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Anticancer and antiproliferative activity of ruthenium complex (II) bearing 3,3’-dicarboxy-2,2’-bipyridine ligand
Pharmacia 70(3): 803–807
DOI 10.3897/pharmacia.70.e111508
Research Article
Anticancer and antiproliferative activity of
ruthenium complex (II) bearing 3,3’-dicarboxy2,2’-bipyridine ligand
Mohamed Saadh1
1 Faculty of Pharmacy, Middle East University, Amman, 11831, Jordan
Corresponding author: Mohamed Saadh (mjsaadh@yahoo.com)
Received 23 August 2023 ♦ Accepted 8 September 2023 ♦ Published 18 September 2023
Citation: Saadh M (2023) Anticancer and antiproliferative activity of ruthenium complex (II) bearing 3,3’-dicarboxy-2,2’-bipyridine
ligand. Pharmacia 70(3): 803–807. https://doi.org/10.3897/pharmacia.70.e111508
Abstract
Even though significant progress has been made in cancer treatment, there is always room for improvement. The experimental drug
Ruthenium Complex II shows promise as a cancer treatment. In this article, the dichloro-3,3’-dicarboxy-2,2’-bipyridyl bis(dimethylsulphoxide)ruthenium(II) [RuCl2(3,3’-dcbpy)(DMSO)2], have been synthesized, characterized, and studied for its anticancer activity against MDA-MB-231 and MRC-5 cell lines, as well as its mechanisms of action and selectivity. According to research, [RuCl2
(3,3’-dcbpy)(DMSO)2], is highly cytotoxic to the MDA-MB-231 and minimum cytotoxic to MRC-5 cell lines, with IC50 values of
5.95 and 579.6 μg/ml, respectively. Ruthenium Complex II is exceptionally effective at destroying cancer cells while causing minimal
harm to healthy cells. RuCl2(3,3’-dcbpy)(DMSO)2] caused apoptosis, which was confirmed by the activation of caspase-3. Ruthenium
complexes hold great promise as powerful anticancer agents. Their unique mechanisms of action, ability to selectively target cancer
cells, and versatility in chemical structure make them attractive candidates for the development of targeted therapies.
Keywords
Anticancer agents, coordination complexes, cytotoxic activity, ruthenium complex, tumor cell lines
Introduction
Cancer, a complex and devastating disease, often demands
innovative treatments due to limitations of traditional
drugs that may cause systemic toxicity and drug resistance
(Abusamra et al. 2015). In contrast, ruthenium complexes
offer promise with targeted mechanisms, tailored design,
and potential to overcome resistance, holding potential for
more effective and selective cancer treatment (Al-Wahish
et al. 2017).
Ruthenium complexes have emerged as intriguing candidates for anticancer therapy due to their unique chemical properties and diverse mechanisms of action (Anitha et
al. 2018). These complexes can interact with biomolecules
such as DNA, proteins, and enzymes, influencing key cellular processes and leading to apoptosis, or programmed
cell death, in cancer cells. Their ability to target specific
cancer cells while sparing healthy ones has sparked interest in their potential as selective anticancer agents.
Ruthenium complexes can inhibit DNA replication and
repair, disrupt cellular signaling pathways, and interfere
with angiogenesis, the process by which tumors develop
new blood vessels (Anitha et al. 2018). Their structural
variability allows for the design of complexes with tailored
properties optimized for specific cancer types. However,
rigorous research is ongoing to assess their toxicity, bio
Copyright Saadh M. This is an open access article distributed under the terms of the Creative Commons Attribution License (CCBY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.
804
Saadh M: Ruthenium complex (II) as anticance agents
distribution, and overall safety profile in order to translate
these findings into effective and safe anticancer treatments
(Chen et al. 2021; Awwadiet al. 2022).
MDA-MB-231 is a widely studied triple-negative breast
cancer cell line known for its aggressive behavior, making it suitable to investigate the potential efficacy of anticancer agents like ruthenium complexes (Csupor‐Löffler
et al. 2009).
In this study, the dichloro-3,3’-dicarboxy-2,2’-bipyridyl
bis(dimethylsulphoxide)ruthenium(II) [RuCl2(3,3’-dcbpy)(DMSO)2], was synthesized, characterized using FT-IR
and X-ray crystallography. This study aims to synthesize a
novel ruthenium complex and characterize it using FT-IR,
UV, and NMR techniques. Subsequently, assess the anticancer potential of a ruthenium complex against MDAMB-231 cells by determining its IC50 value and evaluating
its impact on caspase 3 activity, shedding light on its cytotoxic and apoptotic effects.
ible spectra were generated for 1.0 10-5 M solutions in
CH2Cl2 at 25 °C (Kostova 2006; He et al. 2019).
Materials and methods
MTT cytotoxicity assay
Materials
In brief, incubate 1 × 104 cells with Ru(3,3’-dcbpy)
(DMSO)2 Cl2] at concentrations of 2, 5, 10, 20, 50, and
100 µg/ml for 72 hours. Wells were incubated with MTT
for four hours after exposure. A multi-well plate reader (Bio-Tek Instrument, USA) measured optical density (OD) at 570 nm with a reference wavelength of 630
nm after dissolving MTT crystals in 100 µl of DMSO
solution (Prathima et al. 2023; Saadh et al. 2023). Positive controls: doxorubicin (0.1, 0.5, 1.0, 1.5, 10, 25, 50,
100 µg/ml). (Treated OD/Non-treated OD 100) = 100%
cell growth inhibition (Prathima et al. 2023; Saadh et al.
2023). The IC50 concentration inhibits cell line proliferation 50%.
According to published procedures, 3,3’-dicarboxy-2,2’-bipyridine and [RuCl2 (DMSO)4] were synthesized. Following procedures outlined in the literature,
1,4-dioxane (Merck) was purified and dried (Armarego W
L F, 2017). Ethanol and methanol were redistillated in an
atmosphere of nitrogen. All operations were performed in
the N2 atmosphere. The tested samples were dissolved using distilled water as the solvent.
Preparation
of
dichloro-3,3’-dicarboxy-2,2’-bipyridyl
bis(dimethylsulphoxide)ruthenium(II) [RuCl2(3,3’-dcbpy)(DMSO)2]
A suspension of 3,3’-dcbpy compound (0.121 g,
0.500 mmol) in dry ethanol (20 mL) was added to a suspension of [RuCl2(DMSO)4] (0.242 g, 0.500 mmol) in
dry ethanol (20 mL). Two hours were spent heating the
reaction mixture to reflux under a nitrogen flow. During
this time, the color of the solution changed to a brownred hue. After allowing the reaction to cool to room temperature, it was filtered. The solvents were eliminated in
order to achieve dryness. The residual solid was dissolved
in minimal dry methanol and then filtered. Adding 20 mL
of diethyl ether produced a brown solid. The product was
then filtered, washed with diethyl ether (210 mL), and
vacuum-dried at 60 °C for four h (Kostova 2006; He et al.
2019). Yield 89.5%, m.p. 195–200 °C.
The infrared spectra were recorded on KBr discs using
a Nicolet Impact-400 FT-IR spectrometer. On a Bruker
AVANCE III-500 MHz spectrometer, the 1H and 13C
NMR spectra were acquired. The Philip-Harris melting
point apparatus was used to determine melting points. Using a Cary 100 Bio UV-Vis spectrophotometer, UV-Vis-
Cell culture
MRC-5 and MDA-MB-231 cells were obtained from
ECACC. MCF-7 and MRC-5 cells were cultured in
DMEM supplemented with 10% heat-inactivated fetal
bovine serum, L-glutamine (2 mM), and penicillin/streptomycin (100 U/ml, 100 g/ml) (Prathima et al. 2023) at
37 °C with 5% CO2.
Cell treatment
After 12 hours of attachment, 2.0 ml of fresh medium containing 2, 5, 10, 20, 50, and 100 g/ml [RuCl2(3,3’-dcbpy)
(DMSO)2] was added to six-well plates containing 4 × 104
cells/ml. The biochemistry of cells was evaluated 24 hours
after treatment (Prathima et al. 2023).
Caspase activity assay
RIPA reagent was used to extract total protein from
MDA-MB-231 cells after 48 hours of treatment with
[Ru(3,3’-dcbpy) (DMSO)2 Cl2] (25, 50, and 150 µg/mL).
A commercial kit (KeyGen Biotechnology, Nanjing, China) and an ELISA reader (ELX800, Promega, US) measured caspase-3 activity at 405 nm.
Statistical analysis
SPSS 19.0 performed an unpaired Student’s t-test on
mean standard deviation and P value data. P < 0.05 was
significant.
Results
[RuCl2(3,3’-dcbpy)(DMSO)2]
The new ruthenium complex was made by directly reacting RuCl2(DMSO)4 with 3,3’-dicarboxy-2,2’-bipyridine in
Pharmacia 70(3): 803–807
805
dry ethanol. The [RuCl2(3,3’-dcbpy)(DMSO)2] is formed
by mixing RuCl2(DMSO)4 with one equivalent of ligand
(Equation 1). Microelemental analysis confirmed the
complex formulation. This complex was characterized by
FT-IR and UV-Vis.
[RuCl2(DMSO)4] + 3,3'-dcbpy
Dry EtOH/ Reflux
FT-IR Spectral analysis
The brown complex [RuCl2(3,3’-dcbpy)(DMSO)2]
is soluble in water, methanol, ethanol, acetone,
tetrahydrofuran, and dimethylsulphoxide but not in
dichloromethane, chloroform, petroleum ether, or
diethyl ether.
[RuCl2(3,3'-dcbpy)(DMSO)2]
...(1)
Table 2. [RuCl2(3,3’-dcbpy) (DMSO)2] IC50 values (mean ± SD
μg/ml) from three cytotoxicity assays.
Table 1 and Fig. 1 show the characteristic bands in the
ligand, Ru-DMSO precursor, and newly synthesized
Ru-complex spectra.
100
Cytotoxicity
assay
MTT assay
Treatment
IC50
MDA-MB-231
Fibroblasts
(MRC5)
[RuCl2(3,3’-dcbpy) (DMSO)2]
5.95 ± 0.39
579.6 ± 0.41
Doxorubicin
5.15 ± 0.35
7.45 ± 0.17
95
90
1941.86
558.81
85
80
813.81
424.47
75
671.13
70
760.30
1147.62
2599.48
1016.12
1260.28
40
35
30
1088.87
45
1419.01
50
3076.95
55
2919.24
3422.13
60
1573.63
65
1719.95
25
20
15
4000
3500
3000
2500
2000
1500
1000
500
W avenumbers (c m-1)
Figure 1. FTIR spectrum of [RuCl2(3,3’-dcbpy)(DMSO)2].
Table 1. (N-P), [RuCl2(DMSO)4], and [RuCl2(3,3’-dcbpy)
(DMSO)2] complexes infrared spectral data.
Mode
nO-H
nC-H (Aromatic)
nC-H (Aliphatic)
nC-C (Aromatic)
nC=O
Compounds
[RuCl2(DMSO)4] (3,3’-dcbpy)
[RuCl2(3,3’-dcbpy)
(DMSO)2]
–
3392
3422
–
3073
3076
3002, 2920
–
2919, 2599
–
1578. 1433
1573, 1419
–
1717
1719
nS=O (S-bonded)
1100, 1021
–
1088, 1016
nS=O (O-bonded)
927
–
–
Figure 2. UV-Visible spectrum of [RuCl2(3,3’-dcbpy)(DMSO)2].
Caspase-3 assay
To determine [RuCl2(3,3’-dcbpy)(DMSO)2] cytotoxic
mechanism, caspase3 activity, the apoptosis executor, was
measured. Activation of caspase 3 by [RuCl2(3,3’-dcbpy)
(DMSO)2] (P < 0.05) is dose-dependent (Fig. 3).
UV-Vis spectroscopy.
Ru-complex [RuCl2(3,3’-dcbpy)(DMSO)2] UV-visible spectrum was measured in MeOH solution. Fig. 2 shows three
absorption bands at 381, 302, and 204 nm for the complex.
Antiproliferative and cytotoxicity
The MTT assay assessed doxorubicin’s cytotoxicity. As a positive control, doxorubicin showed IC50 values of 5.15 and 7.45
μg/m against MDA-MB-231 and MRC5. The [RuCl2(3,3’-dcbpy)(DMSO)2] exhibits IC50 values of 5.95 and 579.6 μg/ml
against MDA-MB-231 and MRC5, respectively (Table 2).
Figure 3. Caspase protein activity of MDA-MB-231cells treated
with different concentrations of [RuCl2(3,3’-dcbpy) (DMSO)2].
Values were significantly different compared with the control
group. *P < 0.01.
806
Discussion
Ruthenium complexes exhibit unique chemical properties
that enable targeted interactions with cancer cells, resulting in effective cytotoxicity (Chen et al. 2021; Awwadiet al.
2022). Their versatile coordination chemistry allows for
tailored modifications, enhancing selectivity and reducing off-target effects. This specificity, coupled with their
diverse mechanisms of action and potential to overcome
drug resistance, underscores their promise as valuable
candidates in the development of innovative and potent
anticancer agents (Chen et al. 2021; Awwadiet al. 2022).
Ruthenium exhibits cancer cell specificity, minimizing
impact on normal cells. Its selective behavior holds promise
for targeted therapies with reduced side effects (Sha et al.
2015). This agreement with our study which indicate the
[RuCl2(3,3’-dcbpy)(DMSO)2] has been shown to highly
cytotoxicity against MDA-MB-231 cells in a dose-dependent manner with IC50 ~ 5.95 ± 0.39µg/ml, indicating its
potential as an effective anti-proliferative agent against cancer cells, while exhibiting minimal impact on normal cells.
For example, Ru(bpy)2(dtdpq)2 exhibits potent cytotoxicity
against MCF-7 cells and has the ability to inhibit their proliferation and induce apoptosis, with an IC50 value of 2.3 ±
0.3 μM against MCF-7 cells (Shabani et al. 2023). Also, Ru
(II) complexes inhibit HeLa cells while having minimal effects on normal cells (Valente et al. 2021). Ru (II) complexes
have multiple mechanisms to inhibit cancer cells by generating reactive oxygen species (ROS), inducing apoptosis,
inhibiting DNA repair enzymes, and causing DNA damage
which can damage cancer cells and cause cell death (van
Rijt and Sadler 2009; Sha et al. 2015). The caspase-3 assay
was utilized to elucidate the cytotoxic mechanism of [RuCl2(3,3’-dcbpy)(DMSO)2]. Caspase 3, a pivotal mediator of
apoptosis, exhibited dose-dependent activation triggered
Saadh M: Ruthenium complex (II) as anticance agents
by the ruthenium complex. similarly, ruthenium complex
displays a potential to induce apoptosis in MDA-MB-231
cells, reinforcing its role as an anticancer agent by initiating
programmed cell death pathways. The unique properties of
ruthenium complex, such as its ability to interact with DNA
and proteins, make it an ideal candidate for targeting cancer
cells specifically. When ruthenium complex is introduced
to cancer cells, it interacts with cellular components, triggering a cascade of events that ultimately leads to caspase 3
activation and apoptosis. This targeted approach minimizes damage to healthy cells, making ruthenium complex a
promising candidate for cancer treatment (Yu et al. 2014).
Moreover, the ruthenium complex displays inhibition
against lung cancer (A549) by instigating apoptosis, DNA
damage, and oxidative stress, showcasing its therapeutic potential (Zeng et al. 2017). Similarly, it inhibits colon
cancer (HCT116) cells through apoptosis induction, DNA
damage, and modulation of cellular signaling pathways,
highlighting its promise in colon cancer treatment (Zhang
et al. 2019). Additionally, the complex hinders HeLa cells
by prompting apoptosis, potentially impairing DNA, and
influencing cellular signaling pathways, underscoring its
anticancer capabilities.
Conclusion
In conclusion, [RuCl2(3,3’-dcbpy)(DMSO)2] demonstrated significant cytotoxicity against MDA-MB-231.
Ruthenium exhibits cancer cell specificity, minimizing
impact on normal cells. Moreover, the fact that it activates caspase-3 in a dose-dependent manner suggests an
apoptotic mechanism of action, showing that it could be a
promising anticancer agent. However, we need to do more
research to fully understand its mechanism of action.
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