Cytotoxic activity and apoptosis induction by a series Ag(I)-NHC complexes on human breast cancer cells and non-tumorigenic epithelial cell line

Article abstract of DOI:10.1016/j.molstruc.2020.129462

The main problems encountered in treatment with anticancer drugs, undesired side effects, and toxicity. One of the most important parameters in cell transport is the lipophilic and solubility property of the drug. Enough with the potential effects, side effects with minimal demand for new anticancer compounds, mechanisms of action of the compound can meet because of increased efforts to be clarified. In this case, scientists were encouraged to do new research. In particular, the organometallic compounds are one of the topics focused lately. Ag(I)-NHC complexes are one of the most important classes of organometallic compounds. Although the anticancer activity of Ag(I)-NHC complexes have been known recently times, the anticancer effects of 2-morpholino ethyl substituted benzimidazolium derivative, lipophilic, and solubility properties. Ag(I)-NHC complexes have not unknown yet. Therefore, we aimed to investigate of cytotoxic effect and apoptosis mechanism on breast cancer cell lines (MCF7), breast adenocarcinoma cell lines (MDA-MB-231), and non-tumorigenic epithelium cell lines (MCF 10A) of new Ag(I)-NHC complexes that derivative from morpholine-linked benzimidazole, were synthesized and antimicrobial activity was determined in our previous study. The cytotoxicity was determined by the MTS method, and the apoptosis mechanisms were determined the cell cycle, Annexin V, and caspase-3 analysis. A new benzimidazolium salt bearing morpholino ethyl substituent (2) was synthesized. This benzimidazolium salt was characterized by NMR and FT-IR spectroscopic method and elemental analysis technique. Also, the structure of the new benzimidazolium salt was confirmed by single-crystal X-ray diffraction. Ag(I)-NHC complexes inhibited the growth of MCF7 and MDA-MB-231 cells depending on the dosage and time. The complexes 3a and 3b exhibited a significant difference p < 0.05; p < 0.001; and p < 0.001 level depend on depending on the increase in concentration on cancer cells. All compound induced by apoptosis was associated with stopping the cell cycle in phase G1 and the caspase-3 activity exhibited. The complex 3c was the lowest number of caspase-activating cells (2.1%) compared with both the control and other complexes in MDA-MB-231 cells. But the complex 3a was the highest number of caspase-activating cells (% 9.6). These findings have shown that these new Ag(I)-NHC complexes can be important new anticancer agents for breast cancer treatments.

Full text of DOI:10.1016/j.molstruc.2020.129462

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Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Cytotoxic activity and apoptosis induction by a series Ag(I)-NHC
complexes on human breast cancer cells and non-tumorigenic
epithelial cell line
d
e
e
Türkan Kutlua, Is¸ ıl Yıldırımb,c,∗, Hande Karabıyık , Ayten Kılınçlı , Ibrahim Tekedereli ,
˙
Yetkin Gökf, Miris¸ Dikmeng, Aydın Aktas¸ f,h
a Department of Chemistry/Biochemistry, Faculty of Science, Inonu University, Malatya 44280, Turkey
b Science Institute, Inonu University, Malatya 44280, Turkey
c Vocational School, Pharmacy Services Program, Beykent University, Istanbul 34500, Turkey
d Department of Physics, Faculty of Science, Dokuz Eylul University, Izmir 35390, Turkey
e Department of Medical Biology and Genetics, Medical School, Inonu University, Malatya 44280, Turkey
f Department of Chemistry/Organic Chemistry, Faculty of Science, Inonu University, Malatya 44280, Turkey
g Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskisehir 26210, Turkey
h Health Services Vocational School, Inonu University, Malatya 44280, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
The main problems encountered in treatment with anticancer drugs, undesired side effects, and tox-
icity. One of the most important parameters in cell transport is the lipophilic and solubility prop-
erty of the drug. Enough with the potential effects, side effects with minimal demand for new anti-
cancer compounds, mechanisms of action of the compound can meet because of increased efforts to
be clarified. In this case, scientists were encouraged to do new research. In particular, the organometal-
lic compounds are one of the topics focused lately. Ag(I)-NHC complexes are one of the most impor-
tant classes of organometallic compounds. Although the anticancer activity of Ag(I)-NHC complexes have
been known recently times, the anticancer effects of 2-morpholino ethyl substituted benzimidazolium
derivative, lipophilic, and solubility properties. Ag(I)-NHC complexes have not unknown yet. Therefore,
we aimed to investigate of cytotoxic effect and apoptosis mechanism on breast cancer cell lines (MCF7),
breast adenocarcinoma cell lines (MDA-MB-231), and non-tumorigenic epithelium cell lines (MCF 10A)
of new Ag(I)-NHC complexes that derivative from morpholine-linked benzimidazole, were synthesized
and antimicrobial activity was determined in our previous study. The cytotoxicity was determined by the
MTS method, and the apoptosis mechanisms were determined the cell cycle, Annexin V, and caspase-
3 analysis. A new benzimidazolium salt bearing morpholino ethyl substituent (2) was synthesized. This
benzimidazolium salt was characterized by NMR and FT-IR spectroscopic method and elemental analy-
sis technique. Also, the structure of the new benzimidazolium salt was confirmed by single-crystal X-ray
diffraction. Ag(I)-NHC complexes inhibited the growth of MCF7 and MDA-MB-231 cells depending on the
dosage and time. The complexes 3a and 3b exhibited a significant difference p < 0.05; p < 0.001; and
p < 0.001 level depend on depending on the increase in concentration on cancer cells. All compound
induced by apoptosis was associated with stopping the cell cycle in phase G1 and the caspase-3 activity
exhibited. The complex 3c was the lowest number of caspase-activating cells (2.1%) compared with both
the control and other complexes in MDA-MB-231 cells. But the complex 3a was the highest number of
Received 5 September 2020
Revised 12 October 2020
Accepted 14 October 2020
Available online xxx
Keyword:
Ag(I)-NHC
Morpholine
Benzimidazolium
XRD
MCF7
MDA-MB-231
MCF10A
Apoptosis
Flow cytometry
Abbreviation: Ag(I)-NHC, Silver (I)-N-Heterocyclic Carbene; ATCC, American Type Culture Collection; DMEM/F12, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-
12; DMSO, Dimethyl sulfoxide DNA, Deoxyribonucleic acid; MCF7, Human breast cancer cell line. (ATCC code: HTB-22TM); MCF 10A, immortalized breast cell line (ATCC code:
CRL-10317TM); MEBM, Mammary Epithelial Basal Medium MDA-MB-231,Human breast adenocarcinoma cell line (ATCC code: HTB-26TM); MTS, [3-(4;5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium];FBS, Fetal Bovine Serum FDA, Food Drug Administration; PBS, Phosphate Buffered Saline; WHO, World Health
Organization; 3a, Bromo-[1-(2-morpholinoethyl)-3-propylbenzimidazol-2-ylidene]silver(I); 3b, Bromo [1-isopropyl-3-(2-morpholinoethyl)benzimidazol-2-ylidene]silver(I); 3c,
Bromo-[1-(3-hydroxypropyl)-3-(2-morpholinoethyl)benzimidazol-2-ylidene]silver(I).
∗
Corresponding author.
E-mail address: isil@beykent.edu.tr (I. Yıldırım).
https://doi.org/10.1016/j.molstruc.2020.129462
0022-2860/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: T. Kutlu, I. Yıldırım, H. Karabıyık et al., Cytotoxic activity and apoptosis induction by a series Ag(I)-NHC
complexes on human breast cancer cells and non-tumorigenic epithelial cell line, Journal of Molecular Structure, https://doi.org/10.1016/
j.molstruc.2020.129462

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caspase-activating cells (% 9.6). These findings have shown that these new Ag(I)-NHC complexes can be
important new anticancer agents for breast cancer treatments.
© 2020 Elsevier B.V. All rights reserved.
Although solubility and ionization of the metal sources are
significant parameters to deal with metals in the biological sys-
tem, lipophilicity, and low toxicity are the most important prop-
1. Introduction
Cancer is one of the most important health problems of our
time. A report released in the 2019 year by the Word Health Or-
ganization (WHO) [1] indicated that in 2018, it is estimated that
627.000 women died from breast cancer that is approximately 15
% of all cancer deaths among women. While breast cancer rates
are higher among women in more developed regions, rates are in-
creasing in nearly every region globally. U.S. Breast Cancer Statis-
tics estimated [2]. 42,170 women in the U.S. are expected to die in
2020 from breast cancer. Death rates have been steady in women
under 50 since 2007, but have continued to drop in women over
50. The overall death rate from breast cancer decreased by 1.3 %
per year from 2013 to 2017. These decreases are thought to be the
result of treatment advances and earlier detection through screen-
ing. Moreover, it was reported that breast cancer is the most com-
mon form of cancer in the United States. The National Cancer Insti-
tute (NCI) [3] estimated that the new cases in 2019 approximately
268.000 (15.2 %) women will be diagnosed with breast cancer for
this year, and 41.760 (6.9 %) women will die of the cancer disease.
The new drugs will inevitably be discovered to prevent or cure
cancer with the justification that this rate of cancer growth and
deaths will continue.
erties of cancer drugs. Functional groups including in NHC core
have been shown to greatly affect the toxicity, with lipophilic-
ity and countering playing an important role. It has been estab-
lished that lipophilic character depends on the chain length that
eases the Ag(I)-NHC complexes to penetrate the cell membranes,
where they release silver ions, which interact with cellular or-
ganelles and affect their functionalities [14,10,15]. The research on
this subject is still ongoing. Although the cytotoxic activity of dif-
ferent Ag(I)-NHC complexes have become known recently times,
the cytotoxic effects, lipophilic, and solubility properties of the
benzimidazolium-functionalized Ag(I)-NHC complexes bearing 2-
morpholynoethyl group have not examined as of yet. Therefore,
we aimed to investigate the cytotoxic effect and apoptosis mech-
anism of new Ag(I)-NHC complexes on breast cancer cell lines
(MCF7), breast adenocarcinoma cell lines (MDA-MB-231), and non-
tumorigenic epithelium cell lines (MCF 10A). The new Ag(I)-NHC
complexes were derived from morpholine-linked benzimidazole,
synthesized, and determined in antimicrobial activity in our pre-
vious study.
Cancer is a highly complex disease that associates with sev-
eral processes including sustained proliferative signaling, unlim-
ited replicative potential, and activation of invasion and metasta-
sis. So, development of drugs or agents targeting any of these pro-
cesses is important. Recently, there has been a growing demand
for metal-based compounds in the treatment of cancer. Cisplatin
and other platinum-based drugs are well-known valid anticancer
drugs. However, during chemotherapy, the presence of numerous
side effects and the onset of frequent phenomena of resistance
have pushed many research groups to devise new metal-based
compounds holding improved anticancer properties and fewer un-
desired effects. Amongst the variety of synthesized compounds,
significant antiproliferative effects have been obtained by employ-
ing organometallic compounds, particularly those based on metal
complexes. Recently, Ag(I)-NHCs have been developed the purpose
of potential chemotherapeutic drugs as well. Several possible bio-
logical targets of Ag(I)-NHC complexes have been identified. How-
ever, the mechanisms of action of there are still unclear. Therefore,
further research is required to fully elucidate the mechanism of ac-
tion of these Ag(I)-NHC complexes.
2. Material method
2.1. Material
DMEM/F12 was purchased from Thermo Fischer Scientific.
MEBM were purchased from Lonza. Apoptosis kits were purchased
from BD (Becton, Dickinson and Company, USA), PBS, FBS, antibi-
otic (Penicillin-Streptomycin), Trypsin/EDTA solution and Cisplatin
were purchased from Sigma-Aldrich (Germany). DMSO was pur-
chased from Santacruz (Germany). MTS was purchased Promega
(Germany).
2.2. Used devices
In this study, Nuve mark a laminar flow cabinet, Thermo Scien-
ꢀR
tific mark a CO2 Incubator, and an Emax Plus Microplate reader
was used.
Silver complexes are a good cytotoxic agent against many types
of cancer cells as stated in current literature, and most of them
exhibit lower toxicity than cisplatin. [4] Similarly, it identified that
the cytotoxic properties of Ag(I)-NHC complex on MCF7 and MDA-
MB-231 cell lines [5].
2.3. Experimental
The chemicals used at work including 2-morpholino ethyl
chloride, potassium hydroxide, 3-bromo-1-propanol, diethyl ether,
dimethylformamide (DMF), etc. were purchased from Sigma-
Aldrich, abcr and Merck. All other reagents were obtained com-
mercially from Aldrich and used without further purification. Melt-
ing points were identified in glass capillaries under air with an
Electrothermal-9200 melting point apparatus. On the other hand,
FT-IR spectra assay were recorded in the range 400–4000cm−1 on
Perkin Elmer Spectrum 100 FTIR spectrometer. Carbon (13C) and
Proton (1H) NMR spectra were recorded using either a Bruker 400
Merkur spectrometer operating at 100 MHz (13C), 400 MHz (1H)
in CDCl3 with tetramethylsilane as an internal reference. Elemental
analyses were performed by Inonu University Scientific and Tech-
nology Centrr (Malatya, TURKEY).
It reported that the cytotoxic effects of various Ag(I)-NHC com-
plexes were carried out using MCF7 cells in different studies
[6,7] and several possible biological targets of Ag(I)-NHC com-
plexes have also been identified. The mechanism of action of sil-
ver cations is still not clear; however, it has been thought that sil-
ver cations bind to the cell surfaces and interact with the enzymes
and proteins that are important for the cell wall synthesis [8–10].
Possibly this is the reason that the bonding of silver cations to bi-
ologically compatible ligands increase the bioavailability and ulti-
mately the activity of silver cations as reported that previous study
[11–13].
2

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2.4. Synthesis of 1-(3-hydroxypropyl)-3-(2-morpholinoethyl)
benzimidazolium chloride, 2
model was refined with ShelXL 2018/3 [20] using full matrix least
squares minimization on F2. All non-hydrogen atoms were refined
an isotropically. All H atoms except for H1 were positioned geo-
metrically and refined using a riding model, fixing the aromatic
The
reaction
between
1-(3-hidroxypropyl)benzimidazole
˚
˚
(1.05 g.,
6
mmol) and 4-(2-chloroethyl)morpholine (0.90 g,
C—H lengths at 0.93 A, methylene C—H lengths at 0.97 A. H1 atom
involved in hydrogen bond was found in the difference map and
refined freely.
6 mmol) in acetonitrile (4 mL) gave compound 1. This mixture
was stirred at 80 °C for 24 h. Then, the solvent was evaporated
under vacuum to afford the product as a white solid. This solid
was washed with diethyl ether (2 × 10 mL). The crude product
was recrystallized from ethyl alcohol / diethyl ether (1:3) at room
temperature to give white colored benzimidazolium salt [16].
Yield: 79% (1.54 g); m.p.: 211–212 °C; ν(CN): 1504 cm−1. Anal.
Calc. for C16 H24ClN3O2: C: 58.98; H: 7.42; N: 12.90. Found: C:
58.05; H: 7.34; N: 12.96. 1H NMR (400 MHz, CDCl3) in δ ppm:
2.6. Cell line
MCF7, MDA-MB-231 and MCF 10A were obtained from ATCC.
2.7. Cells culture and treatment
–
–
2.13 (s, 2H -CH2CH2CH2OH); 2.58 (s, 4H -NCH2CH2O -); 2.94
MCF7 and MDA-MB-231 cells were grown in a DMEM: F12
medium, MCF 10A cells were grown in MEBM at a temperature
of 37 °C in a humidified incubator with a 5 % CO2 atmosphere.
The test matters were dissolved in dimethyl sulfoxide (DMSO) in a
stock solution, and then it was diluted to the required concentra-
tions. A total of 70–80 % confluent cells were treated with concen-
trations from 1 μM to 100 μM in all test matters for 24 and 72 h
in the growth medium.
–
–
–
(s, 2H -NCH2CH2 -NC4H8O); 3.61 (s, 2H -NCH2CH2 NC4H8O);
–
–
3.61 (s, 2H -NCH2CH2CH2OH); 3.61 (s, 2H -NCH2CH2CH2OH);
–
–
–
4.74 (s, 4H -NCH2CH2O -); 5.13 (s, 1H -CH2CH2CH2OH); 7.22–
7.76 (m, 4H Ar-H); 10.96 (s, 1H 2-CH). 13C NMR (100 MHz,
–
–
–
CDCl3) in δ ppm: 30.6 ( -CH2CH2CH2OH); 42.7 ( NCH2CH2 -
NC4H8O); 44.8 ( NCH2CH2O ); 52.8 ( NCH2CH2 -NC4H8O); 55.2
–
–
–
–
–
–
–
(−NCH2CH2CH2OH); 57.0 ( CH2CH2CH2OH); 65.0 ( NCH2CH2O );
113.1, 113.6, 127.3, 127.5, 127.6, 131.0, 131.2 and 131.3 (Ar-C); 143.5
(2-C)
2.8. Cell proliferation assay with MTS
2.5. X-ray crystallography
The MTS assay is a colorimetric assay for assessing a cell’s
metabolic activity. The MTS tetrazolium compound is bio-reduced
by cells into a colored formazan product that is soluble in the
culture medium. This conversion is presumably accomplished by
NADPH or NADH produced by dehydrogenase enzymes in metabol-
ically active cells [21].
X-ray single-crystal diffraction data for the benzimidazolium
salt 2 was collected at room temperature on a Rigaku-Oxford
Xcalibur diffractometer with an EOS-CCD detector using graphite-
˚
monochromated MoK radiation λ =0.71073 A. Details of the data
α
collection conditions and refinement processes are summarized in
Table 1. Data reduction and analytical absorption correction was
performed using CrysAlisPro 1.171.40.67a software [17]. The struc-
ture was solved with the ShelXT [18] solution program using dual
methods and by using Olex2 [19] as the graphical interface. The
Measurement of the anti-proliferative effect using the MTS cell
proliferation kit was performed in a 96-well plate using human
breast cell lines. Cells were grown in the appropriate medium and
then seeded in 96 well plates in a 10 μL medium. The presence of
1500 cells in each well was verified and then incubated at 37 °C
in a 5% CO2 atmosphere for 24 h. At the end of the time cycle,
different concentrations from 1 to 100 μM of the test sample were
added. The cells were then incubated under conditions appropriate
for the cell lines ranging from 24 to 72 h. Concentration was ad-
justed from 1 μM to 30 μM both hours. Then, 5μL of MTS reagent
was added to each well and incubated for 1 h. At the end of the
incubation time, the optical density (OD) of the color was mea-
sured using a microplate reader. It found a primary wavelength of
490 nm. The mean absorbance values were calculated and the cell
viability percentages were documented using the Excel Office pro-
gram. Then the IC50 values were calculated. The results are shown
in Table 2. Apoptosis studies were performed according to the de-
termined IC50 values.
Table 1
Crystal data and structure refinement parameters for benzimida-
zolium salt 2.
CCDC number
2026786
C16H24N3O2Cl
325.83
Chemical formula
Formula weight (g/mol)
Temperature (K)
Crystal system
295
Orthorhombic
Pbca, 8
Space group, Z
Crystal size (mm3)
0.45 × 0.32 × 0.23
block, colourless
12.0113(13)
13.5396(10)
21.258(2)
90
Crystal shape/color
˚
a (A)
˚
b (A)
˚
c (A)
α, β, γ (°)
2.9. Apoptosis detection by staining with Annexin V-FITC and
propidium iodide (PI)
3
˚
Volume (A )
Tmin, Tmax
3457.1(6)
0.949, 0.962
1392
F(000)
Abs. Coeff. μ (mm−1
)
0.232
Annexin V-PI FITC apoptosis detection kits were used to detect
the apoptosis method [22] as described by the manufacturer. All
cells were seeded in a 6-well plate with 2 × 105 cells/mL and incu-
bated for 24 h. At the end of the incubation period, test substances
with known IC50 values were added and then allowed to incubate
for another 24 h. The samples were placed in a centrifuge and the
supernatant was discarded. The cells were washed twice with cold
PBS (2 mL), and then the cells were re-suspended in 1 mL 1X Bind-
ing Buffer solution. The solution was then transferred through the
flow tube. Then the cells were re-suspended in 100 μl 1X Bind-
ing Buffer solution. Then 5 μL of Annexin V and 5 μL of PI were
added. The cells were gently mixed and incubated for 20 min in
the dark. 400 μL of 1X Binding Buffer was added to each tube.
Calc. Density (g cm−3
)
1.252
θ range (°)
hmin,hmax
3.158–25.021
−13, 14
kmin, kmax
−16, 10
lmin, lmax
−25, 15
Measured refls
Independent refls
Observed refls
6989
3042
1537
Parameters, restraints
Rint
Goodness of Fit on F2
203, 0
0.0549
0.997
R indices [I>2σ(I)]
R1 = 0.0589, wR2 = 0.1018
0.182, −0.260
<0.0001
−3
)
˚
ρ
max, ρmin (eA
(ꢀ/σ)max
3

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Table 2
Cytotoxicity effects against MCF7, MDA-MB-231 and MCF 10A cells.
IC50 Values μM
Compound
24 h
72 h
MCF7
MDA-MB-231
8
MCF 10A
MCF7
MCF 10A
3a
18
2.12
1.41
7.63
18
14
25
28
2.82
8
6.73
2.51
2.54
14
0.0
1.41
4.87
3b
17
7.5
12
27
0.77
1.69
4.90
2.72
1.14
6.63
7.5
5
15.5
25
3c
23
Cisplatin
>50
>50
1–100 μM NI
NI: No inhibition;
have shown SD. There no found SD values for 3a complex in MDA-MB-231.
The samples were then analyzed by flow cytometry within 1 h. At
least 10,000 cells were analyzed per sample [23].
3. Results
3.1. Synthesis
2.10. Cell cycle analysis
Before this study, we synthesized the salt of "1-(3-
hydroxypropyl)-3-(2-morpholinoethyl) benzimidazolium bromide"
and their Ag(I)-NHC complexes 3a-c [16]. We could not deter-
mine any cytotoxic, antiproliferative, or cytostatic effects of the
benzimidazolium salt we synthesized on breast cancer cells. So,
we worked again to determine the effects of salt on cancer cells
by replacing the halogen in the salt (using chlorine instead of
bromine atoms), and synthesized the 1-(3-hydroxypropyl)-3-(2-
morpholinoethyl) benzimidazolium chloride salt 2 (Scheme 1). But
we could not determine any cytotoxic, antiproliferative or cyto-
static effects of the benzimidazolium salt we synthesized on breast
cancer cells and we took into consideration the silver compounds
associated with the ligands we synthesized earlier. Because a very
low cytostatic activity could be seen in the previous ligands at a
concentration p > 500 μM.
Measuring the DNA content of cells was performed on
a
BD flow cytometer and using the BD Cycle testTM Plus DNA
reagent [24]. Breast cells were seeded on the 6-well plate with
2 × 105 cells/mL and incubated for 24 h. At the end of the incu-
bation period, test substances with known IC50 values were added
and allowed to incubate for 24 h. Cells were harvested from the
plate into 50 mL conical tubes and centrifuged 5 min. The super-
natant was removed. 50 μL of PBS was added onto the pellet and
suspended. The pellet was transferred to the flow tube. The cell
pellets were washed twice with 1 mL of the buffer solution pro-
vided by the manufacture. The pellet was then centrifuged at a
low rpm for 5 min. After removal of the supernatant, the remain-
ing pellet was resuspended again with 1 mL of BD buffer solution.
The cell suspension was incubated in the dark at room tempera-
ture and was analyzed by the BD flow cytometry.
For the synthesis of the new morpholinoethyl substituted
benzimidazolium salt
2 in this study; firstly, we obtained 1-
(3-hydroxypropyl)benzimidazole from the reaction of benzimi-
dazole and 3-bromopropanol. Then, from the reaction of 1-(3-
hydroxypropyl)benzimidazole and 4-(2-chloroethyl)morpholine
were obtained the 1-(3-hydroxypropyl)-3-(2-morpholinoethyl)
2.11. Apoptosis detection by caspase-3 activation
Measurement of the caspase-3 active-cells was performed on
a BD flow cytometer and using a BD FITC Active Caspaz-3 Apop-
tosis kit [25]. Breast cells were seeded into a 6-well plate with
2 × 105 cells/mL and incubated for 24 h. At the end of the incu-
bation period, test substances with known IC50 values were added
and allowed to incubate for 24 h. Cells were harvested from the
plate into 50 mL conical tubes and centrifuged for 5 min. The su-
pernatant was removed. Then the pellet cells were washed twice
with cold 1X PBS solution. Then the pellet cells were re-suspended
in 500 μL of BD Cytofix/CytopermTM solution for permeabilized. In-
cubated cells for 20 min on ice and then centrifuged for 5 min
discarding the BD Cytofix/CytopermTM solution. The samples were
then washed twice with a 500 μL BD Perm/WashTM buffer (1X).
Cells were then incubated with 20 μL of cleaved caspase-3 (Asp
175) BD rabbit polyclonal antibody and incubated for 30 min at
room temperature. Then the pellet was re-suspended again in
1 mL of BD buffer solution and then re-suspended in 0.5 mL BD
Perm/WashTM buffer (1X) and then transferred to the flow tube
and analyzed by flow cytometry. At least 10,000 cells were ana-
lyzed per sample [23].
benzimidazolium chloride
2 (Scheme 2). The new benzimida-
zolium salt 2 was obtained as a white solid in 79 % (1.54 g)
yield. The 1H NMR of benzimidazolium salt in CDCl3 displayed
the -OH proton at 5.13 ppm and the characteristic 2-CH proton at
10.96 ppm. In the 13C NMR, the characteristic 2-C signal appeared
at 143.5 ppm for benzimidazolium salt 2. Other 1H NMR and
13C NMR peaks (Fig. SI1) are compatible with similar bromine
salt [16]. FT-IR spectrum of the benzimidazolium salt 2 showed
a band at 1504 cm−1 corresponding to the vibrations of C N of
=
the benzimidazolium salt [16]. The result of the elemental analysis
demonstrates that the calculated value for benzimidazolium salt
was very close to the found value (Scheme 2).
3.2. Cytotoxicity effect results
Cytotoxicity assays for 3a, 3b, 3c and cisplatin were carried out
in all cell types, IC50 values were calculated for only the silver
complexes 3a-c and apoptotic mechanism studies continued with
IC50 of these complexes.
IC50 values of 3a-c complexes and cisplatin are given in Table 2.
Considering in the Table 2, 3a complex with the 3-propyl sub-
stituent was more cytotoxic in MCF7 cells than compared with the
non-tumorigenic epithelial cell line, (MCF 10A) after 72 h of incu-
bation. The 3a complex was less cytotoxic in MCF7 cells compared
with the MDA-MB-231 adenocarcinoma cells after 24-hours of in-
cubation.
2.12. Statistical analysis
Statistical analysis for cytotoxicity was performed by using the
Excel software program and a comparison between mean values
for cytotoxicity was calculated by Student’s t-tests. Analysis of
variance and difference was considered significant at p < 0.05,
p < 0.001, and p < 0.001.
The 3b complex with the 3-isopropyl substituent was more cy-
totoxic in MDA-MB-231 cells compared with both MCF7 and MCF
10A cells after 24 h of incubation. The 3b complex was more cyto-
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Scheme 1. Synthesis of 2-morpholinoethyl-substituted benzimidazolium Salt 2.
Scheme 2. Structure of 2-morpholinoethyl substituted Ag(I)-NHC complexes 3a-c [16].
toxic effect than cisplatin, complex 3a, and complex 3c in MDA-
MB-231 cells compared with both MCF7and MCF 10A cells af-
ter 24-hours of incubation. In regards to the 72-hour results, in
the MCF7 cells, complex 3b was more cytotoxic compared with
the 3a complex, while was less cytotoxic effect than complex
3c. Compared with MCF 10A cells, complex 3b complex showed
a stronger cytotoxic effect than the MCF 10A cells after 72 h
incubation.
When MCF 10A cells were exposed to the 3a complex at 24 h
then compared with the control group at a concentration of 20 μM
and 30 μM, it showed a significant difference p < 0.05; p < 0.001
respectively. 3a complex for 72 h compared with the control group,
a significant difference was found 15; 20, and 30 M concentrations
(p < 0.05).
When MCF7 cells incubated with 3b complex for 24 h no signif-
icant difference in was observed concentrations of 7.5 and 10 μM
compared with the control group, but 12 μM (p <0.01), and con-
centration of 20 and 30 μM caused a significant difference as a
p < 0.001. A 72-hour incubation with complex 3b was compared
with the control group at a concentration of 7.5 μM (p < 0.01),
while concentrations of 10; 12; 16; 20; and 30 μM showed a sig-
nificant difference at p < 0.001 level.
After 24 h’ incubation, the 3-hydroxypropyl-substituted com-
pound, complex 3c had relatively weak cytotoxic effects compared
with other compounds including cisplatin, with IC50 values of 23,
12, and 25 μM in MCF7, MDA-MB-231, and MCF 10A cells, respec-
tively.
Complex 3c was more cytotoxic than cisplatin, complex 3a, and
complex 3b in MCF7 cells compared with the MCF 10A cells after
72 h of incubation. Complex 3c was more cytotoxic in MCF7 cells
than in MCF 10A cells after both 24 and 72 h of incubation.
Fig. 1 shows that the compounds decreased cell proliferation in
a dose-dependent manner in MCF7, and MCF 10A cells after 24 and
72 h of incubation period and in MDA-MB-231 cells after a 24 of
hour incubation period.
When MDA-MB-231 cells incubation with 3b complex for 24 h,
showed a significant difference p< 0.05 levels at a concentration of
10 μM and 30 μM, but in concentration of 16 and 20 μM p < 0.01
compared with the control groups.
When MCF 10A cells incubated with complex 3b for 24 h, a
significant difference was observed at a concentration of 16 and
20 μM p < 0.01 and at 30 μM concentrations p <0.05 level com-
pared with the control group. When complex 3b complex incu-
bated for 72 h in the concentration of 16 μM showed significant
difference p < 0.01 and in 20 and 30 μM concentrations p < 0.001
level compared to the control group.
When MCF7 cells were incubated with complex 3a for 24 h,
there was no significant difference in cell viability of 2.5, 5, and
10 μM concentrations, but the 15; 20; and 30 μM concentra-
tions were found significantly different in cell viability p < 0.05;
p < 0.01; and p < 0.001 levels, respectively. The complex 3a ex-
hibited a significant difference p < 0.05 level at a concentration of
5 μM. Moreover, in 10; 15; 20, and 30 μM concentrations were sig-
nificant difference at the p <0.001 level when compared with the
control group after 72 hours’ incubation.
When the MCF7 cells incubated with 3c complex for 24 h, a
significant difference was observed at concentration of at 14; 20;
30 μM when compared with the control group (p < 0.05, p < 0.01
p < 0.001 levels, respectively). Incubation of MCF7 cells with com-
plex 3c for 72 h at a concentration of 4 μM, it exhibited a signif-
icant difference p < 0.05 level, while the concentrations of 10 and
14 μM p < 0.01 level; 6; 20; and 30 μM concentrations had signif-
icant difference at p < 0.001 level exhibited.
When MDA-MB-231 cells incubated with complex 3a complex
for 24 h, it a significant difference p < 0.05 at a concentration
of 10 μM at a level of, whereas it showed significant differences
p < 0.01 at 5; 15; 20; and 30 μM concentrations compared with
the control group.
When MDA-MB-231 cells were exposed to complex 3c for 24 h
at a concentration of 14 μM, a significant difference was observed
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Fig. 1. Cytotoxic activity of 3a, 3b, 3c and cisplatin on MCF7, MCF 10A cell after 24, and 72 h of the incubation period. But cytotoxic activity of 3a, 3b, 3c and cisplatin on
MDA-MB-231 was 24 h. Values are represented as mean
SD n = 4).
when compared with control (p < 0.05). A significant difference
was also observed at concentrations of 20 μM (p < 0.01) and
30 μM (p < 0.001).
when compared to the with the control group. (p < 0.01). A sig-
nificant difference was also observed at a concentration of 20 μM
(p < 0.05). A 72 -h incubation with 30 μM of complex 3c showed
a significant difference compared with control (p < 0.001).
When MCF 10A cells were incubated with complex showed 3c
for 24 h at concentrations of 14 and 30 μM, a significant difference
at the level of p < 0.001 at 30 μM concentration was observed
MCF7 cells incubated with cisplatin for 24 h showed a signifi-
cant difference (p < 0.01) at a concentration of 10 μM compared
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Table 3
with the control group. Cisplatin showed significant differences in
MDA-MB-231 cells at concentrations of 5 and 30 μM (p< < 0.01
levels) and at 20 and 25 concentrations of 20 and 25 μM (p < 0.05)
compared to with control group. Cisplatin for a 24 -h incubation
with cisplatin had no significant effect at 1 and 5 μM concentra-
tion, but a significant difference was observed at a concentration of
20 μM, (p < 0.05;) and at concentrations of 25; 30; 35 and 40, and
100 μM concentrations (p < 0.01), and 50 and 100 μM (p < 0.001)
in MCF 10A cells.
˚
Selected bond lengths A, bond and torsion angles (°) and hydrogen bond geom-
etry.
Bond lengths
C1—N1
N1—C2
C2—C7
N2—C8
1.327(4)
1.388(4)
1.379(4)
1.467(4)
C1—N2
N2—C7
N1—C14
O1—C16
1.327(4)
1.390(4)
1.469(4)
1.382(4)
Bond and torsion angles
N1—C1—N2
C1—N2—C7
110.3(3)
C1—N1—C2
108.3(3)
124.7(3)
78.2(4)
3.3. Apoptosis mechanisms
108.0(3)
124.5(3)
−60.1(4)
C1—N1—C14
C1—N2—C8
C1—N2—C8—C9
C1—N1—C14—C15
3.3.1. Determination of apoptotic activity by Annexin V-PI
Annexin A5 (or annexin V) is a cellular protein in the Annexin
group. In flow cytometry, Annexin V is commonly used to de-
tect apoptotic cells by its ability to bind to phosphatidylserine, a
marker of apoptosis when it is on the outer leaflet of the plasma
membrane. Apoptotic cell death was determined by using the An-
nexin V method.
N2—C8—C9—N3
−104.0(4)
O1—H1ꢀꢀꢀCl1 hydrogen bond geometry
O1—H1
H1ꢀꢀꢀCl1
O1ꢀꢀꢀCl1
˚
3.081(3) A
O1—H1ꢀꢀꢀCl1
178(4)°
˚
˚
0.86(4) A
2.22(4) A
Compared with both control and other complexes, complex 3a
achieved the earliest apoptotic cell death when compared with
other cancer cell lines and the non-tumorigenic epithelial cell line
(MCF 10A). showed that complex 3a was the highest early apop-
totic cell with a value of 8.1 % in MCF 10A cells. When the to-
tal number of apoptotic cells (early [Q4] + late [Q2]) was consid-
ered, complex 3a caused the highest degree of apoptotic cell death
deaths of all the cells.
27 % compared with control. The population of MCF 10A cells in
the presence of complex 3c was reduced in all cell-cycle phases
(Figs. 2.1, 3.1, 3.2 and 4.1).
Considering all the results, show that the effects of the tested
compounds in cancer cells are greater than effective than all con-
trol points in the non-tumorigenic epithelial cell line (MCF 10A).
The fact that population of cells shows that does is less damaging
to the non-tumorigenic epithelial cell line (MCF10A).
Complex 3b caused total apoptotic cell death in both MCF 10A
and MDA-MB-231 cells. It showed an early apoptotic cell death of
1.9 % in MCF7 cells. When the total number of apoptotic cells (early
[Q4] + late [Q2]) was considered, complex 3b caused the higher
apoptotic cell death in MDA-MB-231 cells than MCF7 cells.
Complex 3c complex caused early apoptotic cell death by the
second cycle with a value of 7.1 % in MDA-MB-231 cells. In addi-
tion, complex 3c caused early apoptotic cell death by the second
cycle with a value of 4.9 % in MCF 10A cells.
3.3.3. Determination of caspase-3 activity
Compounds 3a-c had caspase-3 activating effects in both MCF
10A cells and MDA-MB-231 cells. The findings show that these
compounds stimulate apoptosis by increasing caspase-3 activity to
a certain degree at the determined IC50 values.
According to Fig. 4, complex 3a had the same effect as a control
in MCF 10A cell. It was the highest number of caspase-activating
cells (9.6 %) compared with both control and other complexes in
MDA-MB-231 cells. According to the results given in Fig. 4, the
3b complex showed a similar effect in both cancer cells and non-
tumorigenic epithelial cell line (MCF 10A).
3.3.2. Cell cycle analysis results
The induction of cell death along with cell-cycle arrest is one
of the foremost mechanisms regulating cell growth. In the human
breast carcinoma cell lines, we investigated these agents for the
relative contribution of these mechanisms to growth inhibition in
culture. The MCF7 cell population in G1 and S phase presence of
the complex 3a increased by 3.2 % and 3.4 %, respectively, when
compared with control. An increased cell population in phase S in-
dicates that cells-maintained DNA replication.
3.3.3.1. Crystal and molecular structure of benzimidazolium salt 2.
X-ray single-crystal diffraction analysis reveals that the molecu-
lar structure of the benzimidazolium salt 2 has a cation molecule
consisting of hydroxypropyl and morpholinoethyl attached N-
benzimidazole ring and a chloride anion linked to the cation
through a hydrogen bond (Fig. 5). Selected bond lengths, bond,
and torsion angles including geometrical details of H-bond are
given in Table 3. The N-benzimidazole ring is planar with a max-
According to Fig. 3, the population of MCF 10A cells in the G1
phase in presence of complex 3a increased to 5.4 % compared with
control group, while the population of cells in the G2 and S phases
decreased to 0.4 % and 0.5 %, respectively. This indicates that com-
plex 3a damages DNA in MCF 10A cells and that these cells are in
the G1 / G0 phase.
˚
imum deviation of 0.024(3) A for C7 atom. C1—N bond lengths of
The cell population for 3a complex decreased by 11.8 % when
compared with the control at the G1 phase in the MDA-MB-231
cancer cells. Although the cell population of MCF 10A cells in the
G1 phase, the population of cells decreased in MDA-MB-231 cells.
The tested have inhibitory effects on cancer cell DNA.
˚
1.327(4) A, and the other C—N bond lengths in benzimidazole ring
˚
agree well with those of a similar structure in literature {1.325(4) A
˚
˚
˚
and 1.328(4) A for C1—N, 1.386(3) A and 1.386(4) A for the other
C—N lengths [26]}.
Although the population increased slightly in the presence of
3b complex compared with the control point during the G1 and
S phases, the population of cells at the G2 / M control point was
reduced by 15.2 % compared to the control group of MCF7 cells.
The population of MDA-MB-231 cells for complex 3b decreased by
11.8 % compared to control at the G1 phase.
The morpholine ring in the benzimidazolium salt 2 adopts
a
chair conformation, with
˚
a total puckering amplitude of
QT = 0.901(1) A. The plane through the C atoms of the morpholine
ring makes a dihedral angle of 67.63(10) ° with the mean plane
of benzimidazole ring. In the absence of intermolecular hydrogen
bonds, the crystal structure of the benzimidazolium salt 2 is stabi-
lized by weak intermolecular interactions. Crystal packing diagram
of the benzimidazolium salt 2 can be found in supplementary in-
formation (Fig. SI2).
The population of MCF7 cells in the G1 phase increased by
6.2 % in the presence of complex 3c compared with control. The
MDA-MB-231 cell count in the G1 phase was further reduced to
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Fig. 2.1. Apoptotic effect of 3a, 3b, 3c complexes on the MCF7, MDA-MB-231 and MCF 10A cells after 24 h of incubation period for IC50 values. NT: Non treatment(or Control
groups) Q1: necrotic cell, Q2: late apoptosis; Q3: live cell; Q4: early apoptotic cells.
Fig. 2.2. 3a, 3b, 3c Annexin V-PI analysis result on the MCF7, MDA-MB-231 and MCF 10A cells at 24 h for IC50 values. NT: Non treatment(or Control groups) Q2: late
apoptosis; Q4: early apoptotic cells.
Fig. 3.1. Cell Cycle analysis histogram data NT: Non-treatment groups (or Control groups); 3a, 3b, 3c complexes on the MCF7, MDA-MB-231 and MCF 10A cells after 24 h of
incubation period for IC50 values.
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Fig. 3.2. 3a, 3b, 3c Cycle analysis result on the MCF7, MDA-MB-231 and MCF 10A cells at 24 h for IC50 values.
Fig. 4.1. 3a, 3b, 3c Caspase analysis result on the MCF7, MDA-MB-231 and MCF 10A cells at 24 h for IC50 values.
ability of the anticancer activity of NHC complexes highly depends
on ring size and substituents. In this study, Ag(I)-NHC complexes
3a-c of the n-propyl, isopropyl and 3-hydroxypropyl substituent
depend on benzimidazole core were used since to be thought to
have low toxicity. It is also important to note that the n-propyl
substituent at position-1, and isopropyl group at position-1 are im-
portant for anticancer activity compared to 3-hydroxypropyl. Be-
cause these groups are lipophilic, whereas the 3-hydroxypropyl
group is high water solubility and hydrophilic properties. The pres-
ence of CH2, and electrostatic interactions, facilitate the activity of
–
complexes, but O -H, which responsible for the H bond, increases
polarity. This situation has seen in the X-ray crystal structure.
In this study, MCF7 and MDA-MB-231 breast carcinoma cells
were preferred selective targets and non-tumorigenic epithelial cell
line, (MCF 10A) was used to determine nontoxic effects. The IC50
values showed that cisplatin reduces cell growth at very high
concentrations in cancer cells. Intake care of Table 2 results, 3a,
3b, 3c complexes with 20 μM concentration, closest to the IC50
value of all compounds were found significantly different p < 0.05;
p < 0.001; and p < 0.01 in MCF7 cells. However, 3a, 3b, 3c com-
plexes with 10 μM concentration, closest to the IC50 value of all
compounds were found significantly different p < 0.05; p < 0.01;
and p < 0.001 in MDA-MB-231 cells. Compared with the cyto-
toxic activity Ag(I)-NHC complexes, although the benzimidazole
core was identical, the cytotoxic effects varied due to the differ-
ence substituents on the benzimidazole core.
Fig. 5. The asymmetric unit of the benzimidazolium salt
2 showing the atom-
labeling scheme. Anisotropic displacement ellipsoids are drawn at the 50% prob-
ability level.
4. Discussion
When Ag(I)-NHC complexes 3a-c were compared with cisplatin,
they were three-fold more active in MCF7 cells after 24 and 72 h
of incubations. Silver complexes 3a-c were more cytotoxic than cis-
platin in MCF7, MDA-MB-23, MCF 10A cells. This cytotoxic effect is
likely due to the lipophilic nature of silver complexes, which aids
To synthesize NHCs, the more common practice is substitutions
of alkyl/aryl groups at the nitrogen of azoles (imidazole, benzim-
idazole, triazole, etc.) to synthesize azolium. There are numerous
ways to generate free NHCs and their metal complexes. The vari-
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the transport of silver cations into the cell and subsequently into
organelles where silver may contribute to toxicity by inhibiting cel-
lular respiration and biomolecule metabolism.
2.1% in MCF 10A cells. This effect is thought to be effective in
other cellular apoptotic mechanisms, such as mitochondria disrup-
tion, cytochrome c release in cancer-cell cytoplasm, and the intra-
cellular production of reactive oxygen species, as well as caspase-
3 activation. Also, it may be thought of targeting Bcl-2proteins by
small molecules or activating alternative pathways to bypass Bcl-
2mediated protection to promote apoptosis. Previously a study, it
was reported that cisplatin treatment overcomes Bcl-2-mediated
protection in MCF7 cells (wt. p53), but not in MDA-MB-231 cells
(mt p53), whereas a Taxane (paclitaxel) or a small-molecule Bcl-2
inhibitor (HA14-1) failed to act similarly. They also find that p53-
dependent Noxa upregulation and lipid peroxidation associate with
this process [33]. The incubation of cells with Ag(I)-NHC complexes
in the presence of inhibitor may help to determine the mecha-
nisms by which apoptotic cell death. In another study reported
that the pro-apoptotic mechanism of action of unsymmetrically
substituted benzimidazolium-functionalized Ag(I)-NHC compounds
using Rhodamine 123 test and found that the tested compound
induces apoptosis via the extrinsic mitochondrial pathway [34].
The literature and our study demonstrate Ag(I)-NHC complexes in-
duce apoptosis by both intrinsic and extrinsic pathways. Support-
ing studies will further validate the importance of the findings of
this study, and It is thought that it will allow the Ag compounds
to be used as anticancer drugs.
Liu and co-workers [27] performed the results of a cytotoxicity
study on a series of silver carbene complexes in MCF7 cells after
72 h of incubation. The IC50 values were range from 3.5 to 9.2 μM,
while IC50 of cisplatin was 1.6 μM after 72 h of incubation. The
values in our study (Table 2) are similar to the study of Liu and
co-workers. Also, Silver compounds 3a-c was more cytotoxic effect
in MCF7 compared with the MCF 10A after 72 hours’ incubation.
A study published in the 2016 year, showed that the IC50 value
of benzimidazole-derived Ag(I)-NHC complex after 24 hours’ incu-
bation on MDA-MB-231 cells was 11.98 μM after 24 hours’ incu-
bation. In this study, complex 3c was less cytotoxic activity, while
complexes 3a, 3b more cytotoxic activity, which is the contrast
to the results of a previous study [28]. The present also investi-
gated the anti-proliferative activity of a series of Ag (I)-NHC com-
plexes and Tamoxifen in MCF7 cells after 24 h of incubation [29].
In this study, complex 3c was lesser cytotoxic activity than pre-
vious studies, but complexes 3a, 3b was parallel to the previous
study (Table 1).
Another study, it was determined that the cytotoxic effect of
bis-NHC coordinated silver(I), and it was found that Ag(I)-NHC
complexes were exhibited a cytotoxic effect 1–5 μM concentra-
tion in the both MCF7 and MDA-MB-231 cells at 24, 48, and 72 h.
Moreover, these complexes were exhibited a high cytotoxic effect
compared to L929 normal cells. Considering these results more ef-
fective than our results. This statue may be associate with the alkyl
chain length [30].
5. Conclusion
Consequently, we have reported the synthesis of a new benzim-
idazolium salt bearing morpholino ethyl group. The new benzimi-
dazolium salt has been characterized by using 1H NMR, 13C NMR,
FTIR spectroscopy, and elemental analysis technique. The molecu-
lar structure of this compound was confirmed by the X-ray diffrac-
tion method. The Ag(I)-NHC complexes 3a-c have been identified
as exhibiting anti-proliferative activity on the MCF7, MDA-MB-231
breast cancer cells. Considering all the time found that these silver
compounds were found to be more active compared with cisplatin
for 24- and 72 -h’ incubation. These compounds may be suggesting
an alternative agent to the cisplatin. All Ag(I)-NHC complexes 3a-c
were the most effective compounds. Biological activity depends on
the nature of the group bound to benzimidazole and cancer cell
line.
The complex 3b exhibits the most cytotoxic activity compared
to other complexes and cisplatin after incubation of 24 h. When
the complexes are compared with each other, the 3a and 3b
complex showed parallel activity, while the 3b complex showed
1.5 times better cytotoxic activity than 3c. Moreover, MDA-MB-
231cells were examined after a 72 -h incubation period and, while
strong inhibition was only observed with concentrations of 20–
30 μM. But the cytotoxic effect was not observed at other con-
centrations.
A cell cycle is a series of events that take place in a cell as it
grows and divides. A cell spends most of its time in what is called
interphase, and during this time it grows, replicates its chromo-
somes, and prepares for cell division. The cell then leaves inter-
phase, undergoes mitosis, and completes its division. In the hu-
man breast carcinoma cell lines, we investigated these agents for
the relative contribution of these mechanisms to growth inhibi-
tion in culture. We determined that Ag(I)-NHC complexes induced
by apoptosis was associated with stopping the cell cycle in phase
G1. Previously study it was found that especially with cis-platin-
treated MCF7 cells, was the formation of numerous micronuclei (in
up to 30 % of the cells) and an increase in the number of binucle-
ate cells (up to 20%) [31].
Apoptosis assays were carried out with all these compounds
and had various degrees of stimulations on the apoptosis in each
cell lines. Although our study is limited, mitochondrial membrane
potential analysis, analysis of genes (Bax, Bcl-2 and caspase-3 and
others such as the activity of caspase-8, caspase-9) that may be
related to PCR, and protein expression levels by the western blot-
ting needs to be done. A more detailed investigation of the study
of the molecular mechanisms of these complexes will allow the
identification of new targets for breast cancer treatment. So, we
may be the future planned objectives of the study. We can pre-
dict what the next period will bring with new studies that will
directly affect us and offer opportunities. One of these mecha-
nisms may be the thioredoxin (Trx) system which is central in
upholding the thiol redox homeostasis within the cell. Also, the
thioredoxin system is involved in a wide range of biological func-
tions within the cell, including ROS scavenging, DNA synthesis,
cell proliferation, apoptosis, and cell signaling. Moreover, high ex-
pression rates of Trx and TrxR in cancer cells may be directly
related to difficult-to-treat carcinomas of the breast. Recognizing
the biological processes underlying in the disease, and the asso-
ciated with molecular mechanisms in the various treatments that
all play an important role in the development of anti-cancer drugs
which can be used to prevent or inhibit the growth of cancerous
cells.
In this study, apoptosis only MDA-MB-231 cells were analyzed
due to caspase-3 deletion in MCF7 cells [32]. Fig. 1 shows that for
the complexes; decreased cell proliferation in a dose-dependent
manner in MDA-MB-231 cells after 24h incubation. According to
the results given Fig. 4, the 3c complex showed lesser effective
caspase activation than the control group and other complexes
3a, 3b with a value of 2.1 % in MCF 10A cells. According to
the results given in Fig. 4, the 3c complex showed mo. In addi-
tion, it had the second-highest number of caspase-activating cells
with 9.6 % compared to both control and other complexes in the
MDA-MB-231 cell. As a result, the apoptotic effect of Ag(I)-NHC
complexes in MDA-MB-231 cells has been determined, and treat-
ment with all compounds caused cancer-cell death via the intrin-
sic apoptotic pathway. Tre lesser effective caspase activation than
the control group and other complexes 3a, 3b with a value of
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Declaration of Competing Interest
[13] M.A. Iqbal, R.A. Haque, S. Budagumpi, M.B. Khadeer Ahamed, A.M.S. Abdul Ma-
jid, Short metal–metal separations and in vitro anticancer studies of a new
dinuclear silver(I)-N-heterocyclic carbene complex of para-xylyl-linked bis-
benzimidazolium salt, Inorg. Chem. Commun. 28 (2013) 64–69, doi:10.1016/
j.inoche.2012.11.013.
[14] M.A. Iqbal, R.A. Haque, M.B.K. Ahamed, A. Majid, S.S. Al-Rawi, Synthesis and
anticancer activity of para-xylyl linked bis-benzimidazolium salts and respec-
tive Ag(I)- Nheterocyclic carbene complexes, Chem. Res. 22 (2013) 2455–2466,
doi:10.1007/s00044-012-0240-6.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgement
[15] E.C. Friedberg, G.C. Walker, W. Siede, R.D. Wood, DNA Repair and Mutagenesis,
American Society for Microbiology Press, Washington, USA, 2005.
[16] I. Yıldırım, A. Aktas, D. Barut-Celepci, S. Kırbag˘, T. Kutlu, Y. Gök, M. Aygün,
Synthesis, characterization, crystal structure, and antimicrobial studies of
2-morpholinoethyl-substituted benzimidazolium salts and their silver(I)-N-
heterocyclic carbene complexes, Res. Chem. Intermed. 43 (11) (2017) 6379–
6393, doi:10.1007/s11164- 017-2995-3.
I would like to thank all authors for their contributions. Stud-
ies were carried out in the Inonu University Molecular Biology and
Genetic Cell Culture Laboratory. This study was supported by Inonu
University Research Project Unit (Project No: I.U.BAP 2016/154).
This work is part of Is¸ ıl Yıldırım’s doctoral dissertation. The authors
acknowledge Dokuz Eylul University for the use of the Rigaku Ox-
ford Xcalibur Eos Diffractometer (purchased under University Re-
search Grant No. 2010.KB•FEN.13).
[17] CrysAlisPro Software System, Rigaku Oxford Diffraction, 2019.
[18] G.M. Sheldrick, SHELXT
- integrated space-group and crystal-structure
determination, Acta Crystallogr. Sect.
S2053273314026370.
A
71 (2015) 3–8, doi:10.1107/
[19] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H. Puschmann, OLEX2:
a complete structure solution, refinement and analysis program, J. Appl. Cryst.
42 (2009) 339–341, doi:10.1107/S0021889808042726.
Supplementary materials
[20] G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Crystallogr.
Sect. C 71 (2015) 3–8, doi:10.1107/S2053229614024218.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2020.129462.
[21] M.V. Berridge, A.S. Tan, Characterization of the cellular reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellu-
lar localization, substrate dependence, and involvement of mitochondrial elec-
tron transport in MTT reduction, Arch. Biochem. Biophys. 303 (1993) 474–482.
[22] (2008). http://www.bdbiosciences.com/ds/pm/tds/556547.pdf.
[23] M. Dikmen, Z. Cantürk, Y. Öztürk, Y.I. Tunali, Investigation of the apoptotic ef-
fect of curcumin in human leukemia HL-60 cells by using flow cytometry, Can-
cer Biother. Radiopharm. 25 (6) (2011) 749–755 10.1089/cbr.2010.0822.
[24] (2015). http://www.bdbiosciences.com/ds/is/tds/23-2923.pdf.
References
[1] (2019). http://www.WHO.com.
[2] (2019).
https://www.breastcancer.org/symptoms/understand_bc/statistics,
https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm567309.
htm.
[25] (2015). http://www.bdbiosciences.com/ds/pm/tds/550480.pdf.
[26] A. Aktas¸ , D. Barut Celepci, Y. Gök, Synthesis, crystal structures, spectral FT-IR,
NMR and UV–vis investigations and Hirshfeld surface analysis of two new 2-
hydroxyethyl-substituted N-heterocyclic carbene precursors, J. Chin. Chem. Soc.
66 (10) (2019) 1389–1396, doi:10.1002/jccs.201900020.
[3] (2020). https://seer.cancer.gov/statfacts/html/breast.html.
[4] U. Kalinowska-Lis, A. Felczak, L. Checinska, I. Szabłowska-Gadomska, E. Patyna,
M. Małecki, K. Lisowska, J. Ochocki, Antibacterial activity and cytotoxicity of
silver(I) complexes of pyridine and (Benz)imidazole derivatives. X-ray crystal
structure of [Ag(2,6-di(CH2OH)py)2]NO3, Molecules 21 (2) (2016) 87 10.3390/
molecules21020087.
[5] C.H. Wang, W.C. Shih, C.H. Chang, Y.Y. Kuo, W.C. Hung, T.W. Ong, W.S. Li,
Preparation and characterization of amino-linked heterocyclic carbene pal-
ladium, gold, and silver complexes and their use as anticancer agents that
act by triggering apoptotic cell death, J. Med. Chem. 54 (2011) 5245–5249,
doi:10.1021/jm101096x.
[27] W. Liu, K. Bensdorf, A. Hagenbach, U. Abram, B.A. Niu, A. Maripapan, R. Gust,
Synthesis and biological studies of silver N-heterocyclic carbene complexes de-
rived from 4,5-diarylimidazole, Eur. J. Med. Chem. 46 (12) (2011) 5927–5934,
doi:10.1016/j.ejmech.2011.10.002.
˙
˙
[28] S. Akkoç, I.Ö. Ilhan, Y. Gök, P.J. Upadhyay, V. Kayser, In vitro cytotoxic activities
of new silver and PEPPSI palladium N-heterocyclic carbene complexes derived
from benzimidazolium salts, Inorg. Chim. Acta 449 (2016) 75–81, doi:10.1016/
j.ica.2016.05.001.
[6] R.A. Haque, S.F. Nasri, M.A. Iqbal, S. Al-Rawi, S.F. Jafari, M.B.K. Ahamed, A. Ma-
jid, Synthesis, characterization, and crystal structures of bis-imidazolium salts
and respective dinuclear Ag (I) N-heterocyclic carbene complexes: in vitro an-
ticancer studies against ‘‘human colon cancer’’ and ‘‘breast cancer’’, Hindawi
Publ. Corp. J. Chem. 2013 (2013) 1–11, doi:10.1155/2013/804683.
[7] W. Streciwilk, J. Cassid, F. Hackenberg, H. Müller Bunz, F. Paradisi, M. Tacke,
Synthesis, cytotoxic and antibacterial studies of p-benzyl-substituted NHC-
silver(I) acetate compounds derived from 4,5-di-pdiisopropylphenyl-or 4,5-di-
p-chlorophenyl-1H-imidazole, J. Organomet. Chem. 749 (2014) 88–99, doi:10.
1016/j.jorganchem.2013.09.033.
[29] S.Y. Hussain, R.A. Haque, P.O. Asekunowo, A.M.S. Abdul Majid, M.T. Agha,
M.R. Razalli, Synthesis, characterization and anti-proliferative activity of propy-
lene linked bis-benzimidazolium salts and their respective dinuclear silver(I)-
N-heterocyclic carbene complexes, J. Organomet. Chem. 840 (2017) 56–62,
doi:10.1016/j.jorganchem.2017.04.011.
[30] E. Çevik-Yıldız, N. S¸ ahin, S. S¸ ahin-Bölükbas¸ ı, Synthesis, characterization and
investigation of antiproliferative activity of novel Ag(I)-heterocyclic carbane
(NHC) complexes, J. Mol. Struct. 1199 (2020) 126987, doi:10.1016/j.molstruc.
2019.126987.
[8] M.A. Iqbal, R.A. Haque, M.B.K. Ahamed, M.A. Abdul, S. Al-Rawi, Synthesis and
anticancer activity of para-xylyl linked bis-benzimidazolium salts and respec-
tive Ag(I)-Nheterocyclic carbene complexes, Med. Chem. Res. 22 (5) (2013)
2455–2466, doi:10.1007/s00044-012-0240-6.
[31] A.M. Otto, R. Paddenberg, S. Schubert, H.S. Mannherz, Cell-cycle arrest, mi-
cronucleus formation, and cell death in growth inhibition of MCF-7 breast can-
cer cells by tamoxifen and cisplatin, J. Cancer Res. Clin. Oncol. 122 (1996) 603–
612, doi:10.1007/BF01221192.
[9] C. Graham, The role of silver in wound healing, J. Nurs. 14 (19) (2005) 22–24,
doi:10.12968/bjon.2005.14.Sup5.19954.
[32] R.U. Jänick, M.L. Sprengart, M.R. Wati, A.G. Porter, Caspase-3 is required for
DNA fragmentation and morphological changes associated with apoptosis, J.
Biol. Chem. 273 (1998) 9357–9360, doi:10.1074/jbc.273.16.9357.
[33] O. Kutuk, E.D. Arisan, T. Tezil, M.C. Shoshan, H. Basaga, Cisplatin overcomes
Bcl-2-mediated resistance to apoptosis via preferential engagement of Bak:
critical role of Noxa-mediated lipid peroxidation, Carsinogenesis 30 (9) (2009)
1517–1527, doi:10.1093/carcin/bgp165.
[10] A. Kascatan-Nebioglu, M.J. Panzner, C.A. Tessier, C.L. Cannon, W.C. Youngs, N-
Heterocyclic carbene-silver complexes: a new class of antibiotics, Coord. Chem.
Rev. 251 (5–6) (2007) 884–895, doi:10.1016/j.ccr.2006.08.019.
[11] R.A. Haque, M.A. Iqbal, M.B. Khadeer Ahamed, A.A. Majid, Z.A. Abdul
Hameed, Design, synthesis and structural studies of meta-xylyl linked bis-
benzimidazolium salts: potential anticancer agents against ’human colon can-
cer’, Chem. Cent. 6 (1) (2012) 68, doi:10.1186/1752-153X-6-68.
[34] A. Habib, V.M. Nazari, M.A. Iqbal, H.N. Bhatti, M.B.K. Ahmed, A.M.S.A. Majid,
Unsymmetrically substituted benzimidazolium based silver(I)-N-heterocyclic
carbene complexes: synthesis, characterization and in vitro anticancer study
against human breast cancer and colon cancer, J. Saudi Chem. Soc. 23 (7)
(2019) 795–808, doi:10.1016/j.jscs.2019.03.002.
[12] R.A. Haque, M.Z. Ghdhayeb, S. Budagumpi, A. Salman, M.B. Khadeer Ahamed,
A.M.S. Abdul Majid, Non-symmetrically substituted N-heterocyclic carbene–
Ag(I) complexes of benzimidazol-2-ylidenes: synthesis, crystal structures, anti-
cancer activity and transmetallation studies, Inorg. Chim. Acta 394 (1) (2012)
519–525, doi:10.1016/j.ica.2012.09.013.
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