs square root of scan
rate for first (open circles), second (open squares), and third
reduction (solid squares) of CoQ3in anhydrous DMF. Figure 7. Cytotoxic effect of CoQ3on MCF-7 human breast cancer
cells after exposure for 24 h.
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changes observed during the process of cell death. The dual
staining method of AO/EB detects such morphological
changes. Figure 8 corresponds to AO/EB staining of
control/non-treated and CoQ3-treated MCF7 breast cancer
cells. Based on fluorescence emission and nucleus morphology,
cells were distinguished to have viable, apoptotic, or necrotic
characteristics. The viable cells were observed to have uniform
green-colored nuclei with a typical cell morphology and intact
membrane. On the other hand, apoptotic cells showed
irregular cell morphologies with orange to red condensed
chromatin and/or fragmented nuclei. Furthermore, the large
orange to red fluorescent swollen cells with no fragmented
nuclei were differentiated as necrotic cells. The results from
AO/EB staining reveal that the control group contains more
viable cells and a few apoptotic and necrotic cells. In contrast,
CoQ3-treated MCF7 breast cancer cells induced majority of
cell death through the apoptosis mode and actually very few by
necrosis. Furthermore, condensed and fragmented morpholo-
gies were mostly observed in the CoQ3treatment group. The
results of calculating the percentage of apoptotic cell death
induced by CoQ3and analyzed by fluorescent images of AO/
EB staining revealed that AQS-treated cells induced a higher
percentage of apoptotic cells and a lower percentage of
necrotic cells than untreated cells (Figure 9). The graph
depicts a percentage count of apoptotic normal and abnormal
cells. The error bar represents the standard deviation across
three replicates.
3. CONCLUSIONS
A Co(III) complex of 1-amino-4-hydroxy-9,10-anthraquinone
(QH) with the molecular formula CoQ3was synthesized and
characterized by different methods. The optimized molecular
structure of CoQ3was estimated using computational
methods. The HOMO and LUMO of CoQ3were also
characterized by this method. Electrochemical properties of
CoQ3were studied in anhydrous DMSO and anhydrous DMF
media using cyclic voltammetry, and the mechanism of
reduction was established. It showed that different reduced
anions of CoQ3are stabilized in a metal surrounding
environment and that reductions would therefore be delayed.
Polarity of the solvents also affects stability of the reduced
anion. A significant modification of electrochemical properties
of QH was also seen when it was bound to Co(III) in CoQ3.
The IC50 value of CoQ3for 24 h of incubation corresponding
to cytotoxicity of CoQ3on human breast cancer cells MCF-7
was evaluated as 95 ±0.05 μg/mL. The study revealed that
such cancer cells underwent both early and late apoptosis due
to CoQ3.
4. EXPERIMENTAL SECTION
4.1. Materials. 1-Amino-4-hydroxy-9, 10-anthraquinone
(QH) (Scheme 1) (96%) purchased from Alfa Aesar, Germany
was recrystallized from an ethanol−methanol mixture and
characterized as mentioned earlier.
6−9
The quinone moiety
being sensitive to light, solutions were prepared either just
before an experiment or very carefully stored in the dark.
CoCl2·6H2O purchased from Merck, India was used to prepare
the Co(III) complex. KCl and tetrabutyl ammonium bromide
[TBAB] (both are AR grade, Spectrochem, India) were used as
supporting electrolytes in aqueous and non-aqueous media,
respectively.
Dimethyl sulfoxide (DMSO) (99.0%, Spectrochem, India)
was first dried over fused CaCl2for 3−4 days, decanted, and
then distilled under reduced pressure.
51
The distilled sample
was preserved in a well-stoppered Jena bottle in desiccators
and redistilled again before use. N,N-Dimethyl formamide
(DMF) (99.5%, Spectrochem, India) (LR, BDH) was
purified
52
first by distilling under reduced pressure in a N2
atmosphere and then preserving the distillate over dry K2CO3
(Merck) for a week. Then, the DMF was decanted and allowed
to mix with dry P2O5(Riedel) and distilled again to be able to
use it under anhydrous conditions. Anhydrous DMF and
DMSO were used as solvents in electrochemistry experiments.
All aqueous solutions were prepared in triple-distilled water.
4.2. Synthesis of CoQ3.An aqueous solution of 0.5 mmol
CoCl2·6H2O and a solution of 1.5 mmol QH in acetonitrile
were mixed and stirred for about 6 h using a magnetic stirrer.
Co(II) was oxidized to Co(III) by purging air into the reaction
media. The solution was kept for 7 days in air to allow it to
evaporate till it was almost 5 mL. A violet-colored complex was
separated by filtration followed by washing with acetonitrile.
The complex was recrystallized from a methanol−acetonitrile
mixture and dried in air. Results of elemental analysis showed
that it has the formula CoQ3. Found: C, 65.09%; H, 3.08%; N,
5.51%. Calculated: C, 65.13%; H, 3.10%; N, 5.43%. In 25%
aqueous ethanol solution, 0.1 mM CoQ3showed a
conductance less than 5 μS/cm at 298.15 K, indicating that
it is neutral.
4.3. Computational Studies. The structure of CoQ3was
optimized using DFT with Ahlrich SV basis
53,54
and B3LYP
functional
55−57
using the Orca program suite.
58
Electronic
transitions were calculated by the time-dependent DFT
method with the same basis set and functional using Orca.
Pictures of molecular orbitals (MOs) were generated with the
same basis set and functional using Gaussian 09W
59
and MaSK
software.
60
4.4. Analytical Methods. With the help of a Perkin-Elmer
2400 II elemental analyzer, the carbon, hydrogen, and nitrogen
Figure 8. AO/EB staining of the control and CoQ3-treated MCF-7
human breast cancer cells.
Figure 9. Comparison of percentage of cells in apoptotic death
compared to healthy cells and necrotic death.
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analyses were done. FTIR analysis was performed on a Perkin
Elmer RX-I spectrophotometer. Spectra were obtained using
KBr pellets in the range 4000−400 cm−1. The mass spectrum
was recorded on Micromass Q-Tofmicro, Waters Corporation.
CoQ3was dissolved in an anhydrous acetonitrile solvent, and
the MS data were recorded by using ESI positive mode. The
instrument applies a focusing voltage to the electrospray probe
to promote mobile phase evaporation as part of the ionization
process. PXRD data was collected on a Bruker AXS D8 powder
diffractometer using Cu Kαradiation (λ= 1.548 Å) generated
at 40 kV and 40 mA. UV−visible spectroscopy was done on a
spectrophotometer (model: MECASYS OPTIZEN POP).
Experiments related to cyclic voltammetry were performed
using the conventional three-electrode system. The temper-
ature was maintained at 25 °C with the help of a circulating
water bath. The working electrode was glassy carbon, the
surface area of which was 0.07065 cm2, the counter electrode
was a platinum wire, and the reference electrode was Ag/AgCl
in satd. KCl. Using a potentiostat (model DY2312, Digi-Ivy),
all electrochemical studies were performed. The range of
concentrations of different solutions was 5 ×10−5moldm−3to
1.5 ×10−3moldm−3. Before the solutions were subjected to
cyclic voltammetry, they were degassed for nearly30 min using
highly pure Ar.
4.5. Cell Culture. MCF7 human breast cancer cells were
procured from National Center for Cell Science, Pune, India.
Cells were cultured and maintained in DMEM high-glucose
medium (Sigma-Aldrich, USA) supplemented by 10% fetal
bovine serum (Gibco, Thermo Fisher, USA) and 20 mL of
penicillin/streptomycin as antibiotics (Gibco, Thermo Fisher,
USA), and incubated at 37○C with 5% CO2in a CO2
incubator (Thermo scientific, USA). All experiments were
carried out using cells from the passage of 15 or less.
4.6. Cell Viability Assay. CoQ3was dissolved in DMSO
and a stock solution was prepared. It was then diluted to obtain
different concentrations of the compound in the range 0−200
μg/mL. Two hundred microliters of such solutions was added
to wells containing 5 ×103MCF-7 cells per well of a 96-well
culture plate. DMSO was used as the control solvent. Twenty
microliters of MTT solution (5 mg/mL in PBS) was
transferred to each well following 24 h of incubation, and
the plate was incubated at 37 °C for 4 h in the dark. To
dissolve formazan crystals, 100 μL of DMSO was added to
each well and the absorbance of the final solution was
measured at 570 nm using a microplate reader (Bio-Rad,
iMark, USA). Data was collected for three replicates each, and
the respective mean was used in the following formula to
calculate percentage inhibition:
percentage inhibition = ([mean OD of untreated cells
(control) −mean OD of treated cells (treated)] ×100)/
(mean OD of untreated cells (control))
4.7. Acridine Orange (AO) and Ethidium Bromide (EB)
Staining. CoQ3-induced apoptosis was examined using the
fluorescent-based dual staining method AO/EB as defined by
Spector et al.
61
with some modifications. In brief, cells treated
for 24 h with the IC50 concentration of CoQ3were harvested
and washed with cold PBS. Cell pellets were resuspended and
diluted with PBS. The cell suspension (5000 in number) was
mixed with AO/EB solution (3.8 μM AO and 2.5 μMEBin
PBS) and transferred to a clean microscope slide. Morpho-
logical features of the cells were examined under a fluorescent
microscope (Carl Zeiss, Axioscope2plus) with a UV filter
(450−490 nm).
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.1c06125.
(Figure S1) Mass Spectrum of CoQ3, (Figure S2) IR
spectrum of QH and CoQ3, (Figure S3) energy level
diagram of QH and CoQ3, (Figure S4) fluorescence
spectra of QH and CoQ3in aqueous ethanol, and (Table
S1) structural parameters of CoQ3(PDF)
■AUTHOR INFORMATION
Corresponding Authors
Saurabh Das −Department of Chemistry, Jadavpur University,
Kolkata 700032, India; orcid.org/0000-0002-0455-
8760; Phone: +91 9123865911; Email: dasrsv@yahoo.in,
saurabh.das@jadavpuruniversity.in; Fax: +91 33
24146223
Asoke Prasun Chattopadhyay −Department of Chemistry,
University of Kalyani, Nadia 741235 West Bengal, India;
orcid.org/0000-0002-2411-1384;
Phone: +919836156800; Email: asoke@klyuniv.ac.in;
Fax: : +91 33 2582 8282
Partha Sarathi Guin −Department of Chemistry, Shibpur
Dinobundhoo Institution (College), Howrah 711102 West
Bengal, India; orcid.org/0000-0001-9258-9227;
Phone: +91 9330083036; Email: parthasg@gmail.com;
Fax: +91 33 2688 0344
Authors
Somenath Banerjee −Department of Chemistry, Shibpur
Dinobundhoo Institution (College), Howrah 711102 West
Bengal, India; Department of Chemistry, Jadavpur
University, Kolkata 700032, India
Sanjay Roy −Department of Chemistry, Netaji Subhas Open
University, Nadia 741235, India; orcid.org/0000-0001-
6841-4961
Dhanasekaran Dharumadurai −Department of Microbiology,
School of Life Sciences, Bharathidasan University,
Tiruchirappalli 620 024, India
Balaji Perumalsamy −National Centre for Alternatives to
Animal Experiments, Bharathidasan University,
Tiruchirappalli 620 024, India
Ramasamy Thirumurugan −National Centre for Alternatives
to Animal Experiments, Bharathidasan University,
Tiruchirappalli 620 024, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.1c06125
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
P.S.G. is grateful to UGC, New Delhi, India, for the financial
support through the Major Research Project (file no. 41-225/
2012(SR) dated 18 July 2012).
■ABBREVIATIONS
QH, 1-amino-4-hydroxy-9,10-anthraquinone; CoQ3, Co(III)
complex of 1-amino-4-hydroxy-9,10-anthraquinone; TBAB,
tetrabutyl ammonium bromide; DMSO, dimethyl sulfoxide:;
DMF, N,N-dimethyl formamide
ACS Omega http://pubs.acs.org/journal/acsodf Article
https://doi.org/10.1021/acsomega.1c06125
ACS Omega XXXX, XXX, XXX−XXX
G

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