Ruthenium (II)/allopurinol complex inhibits breast cancer progression via multiple targets

Article abstract of DOI:10.1007/s00775-021-01862-y

Abstract: Metal complexes based on ruthenium have established excellent activity with less toxicity and great selectivity for tumor cells. This study aims to assess the anticancer potential of ruthenium(II)/allopurinol complexes called [RuCl₂(allo)₂(PPh₃)₂] (1) and [RuCl₂(allo)₂(dppb)] (2), where allo means allopurinol, PPh₃ is triphenylphosphine and dppb, 1,4-bis(diphenylphosphino)butane. The complexes were synthesized and characterized by elemental analysis, IR, UV–Vis and NMR spectroscopies, cyclic voltammetry, molar conductance measurements, as well as the X-ray crystallographic analysis of complex 2. The antitumor effects of compounds were determined by cytotoxic activity and cellular and molecular responses to cell death mechanisms. Complex 2 showed good antitumor profile prospects because in addition to its cytotoxicity, it causes cell cycle arrest, induction of DNA damage, morphological and biochemical alterations in the cells. Moreover, complex 2 induces cell death by p53-mediated apoptosis, caspase activation, increased Beclin-1 levels and decreased ROS levels. Therefore, complex 2 can be considered a suitable compound in antitumor treatment due to its cytotoxic mechanism. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00775-021-01862-y

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-021-01862-y
ORIGINAL PAPER
Ruthenium (II)/allopurinol complex inhibits breast cancer progression
via multiple targets
Ingrid O. Travassos1 · Francyelli Mello‑Andrade
1,2
· Raíssa P. Caldeira · Wanessa C. Pires ·
1
1
1
3
3
4
5
Paula F. F. da Silva · Rodrigo S. Correa · Tamara Teixeira · Alisson Martins‑Oliveira · Alzir. A. Batista ·
Elisângela P. de Silveira‑Lacerda1
Received: 26 November 2020 / Accepted: 8 March 2021
©
Society for Biological Inorganic Chemistry (SBIC) 2021
Abstract
Metal complexes based on ruthenium have established excellent activity with less toxicity and great selectivity for tumor cells.
This study aims to assess the anticancer potential of ruthenium(II)/allopurinol complexes called [RuCl (allo) (PPh ) ] (1) and
2
2
3 2
[
RuCl (allo) (dppb)] (2), where allo means allopurinol, PPh is triphenylphosphine and dppb, 1,4-bis(diphenylphosphino)
2
2
3
butane. The complexes were synthesized and characterized by elemental analysis, IR, UV–Vis and NMR spectroscopies,
cyclic voltammetry, molar conductance measurements, as well as the X-ray crystallographic analysis of complex 2. The
antitumor eꢀects of compounds were determined by cytotoxic activity and cellular and molecular responses to cell death
mechanisms. Complex 2 showed good antitumor proꢁle prospects because in addition to its cytotoxicity, it causes cell cycle
arrest, induction of DNA damage, morphological and biochemical alterations in the cells. Moreover, complex 2 induces
cell death by p53-mediated apoptosis, caspase activation, increased Beclin-1 levels and decreased ROS levels. Therefore,
complex 2 can be considered a suitable compound in antitumor treatment due to its cytotoxic mechanism.
Graphic abstract
Keywords DNA damage · Cell death · ROS · Triple-negative breast cancer
Extended author information available on the last page of the article
Vol.:(0
1
123456789)
3

JBIC Journal of Biological Inorganic Chemistry
Introduction
for complexes 1 and 2 were recorded on a Hewlett Packard
8
452A diode array spectrophotometer. The microanalyses
Several ruthenium (Ru) complexes have been identiꢁed and
widely studied in an antitumor preclinical assay. Ru complex
exhibits interesting physico-chemical and biochemical proper-
ties [1–3], such as binding to transferrin [4]; interaction with
DNA [5], inhibiting topoisomerase [6], inducing oxidative
stress and caspase-mediated apoptosis [7], as well as in vivo
inhibiting tumor progression [8] associated with low toxicity
proꢁles [9, 10]. Due to its properties, ruthenium can be coor-
dinated with several diꢀerent ligands, given many possibilities
to improve its application in cancer therapy. The mechanism
of action of this class of complexes can vary depending on
their composition or depending on the structure of the ligands
bound to the metal. In fact, currently, new concept in the anti-
tumor drug design is based on the combination of diꢀerent
bioactive substances to produce an anticancer hybrid com-
pound that exerted its cell-killing eꢀects by interacting with
multiple intracellular targets.
were performed with an EA 1108 CHNS microanalyser
(Fisons Instruments). Molar conductivity values were
–
3
obtained at 293 K using 10 M solutions of the complexes
in CH2Cl2 by using a Meter Lab CDM2300 instrument. The
spectra in the infrared region (IR) were recorded on an FT-IR
−
1
Bomem-Michelson 102 spectrometer in the 4000–250 cm
region (Figs. 7S-8S), using cesium iodide (CsI) pellets. The
1
31
1
H and P{ H} NMR spectra of complexes 1 and 2 were
recorded on a Bruker DRX 400 MHz. Cyclic voltammetry
experiments were carried out at room temperature in CH2Cl2
containing 0.10 M Bu4NClO4 (TBAP) (Fluka Purum), with
a BAS-100B/W electrochemical analyzer (Bioanalytical
Systems Inc.). The working and auxiliary electrodes were
stationary Pt foils; a Luggin capillary probe was used and
the reference electrode was Ag/AgCl. Ferrocene (Fc) was
used for calibrating the electrochemical system and the
redox potential and under these conditions the ferrocene was
+
In this frame, Ru complexes with the allopurinol ligand
were designed and synthesized. Allopurinol comprises a class
of medications called xanthine oxidase (XO) inhibitors and is
used in clinical practice for its protective eꢀects against inꢂam-
matory diseases, chronic heart failure [11], and as concomitant
medication in the treatment of leukemia [12] and in the tumor
lysis syndrome [13].
oxidized at 0.43 V (Fc /Fc). Crystals of complex 2 were
obtained by slowly evaporating the dichloromethane/hex-
ane solution. The crystal was mounted on an Enraf–Nonius
Kappa-CCD diꢀractometer with graphite monochromated
MoKα (λ=0.71073 Å) radiation. The ꢁnal unit cell param-
eters were based on all reꢂections. The structure was solved
by direct methods and reꢁned by full-matrix least-squares on
2
Allo molecule is an inhibitor for xanthine oxidase (XO).
Reactive oxygen species (ROS) are produced as a consequence
of several metabolic processes, including oxidation of xanthine
by xanthine oxidase (XO) [14]. In a previous report, Ru com-
plex was shown with allopurinol ligand ability to act as a selec-
tive and competitive tight-binding inhibitor of XO [15]. Some
studies have shown that XO-derived ROS are implicated in the
oncogenesis process. In addition, prior reports have indicated
that XOR-derived ROS may be involved in the mechanisms
associated with breast cancer pathogenesis [16]. However,
there are no studies on the cytotoxic eꢀects of Ru/allo com-
plexes in breast cancer cells, including ROS level induction.
Aiming to develop new antitumor agents with improved
cytotoxicity against tumor cells, in this study, we present the
synthesis, characterization and a detailed study on cytotoxic
activity against breast tumor cells of two new Ru complexes
with allo ligand called complexes [RuCl (allo) (PPh ) ]
F using SHELXL-97. All hydrogen atoms were positioned
stereochemically and reꢁned using a riding model and all
non-hydrogen atoms were reꢁned considering their aniso-
tropic thermal displacements. More information about the
crystal structure and reꢁnement data is depicted in Table S2.
Supplementary crystallographic data were deposited under
number CCDC 1571686 and can be obtained free of charge
from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/structures.
Synthesis and characterization
[
(
RuCl (allo) (PPh ) ] (1). In a Schlenk ꢂask, allo ligand
2
2
3 2
30 mg, 0.22 mmol) was dissolved in 80 mL of methanol.
Afterwards, 100 mg (0.104 mmol) of [RuCl (PPh ) ] pre-
2
3 3
cursor solubilized in 20 mL of CH Cl was added to the
2
2
2
2
3 2
allo solution. The mixture was kept under stirring at reꢂux
for 4 h. Then, the volume was reduced to ca. 2 mL and
a yellow solid was precipitated and separated by ꢁltration,
washed in methanol, diethyl ether and dried under vacuum
(
1) and [RuCl (allo) (dppb)] (2), where PPh means triph-
2
2
3
enylphosphine and dppb is 1,4-bis(diphenylphosphino)butane.
to yield 80 mg (80%). Anal. Calc. for [RuC H O N P ]:
Materials and methods
46 38
2
8 2
exp. (calc) C 56.80(56.91); H 3.93 (4.15); N 11.47 (11.54)
2
–1
–1
%
. Molar conductance (S cm mol , CH Cl ) 3.4. IR (cm ):
RuCl ∙3H O and 1,4-bis(diphenylphosphino)butane (dppb),
2 2
3
2
3
1
9
173, 3059, 1710, 1682, 1595, 1556, 1481, 1433, 1367;
393, 1282, 1242, 1215, 1159, 1190, 1090, 1028, 999, 956,
triphenylphosphine (PPh ) and allopurinol ligands were
3
obtained from Sigma-Aldrich. The UV–Vis spectra obtained
3
1
1
14, 848, 790, 783, 746, 696, 606, 522, 418, 320. P{ H}
1
3

JBIC Journal of Biological Inorganic Chemistry
NMR (162 MHz, CDCl , 298 K): δ(ppm) 38.6 (s). UV–Vis
collected using a sterile syringe and EAC cells were cul-
tured at one passage in vitro prior to use. MDA-MB-231
(human breast carcinoma) and L-929 (murine breast adipose
ꢁbroblast) were purchased from BCRJ (Banco de Células
do Rio de Janeiro). EAC cells were cultured in RPMI 1640
medium (Gibco, Invitrogen, Carlsbad, CA, USA), (pH
7.2–7.4); L-929 and MDA-MD-231 cells were cultured in
DMEM medium (Gibco, Invitrogen, Carlsbad, CA, USA),
3
–
4
–1
–1
(
CH Cl , 1.6×10 M): λ/nm (ε/M cm ) 350 (14470).
2
2
[
RuCl (allo) (dppb)] (2). In a Schlenk flask, 16 mg
2
2
(
0.118 mmol) of allo was dissolved in 5 mL of methanol.
Afterwards, 25 mL of CH Cl were added to this solution
2
2
containing 50 mg (0.058 mmol) of [RuCl (dppb)(PPh )]
2
3
reactant. After stirring for 30 min. under room tempera-
ture, the color mixture changed from a greenish to a yel-
lowish suspension. The solvent was removed under reduced
pressure and the yellowish solid was obtained after leav-
ing the solution in the refrigerator. The powder was ꢁl-
tered and washed in methanol and diethyl ether and then
dried under vacuum to yield 35 mg (70%). Anal. Calc. for
−
1
(pH 7.2–7.4), and routinely subcultured using 2.5 g L
trypsin-ethylenedinitrile tetraacetic acid (EDTA) solution.
−
1
Both mediums were supplemented with 100 U mL peni-
−
1
cillin G, 100 µg mL streptomycin, and 10% fetal bovine
serum (FBS) in an incubator (Thermo Scientiꢁc) at 37 °C
[
(
RuC H O N Cl P ]: exp. (calc) C 52.35(52.42); H 4.08
with 5% CO . The protocol (039/2012) of the EAC culture
3
8
36
2
8
2
2
2
2
–1
4.16); N 12.78(12.97) %. Molar conductance (S cm mol ,
was approved by the Ethics Committee at the Federal Uni-
–
1
CH Cl ) 4.7. IR (cm ) 3142, 3063, 2951, 2924, 2870, 1709,
versity of Goias.
2
2
1
1
8
690, 1597, 1556, 1483, 1435, 1356, 1400, 1215, 1254,
283, 1157, 1122, 1095, 1070, 1028, 999, 905, 955, 879,
MTT assay
3
1
1
14, 785, 742, 721, 696, 630, 611, 511, 422, 312. P{ H}
NMR (162 MHz, CDCl , 298 K): δ(ppm) 41.1 (s). UV–Vis
The cytotoxic activity of complexes 1 and 2 was measured
3
–
5
–1
–1
(
CH Cl , 6.8×10 M): λ/nm (ε/M cm ) 310 (2547).
by 3-(4,5 dimethylthiazol-2yl)-2,5diphenil tetrazolium bro-
2
2
5
mide (MTT), as described by [18]. Brieꢂy, 1×10 of EAC,
DNA titration experiments
MDA-MB-231, and L-929 cells were plated in 96-well tis-
sue culture plates and treated with diꢀerent concentrations
of tested compounds and cisplatin (0.2 to 200 µM) for 48 h.
The calf thymus DNA (ctDNA) solution from Sigma-
Aldrich was prepared in a Tris–HCl buꢀer (5 mM Tris–HCl,
pH 7.2). The concentration of this standard solution was
measured from its absorption intensity at 260 nm using the
−
1
After treatment, the MTT solution (1 mg mL ) was added
to each well, and plates were incubated at 37 °C for 3 h.
SDS solution was used to dissolve formazan crystals and the
absorbance was determined at 545 nm using a Stat Fax 2100
microplate reader (Awareness Technology, Palm City, FL,
USA). Cell viability was expressed as a percentage relative
to control (100% viability) and was calculated as follows:
viability (%)=(Absorbance of the treated wells)/(Absorb-
ance of the control wells) × 100. Each concentration was
tested in three diꢀerent experiments run in triplicate. The
IC values were obtained from dose–response curves using
−
1
−1
molar absorption coeꢃcient value of 6600 M cm . The
ctDNA solution is protein-free, given that the ratio of UV
absorbance at 260 and 280 nm is about 1.8:1. The solu-
tion of Ru complexes 1 and 2 used in the experiments was
prepared in a Tris–HCl buꢀer containing 2% of DMSO. In
the titration experiments, diꢀerent concentrations of the
ctDNA were used while the Ru complex was at 25 µM. The
intrinsic equilibrium binding constant (Kb) of the com-
plexes to ctDNA was obtained by monitoring changes in
the absorption intensity with an increasing concentration
of ctDNA and it was analyzed by regression analysis. Bind-
ing constants, Kb, were calculated according to equation
5
0
GraphPad Prism 7.0 for Windows (GraphPad Software, San
Diego, CA, USA). The IC values obtained from the MTT
5
0
assay were used to determine the most promising compound
and progress for further tests.
[
17]: [ctDNA]/(εa−εf)=[ctDNA]/(εb−εf)+1/Kb(εb−εf), in
The Selectivity Index (SI) indicates the selectivity of
the compounds and their potential use for preclinical tests,
approaching safety data. The index was determined by the
ratio of IC values obtained in non-tumor and tumor cell
which [ctDNA] is the concentration of ctDNA in base pairs,
εa is the ratio of the absorbance/[Ru], εf is the extinction
coeꢃcient of the free Ru(II) complex, and εb is the extinc-
tion coeꢃcient of the complex in the fully bound form. The
stability of the complexes was evaluated in these conditions
5
0
lines for the two complexes of Ru (II) and allo.
3
1
using the P NMR technique and they showed to be stable.
Clonogenic cell survival assay
Cell culture
The ability for MDA-MB-231 cells to form colonies was
tested after exposition to complex 2 at 9 µM, 4.5 µM and
2.25 µM as control untreated cells. Cells were seeded in
Murine breast carcinoma cells, EAC, were maintained in
ascitic form by sequential passages in Swiss mice. Seven
days after cell inoculation in Swiss mice, ascitic ꢂuid was
2
6-well plates at a concentration of 3×10 cells/well in dupli-
cate of three independent experiments. Afterwards, cells
1
3

JBIC Journal of Biological Inorganic Chemistry
were exposed to complex 2 for 48 h, then the plates were
washed with PBS, the medium was replaced and cells were
cultured for 8 days.
S, G2/M and sub-G1 was analyzed using the ModFit LT
software (Verity Software House, Topsham, ME, USA).
The quantities of colonies formed were visually observed
to determine the end of the experiment. The medium was
carefully removed and the plates were washed in PBS twice
before, ꢁxing the colonies with 3 mL 10% neutral buꢀered
formalin solution for 15 min. After removing the forma-
lin solution, colonies were stained with INSTANT PROV
Comet assay
DNA damage was assessed using the single-cell gel elec-
5
trophoresis (SCGE) assay according to [21]. Brieꢂy, 2×10
EAC cells were seeded in a 12-well cell culture plate and
incubated for 24 and 48 h in the absence or presence of
complex 2 at concentrations of 9 and 18 µM. After incuba-
tion, the cell suspension was homogenized with 100 µL of
low-melting point agarose (0.5%) and then plated on slides
that already contained standard agarose (1.5%). Cell lysis,
electrophoresis, neutralization, and staining were carried
out as described by [22]. The experiment was prepared in
duplicate, and > 100 cells were screened per sample. The
images were acquired by a ꢂuorescence microscope (Leica
Microsystems, Wetzlar, Germany) and analyzed using Com-
etScore software (Comet Scoring Software, Version 1.5,
TriTek Corp.) The nucleus was classiꢁed visually accord-
ing to the migration of the fragments, as previously proposed
by [23], as follows: class 0, 1, 2, 3 and 4. The damage index
(DI) was assessed for each comet according to the following
criteria: DI=(0×n0)+(1×n1)+(2×n2)+(3×n3)+(4×n
4), where n=number of cells in each class analyzed. Thus,
DI ranged from 0 (completely undamaged: 100 cells×0) to
400 (with maximum damage; 100 cells×4).
(
NEWPROV®), reagents II and III; plates were left to dry
for 24 h at room temperature. A stereomicroscope (Leica
Microsystems, EZ4 HD) coupled to a camera was used for
capturing images and counting colonies. Data acquired
were analyzed according to the plating eꢃciency (PE) and
survival fraction (SF) parameters described by [19]. Plat-
ing eꢃciency is the ratio of the number of colonies to the
number of cells plated: PE = Number of colonies formed/
number of cells plated × 100. The survival fraction is the
number of colonies that arise after treating the cells. SF=PE
of treated cells/PE of control×100. The dose-survival curve
was obtained using GraphPad Prism Software.
Migration assay
The cell migration assay was used to evaluate the activity
of complex 2 concerning the aggregation of cell–cell inter-
actions after carrying out a section in the cell monolayer.
5
The experiment was conducted culturing 2×10 of MDA-
MB-231 cells in 24 well plates. After the cells acquired a
conꢂuence of 70%, a vertical section was performed on the
cell monolayer using a pipette tip of 200 µL. Before acquir-
ing the images, the cells were gently washed in PBS twice.
The cells were treated with complex 2 at concentrations of
Determination of reactive oxygen species (ROS)
generation
The production of superoxide anion was assessed by dihy-
droethidium (DHE) staining. The ꢂuorochrome DHE is
oxidized by superoxide to ꢂuorescent ethidium that inter-
calates with nuclear DNA, staining the nucleus with bright
4
.5 µM and 9 µM at 24 and 48 h. For each time (at the
beginning, 24 and 48 h), we recorded at least four photo-
graphic images using a conventional camera coupled to a
microscope (Leica Microsystems, DM IL LED). Acquired
images were analyzed by TScratch 1.0 software (MATLAB,
The MathWorks, Inc., Natick, Massachusetts, USA) [20] and
GraphPad-Prism software in order to evaluate the migration
cell section compared to the control.
7
red ꢂuorescence. Brieꢂy, MDA-MB-231 cells (1×10 cells/
well) were plated in circular glass blades inside well plates
for 24 h. Then, they were treated with complex 2 at 9 µM for
24 and 48 h. Then, after the treatment, the cells were washed
once in iced PBS and incubated for 30 min at 37 °C in the
dark with (DHE, 5 µM) (Molecular Probes, Life Technolo-
gies, NY, USA). A rhodamine ꢁlter with an excitation wave-
length of 596 nm and an emission wavelength of 620 nm was
used for DHE analysis. All the images were acquired using
Leica Microsystems, (Wetzlar, Germany). For each circular
glass blade, images from three random ꢁelds were taken.
Then, the total red ꢂuorescence intensity in each ꢁeld was
quantiꢁed by ImageJ software (http://imagej.net/Welcome).
Afterwards, the red ꢂuorescence was normalized to a total
number of cells per ꢁeld to calculate the amount of cells pro-
ducing ROS [24]. Three ꢁelds per condition were analyzed.
All images presented are×200 magniꢁcation.
Cell cycle analysis
In this assay, EAC cells were treated with complex 2 at
5
1
8 µM in 24 h. A quantity of 5×10 cells was harvested and
washed with phosphate-buꢀered saline (PBS), ꢁxed with
0% (v/v) cold ethanol, and stored overnight at −20 °C. The
7
ꢁ
xed cells were washed in PBS and incubated with PI con-
taining 0.05% RNAase. The samples were incubated at 4 °C
in the dark and analyzed by ꢂow cytometry (FACSCalibur,
BD Biosciences). The percentage of cells in phases G0/G1,
1
3

JBIC Journal of Biological Inorganic Chemistry
Apoptosis analysis by ꢀow cytometer
The analysis used Annexin V-FITC and propidium iodate
Western blot assay
Detecting cell death proꢁle proteins triggered by complex 2
(
PI) for double staining by flow cytometer discriminate
was performed using Western Blot analysis. The EAC cells
6
cells by dyeing, size, and granulation which displays the
at 2×10 were tested in the absence and presence of com-
cell death profile. EAC cells were treated with complex
plex 2 at 18 µM for 1 h and 2 h. After treatment, cells were
lysed using an RIPA buꢀer solution and protein content was
determined using the Bradford assay. Proteins (40 µg per
sample) were loaded in 12% SDS-PAGE at 100 V for 2 h,
followed by electroblotting to nitrocellulose membranes
(0.45 mm) (Bio-Rad Laboratories, Inc.) at 400 mA for 1.5 h.
The membranes were blocked with 3% BSA in Tris-buꢀered
saline with Tween (TBS-T) for 1 h and then incubated over-
night at 4 °C with 2% BSA in TBS-T containing a 1:1000
dilution of primary antibodies against GAPDH, caspase-3,
-7, Beclin-1, Bax and Bak (Santa Cruz Biotechnology Inc.)
The membranes were washed in TBS-T and probed with
horseradish peroxidase-conjugated mouse anti-rabbit sec-
ondary antibodies (Santa Cruz Biotechnology Inc.,). Bound
antibodies were revealed using a BCIP/NBT solution AP
colorimetric system. The optical density of target protein
bands was measured using IMAGE J software downloaded
from the NIH website (http://rsb.info.nih.gov.ij/).
5
2
at 18 µM for 24 and 48 h. Briefly, a quantity of 5 × 10
cells was harvested and washed with cold PBS. The
cells were re-suspended in 500 µL of Annexin V binding
buffer. Afterwards, Annexin V-FITC and PI dyes were
added. Flow cytometric analysis was performed imme-
diately after staining. Data acquisition and analysis were
performed on flow cytometer (FACSCalibur, BD Bio-
sciences) using Cell Quest™ software.
To determine the number of death cells, we
used the following formula: Percent of cell
TREATED
death = [(Q1 + Q2 + Q4)
/(Q3 + Q1 + Q2 + Q4)]
UNTREATED
–
[(Q1 + Q2 + Q4)
/(Q3 + Q1 + Q2 + Q4)] × 100
[
25].
Morphological analysis by ꢀuorescence microscope
The morphologic profile of EAC cells induced by com-
plex 2 was evaluated by morphological technique detec-
tion using PI, Hoechst 33,342 (HO) and Annexin V-FITC
dyes in fluorescence microscopy [26]. EAC cells were
grown in 6 well tissue culture dishes and tested in the
presence and absence of complex 2 at a concentration
of 18 µM. After 24 and 48 h, cells were harvested and
Statistical analysis
Statistical analysis of the results was performed by using
one-way or two-way ANOVA followed by Bonferroni or
Dunnett’s post hoc test for multiple comparisons with con-
trol. A probability of 0.05 or less was deemed statistically
signiꢁcant. All statistical analyses were performed using sta-
tistical software GraphPad Prism, version 7.0 for Windows,
GraphPad Software, San Diego California USA.
5
washed with PBS to a density of 2 × 10 cells. The cells
were suspended in a 100 µL Annexin V binding buffer
with 2 µL HO and 10 µL Annexin V-FITC at 37 °C in the
dark. After 10 min, cells were then incubated with 2 µL
PI for another 5 min in the same conditions. Aliquots of
3
0 µL cell suspension were placed on glass microscope
Results
slides. The slides were observed immediately under a
fluorescence microscope (Olympus BX43, Japan) and
analyzed in three color channels using CellSens Stand-
ard 1.6 software (Olympus corporation, LEADTOOLS©
Synthesis and characterization of Ru/allo complexes
As can be seen in Scheme 1, the synthetic route of the Ru
(II) complexes is straightforward, providing good yields and
analytically pure complexes. The two compounds were char-
acterized by spectroscopic and electrochemical techniques
and the crystal structure of complex 2 was determined by the
X-ray crystallographic technique, conꢁrming the composi-
tion and stereochemistry of the complexes. Both complexes
1 and 2 are neutral, presenting practically no conductivity
1
991–2009, LEAD technologies, North Carolina, USA).
Ten to twenty images (40× objectives) were acquired
using a field-of-view of 20 µm from each sample. EAC
cell images acquired after treatment with complex 2 at
1
8 µM for 24 and 48 h were selected for photographic
documentation. The percentages of apoptotic and necrotic
cells were determined from the total number of cells,
where: LN are considered live cells with a regular nucleus
and blue chromatin, LC are live cells with condensed or
a fragmented nucleus and blue chromatin, DC are dead
cells with a fragmented or condensed nucleus and pink
chromatin and DD are dead cells with a diffuse nucleus
and pink chromatin [26–33].
−
1
in CH Cl (3.4 and 4.6 μS.cm , for 1 and 2, respectively).
2
2
The main bands in the infrared spectra of complexes 1
and 2 conꢁrm the presence of the allopurinol ligand coordi-
nated to the metal centre (Table 1S and Figs. 6S/7S; Supple-
mentary information). Electronic spectra of complexes 1 and
2 show a broadband at around 350 and 310 nm, respectively.
1
3

JBIC Journal of Biological Inorganic Chemistry
Scheme 1 Synthetic route to obtain complexes 1 and 2
The redox behavior of the complexes was studied, in
CH Cl , at room temperature with a potential range from 0
Fig. 1 Crystal structure of complex 2 with main atoms labelled and
ellipsoids at 30% probability
2
2
to+1.5 V, with a Pt disc electrode versus an Ag/AgCl refer-
ence electrode. The cyclic voltammograms of complexes 1
and 2 exhibit a one-electron wave for a Ru(II)/Ru(III) redox
for allo free. As described in Table 1, complex 2 showed
couple, with an anodic peak, E , at + 776 and + 724 mV,
an IC value lower than complex 1 for tumor cells and the
pa
50
3
1
1
respectively. In addition, the P{ H} NMR spectra of com-
plexes 1 and 2 exhibit just one singlet signal at 38.6 ppm
for complex 1, and 41.1 ppm for complex 2. The crystal
structure of complex 2 is depicted in Fig. 1. Crystal data col-
lection and structure reꢁnement parameters are summarized
in Table 2S.
highest SI. Complex 1 is more sensitive to cancer cells than
non-tumor cells. Moreover, the cytotoxic activity (IC ) of
5
0
complex 2 against the MDA-MB-231 cell was 9.1± 0.9 μM,
which is approximately 9 times better than that of cispl-
atin (87.5 ± 10.6 μM). Allo free of metal did not demon-
strate cytotoxic activity and the IC values were more than
5
0
2
00 μM. Although both complexes (1 and 2) have similar
DNA binding: UV–Vis spectrophotometric studies
chemical structures, complex 2 was selected for further
assessments, considering the IC and SI values, as well as
5
0
Complexes 1 and 2 exhibit similar spectral behavior, in
which the absorption spectra decrease when ct-DNA ali-
quots are added. The ratio of the slope to the intercept in
the plot of [DNA]/(εa-εf) vs. [DNA] gives the value of
Kb, which was calculated from the metal to ligand charge
transfer (MLCT) absorption band (λmax) at around 292 and
higher solubility in DMSO and water. The stability of the
Table 1 Values of IC (inhibits 50% of cell viability) and SI (Selec-
50
−1
tivity Index) of complexes 1, 2, allopurinol in μmol L
Compound
IC50
SI4 SI5
3
50 nm for complexes 1 and 2, respectively. Slight changes
L9291
EAC2
MDA-MB-2313
were observed in the absorption spectra by adding ct-DNA
Cisplatin
(1)
(2)
29.0± 1.9 28.0± 1.5 87.5± 10.6
65.0± 3.1 98.2± 2.3 87.4± 7.1
24.6± 1.4 18.0± 1.4 9.1± 0.9
1.0 0.3
0.7 0.7
1.4 2.7
(
low hypochromism of c.a. 10%). DNA-binding constants,
3
Kb, were observed, presenting values of 5.2 × 10 and
3
−1
1
.5×10 M for complexes 1 and 2, respectively.
Allopurinol >200
>200
>200
*
*
Cellular viability
Results obtained by enzymatic reduction MTT assay after 48 h of the
cells exposure to compounds in concentrations from 0.2 to 200 µM.
Data show the mean of IC ± standard deviation of three independent
The cytotoxicity of the two Ru/allo complexes was measured
for two diꢀerent breast cancer cell lines, EAC and MDA-
MD-231, and non-tumor cell line, L-929, determining the
values of IC and SI for each compound tested, as well as
50
1
2
3
experiments. murine ꢁbroblast; Ehrlich ascites carcinoma; Human
4
metastatic breast adenocarcinoma; Selectivity Index (IC L-929/
5
0
5
IC EAC); Selectivity Index (IC L-929 / IC MDA-MD-231); *
5
0
50
50
Selectivity not calculable
5
0
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JBIC Journal of Biological Inorganic Chemistry
complexes in DMSO/culture media was carried out after
DNA damage evaluation
3
1
the incubation period of 6 h and the P NMR spectra are
represented in Figs. S15-S16.
Visually, comets can be distinguished by the head and tail
and qualitatively classiꢁed according to the DNA fragmenta-
tion pattern [34, 35]. As shown in Fig. 2, breast cancer cells
treated with 9 and 18 µM of complex 2 present diꢀerent
degrees of DNA fragmentation. In 24 h, the basal levels
of the DNA damage index determined for EAC cells were
13, while for 9 and 18 µM of complex 2, they were 206
(p<0.01) and 265 (p<0.01), respectively. Complex 2 at 9
and 18 µM caused a DNA damage index of 210 (p <0.01)
and 367 (p<0.01) in 48 h. As expected, the untreated con-
trol showed a low DNA damage index in 48 h (16).
Clonogenic cell survival assay
Our results demonstrated that complex 2 promoted a signiꢁ-
cant decrease in cell reproductive capacity with a reduction
in breast cancer cell survival in 95.3% at 9 µM, 87.39% at
4
.5 µM and 56.95% at 2.25 µM, when compared to untreated
cells (Fig. 18S). As demonstrated in Fig. 18S, inhibition
of cell proliferation occurred in a compound concentration-
dependent in an 8-day experiment period after 48 h of treat-
ment. This experiment also demonstrated that complex 2
caused the cells to be unable to generate clones from single
cells.
Assessment of superoxide generation
To establish the eꢀect exerted by complex 2 in the gener-
ation of superoxide in cancer cell lines, we analyzed the
changes induced by complex 2 treatments on MDA-MB-231
cells in 24 and 48 h. The time-dependent eꢀect promoted by
complex 2 on the DHE signal is shown in Fig. 3. Untreated
cells showed an increase in the number of DHE-positive
cells in a time-dependent manner. Strikingly, we found that
the treatment with complex 2 at 9 µM was suꢃcient to elicit
a signiꢁcant time-dependent reduction in the superoxide
production in 24 h (p<0.01) and 48 h (p<0.001) (Fig. 3c,
d, respectively) when compared with the untreated control.
Migration inhibition
As can be observed in Fig. 19S, the experiment was con-
ducted with MDA-MB-231 cells treated with complex 2.
The results demonstrated an impediment to the reconstruc-
tion of the cell monolayer after the scratch, as well as pro-
moting detachment and loss of cell–cell and cell-extracel-
lular matrix adhesion. These results were obtained for both
concentrations of complex 2, 4.5 µM and 9 µM, and both
times, 24 and 48 h. Until the beginning of the experiment
(
time 0 h), a well-deꢁned gap was observed, however after
2
4 h of treatment with complex 2, the open gap increased by
.54% at 4.5 µM of complex 2 and 28.75% at 9 µM of com-
Proꢁle cell death by ꢀow cytometry
6
plex 2. After 48 h, an increase in the open area of 48.67%
at 4.5 µM and 72.18% at 9 µM was observed compared to
the control.
The cell death proꢁle and number of death EAC cells were
evaluated using Annexin V-FITC and PI double staining
by ꢂow cytometry. The population of untreated and treated
cells with complex 2 at 24 and 48 h is represented in gated
regions in Fig. 4. The quantiꢁcation of dead cells was dem-
onstrated by the sum of gated regions in the dot plot, result-
ing in 40.8% of dead cells for 24 h and 45.9% for 48 h after
the treatment [25].
Cell cycle arrested
The cell cycle distribution of EAC cells was analyzed by
ꢂow cytometry to investigate which cell cycle phases were
arrested after treatments. PI dye binds to the DNA molecule
emitting ꢂuorescence, which is detected by the ꢂow cytom-
eter, making it possible to analyze the amount of DNA in the
sample. Figure 20S shows the results of 24 and 48 h analyses
of histogram distribution.
Gated cells were analyzed by diꢀerent parameters after
treatment with complex 2: cells marked Annexin V-/PI- are
considered as viable (46.0% at 24 h and 43.6% at 48 h),
respectively in Fig. 4e, f. Cells marked with Annexin V+/
PI−are commonly classiꢁed as early apoptosis (4.3% at 24 h
and 5.5% at 48 h). In addition, cells marked Annexin V+/
PI + can be classiꢁed as either late apoptosis as necrop-
tosis—where higher FSC/SSC consists of an increase
of the size and grain. The parameters for necrotic cells
are described as Annexin V + /PI + and lower FSC/SSC.
Annexin V−/PI+has been described for necrosis and also
DNA fragments [27, 36, 37].
Compared to the untreated control, in 24 h in cells treated
with complex 2, a 3.4-fold increase in arrested cells was
observed in the GO/G1 phase and a 2.5 decrease in the S
phase. In 48 h, the data showed that complex 2 maintains the
cell cycle arrested in the G0/G1 phase about twofold more
compared to the control, avoiding the normal progression of
cell kinetics. We observed that complex 2 induced a pattern
change in the cell cycle, both at 24 h (G0/G1: 46.2%± 4.9,
S: 32.6% ± 5.4, G2/M: 21.1% ± 2.2) as at 48 h (G0/G1:
For both times, the analysis of dot plots exhibited fea-
tures that indicate a large amount of cells in late apoptosis
(21.2% at 24 h and 28.4% at 48 h) due to a lack of membrane
4
6.5%± 5.6, S: 31.9%± 5.4, G2/M: 20.8%± 3.2).
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JBIC Journal of Biological Inorganic Chemistry
Fig. 2 EAC cell comet
images were acquired using a
ꢂuorescence microscope (400×
objectives) after cell staining
with ethidium bromide. Images
were evaluated using the Comet
Score Software, according to
DNA fragmentation, size of
the comet “head” and “tail”.
a refers to the representative
image of treated cells with 2
at 9 µM in 24 h and b at 48 h.
c shows representative images
of treated cells with complex
2
at 18 µM in 24 h and d in
4
8 h. The bar graph shows
DI comparison between EAC
cells treated with complex 2
at 9 µM, 18 µM and untreated
control after 24 and 48 h. Data
are shown as the mean± S.D.
*
*p<0.001 versus control. The
p value was determined by the
ANOVA test followed by the
Bonferroni multiple- compari-
son test
integrity marked with Annexin V-FITC, as well as when
only DNA content was marked by PI, followed by cells pre-
senting lower FSC/SSC (24.1% at 24 h and 26.5% at 48 h).
and fragmentation, followed by bleb formation and mem-
brane rupture (DC), or followed by a second stage, for loss
of cytoskeletal structure, dissolution of cellular content and
cytoplasmic content externalization (DD).
Morphological features
Western blotting
The percentage of cells was evaluated in Table 4S accord-
ing to features summarized in Fig. 5. After treatment
with complex 2, most cells found at 24 (DC: 49.9± 6.2%,
DD: 39.8 ± 6.7%) and 48 h (DC: 58.5 ± 4.6%, DD:
To identify crucial proteins associated with cell death
machinery initiated by this novel Ru (II) complex, we ana-
lyzed the protein expressions of eꢀector caspases (3 and
7) and initiator caspases (8 and 9). The expressions of
3
3.5 ± 3.8%) initially presented chromatin condensation
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JBIC Journal of Biological Inorganic Chemistry
Fig. 3 Complex 2 suppresses
ROS production induced by
MDA-MD-231 cells. The
images were acquired using
ꢂuorescence microscopy after
cell staining with ROS Fluores-
cent Probe-DHE. Panels (a, b)
refer to the representative image
of untreated cells for 24 h and
4
8 h, respectively. Panels c, d
refer to the representative image
MDA-MB-231 treated with 2
at 24 h and 48 h, respectively.
Graph e shows a comparison
between MDA-MB-231 cells
treated with 2 versus untreated
control after 24 and 48 h of
3
independent experiments.
Data are shown as mean± S.D.
*
*
p<0.01 versus control;
*p<0.001 versus control.
The p value was determined by
ANOVA
pro-apoptotic proteins Bax, Bak, Bim and Beclin-1 were
also analyzed.
well as the Bax and Bak proteins were not obtained (data
not shown).
Cells treated with complex 2 at 18 µM demonstrated an
increase in the expression of Beclin-1 (23.4% in 1 h and
3
4.4% in 2 h), Caspases-7 (17.4% in 1 h and 28.0% in 2 h),
Discussion
Caspase-3, (7.7% in 1 h and 43.8% in 2 h), p53 (32.7% in 1 h
and 26.0% in 2 h) and Bim (57.1% in 1 h and 70.7% in 2 h),
when compared to the untreated control as demonstrated in
Fig. 6. At 1 and 2 h, the expression of caspases 8 and 9, as
In this present study, we report on the synthesis, char-
acterization and cytotoxicity of two Ru/allo complexes
against breast cancer cells. As previously mentioned, the
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3

JBIC Journal of Biological Inorganic Chemistry
Fig. 4 EAC cells were evaluated using ꢂow cytometer using annexin
V-FITC/PI double staining. The percentages of gated cells are
described in representative experiments on panels a, b (control) and
treated cells with complex 2 at 18 µM on (c, d). Using a dot plot
graph, four diꢀerent cell phenotypes can be distinguished: annexin
V−/PI−(lower left quadrant, Q3); annexin V+/PI−, (lower right
quadrant, Q4); annexin V−/PI+(upper left quadrant, Q1), and
annexin V+/ PI+(upper right quadrant, Q2). Bar graphs (e, f) repre-
sent the results obtained in a percentage of cells considered as viable,
in early and late apoptosis and necrosis treated with complex 2 in 24
and 48 h. Data are shown as the mean± S.D. **p<0.001 versus con-
trol. The p value was determined by the ANOVA test followed by the
Bonferroni multiple- comparison test
1
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JBIC Journal of Biological Inorganic Chemistry
cytotoxicity and mechanism of action of metal complexes
can vary according to the coordination sphere of the metal
center and the structure of the complexes. Thus, the cyto-
toxicity of the Ru complexes against tumor cells was com-
pared with the metallodrug cisplatin [4, 7, 10, 38–41], a
commonly used chemotherapeutic drug to treat several types
of cancer, including breast cancer, including HaCat (nor-
mal human skin keratinocytes) [10] and MCF-10A (human
breast epithelial cell line) [7, 39]. Although both complexes
1
and 2 have similar chemical structures, our data indicate
that complex 2 is more cytotoxic against tumor cells than
non-tumor cells in a minor concentration, and comparing
values of IC and selectivity, complex 2 showed more sig-
5
0
niꢁcant activity against both tumor cells than complex 1 (see
Table 1). Moreover, complex 2 evaluated here showed to be,
in general, more cytotoxic for EAC [38] and MDA-MD-231
cells [39] than other Ru complexes [40–42].
Fig. 5 EAC cell images of untreated control and treated with complex
2
at 18 µM were captured during experimentation using ꢂuorescence
The two neutral complexes, 1 and 2, were obtained
from the precursors [RuCl (PPh ) ] and [RuCl (PPh )
microscope of triple channel and discriminated according to stain-
ing and morphology features: LN—Live cells show dispersed and
regular chromatin, intact membrane and stained with HO; LC—Early
apoptosis can present condensation/fragmentation/marginalization of
chromatin, intact or disturbance in membrane or bleb formation and
stained with HO/An-V FITC; DC—Late apoptosis shows karyorhexis
with membrane disruption and budding formation and stained with
HO/An-V FITC/PI; DD—Secondary necrosis shows karyorhexis/kar-
yolysis with membrane rupture, and stained with HO/An-V FITC/ PI
2
3 3
2
3
(
dppb)], respectively, by exchanging one PPh ligand
3
and coordinating two allo molecules as the monoden-
tate ligand. In the design of the complexes used in this
work, phosphines were used to stabilize the metal center
and to provide good hydrophobic properties for the
compounds. The chlorido ligands were used to neutral-
ize the charge of the Ru (II) ion. Thus, the proposed
Fig. 6 Expression of Beclin-1, Caspase-7, Caspase-3, p53 and Bim
proteins in Western blot analysis shows EAC untreated and treated
cells with complex 2, at 18 µM and times of 1 and 2 h of 2 independ-
ent experiments. The GAPDH expression was used as the control.
The bar graph shows the relative level (%) of proteins compared to
the control. Data are shown as mean± S.D. *p<0.01 versus control;
**p<0.001 versus control. The p value was determined by ANOVA
1
3

JBIC Journal of Biological Inorganic Chemistry
general formula is [RuCl (allo) (PPh ) ] for complex
We evaluated the stability of complexes 1 and 2 in
DMSO/culture media, in which complex 2 was slightly more
stable than complex 1 (see Figs. 15S/16S). We also studied
the hydrolysis of chloride by molar conductivity (Fig. 17S),
but no signiꢁcant changes were observed after 48 h. Com-
plex 1 may be more unstable because it presents all ligands
2
2
3 2
1
and [RuCl (allo) (dppb)] for complex 2. In the FT-IR
2
2
spectra of the complexes, the carbonyl stretching vibration
−
1
occurs in the range of 1710–1709 cm , meanwhile vC=O
−
1
stretching vibration of the ligand free is 1703 cm . This
very small shift suggests that carbonyl is not coordinated
to ruthenium.
3
1
as monodentate. As can be seen in Fig. 15S, the P NMR
The bands of the UV–Vis spectrum of complexes 1 and
signal at around -6 ppm suggests the labilization of the PPh
3
2
are assigned to the charge transfer from the Ru(II) atom
ligand. On the other hand, analyzing Fig. 16S, the singlet
related to complex 2 (close to 42 ppm) with high intensity
emphasizes that this complex is more stable. The stability
can be assigned to the presence of the dppb ligand as biden-
tate and it is not labilized given that there are no additional
to the allo and phosphine ligands. Complex 2 exhibits a
redox process in a slightly lower potential than complex 1,
probably because the PPh ligand acts as a slightly better
3
π–electron acceptor ligand when compared with the dppb
3
1
one. The same stereochemistry around metal in complexes
P NMR signals related to free diphosphine. Therefore,
31
1
1
and 2 can also be highlighted by the P{ H} NMR spectra
complex 2 was selected for more in-depth studies. Compar-
of complexes 1 and 2. The singlet signals at around 40 ppm
ing the cytotoxicity of both complexes (Table 1), compound
may indicate the magnetic equivalence of the two phospho-
2 is the most active one. Probably, the exchange of PPh in
3
rus atoms of PPh and dppb, respectively, which are trans
complex 1 may inactive this compound, suggesting that the
presence of the coordinated phosphine ligand in complex 2
plays a key role in the biological activity.
3
positioned to the nitrogen atom of the allo ligand. This same
behavior was also observed in a previous report with phos-
3
1
phorus atoms trans to N-heterocyclic ligands [43]. The
P
The MDA-MB-231 cell line is a highly metastatic breast
carcinoma and is characterized as a triple-negative pheno-
type by the absence of estrogen receptor (ER), progesterone
receptor (PR), human epidermal receptor 2 (HER-2) and
increased expression of ferritin levels [49, 50]. The clono-
genic assay has been considered useful in in vitro tests to
verify the survival of tumor cells and the maintenance of
their reproductive viability after exposure to a cytotoxic
agent [19]. While cell migration assay is a screening tool
used to evaluate antimetastatic properties [51]. Complex 2
inhibits cell survival ability of a single cancer cell to grow
into a colony (Fig. 18S) and cell migration (Fig. 19S) of
MDA-MB-231 cells. This is important information as the
metastasis involves a complex series of tumor process initia-
tion and metastatic development that begins from implanta-
tion and multiplication of single cells in a secondary site, far
from the primary tumor, giving rise to tumorigenesis [52].
Thus, based on our results it can be suggested that this Ru(II)
complex can become a novel antimetastatic agent.
NMR technique was used to evaluate the stability of the
complexes in DMSO and DMSO/H O solution, where both
2
of them showed to be stable for at least 48 h.
Complex 2 was also characterized by single-crystal X-ray
diꢀraction, revealing that the allo ligand is coordinated to
Ru by the N8 atom as the monodentate ligand. In the litera-
ture, this same behavior is observed for ꢁrst-row transition
metals [44]. Comparing molecular geometry of complex
2
with the free allo, the coordination to Ru leads to small
variations in the C–N, N=N and C=O bond lengths [45].
In free allo, the N8-N9 and C7-N8 bond lengths are 1.374
and 1.325 Å, respectively. As a result of coordination, in
the complex these two bonds present values of 1.339(9)
and 1.300(10) Å. As observed in Fig. 1, the ꢁve-membered
ring is involved in an intramolecular π–π interaction with
the adjacent phenyl group of the dppb ligand, stabilizing
complex 2 [Interplanar distance of 3.530 Å]. The crystal
self-assembly of Ru complex 2 is stabilized mainly by well
orientated intermolecular contacts, involving the N1–H1…
O6 atoms [distance of 1.915 Å] that form an inꢁnite chain
along the c axis (see Fig. 1S). In addition, this chain is con-
nected by a centrosymmetric H-bond involving the N9-H9…
N3 atoms with a separation of 2.525 Å.
The cellular mechanisms that lead to DNA fragmenta-
tion can be initiated by a very wide range of factors, both
external and internal [53]. The results of the cell cycle
kinetics and comet assay, but not the ROS detection test,
show the interaction of complex 2 with DNA. As can be
seen in Fig. 2, after exposure to complex 2 within 24 h,
there was no evidence of cell repair mechanism, since
complex 2 maintained the cell synthesis phase (S) inhib-
ited, and consequently the reduction of the G2/M phase.
The increase in cells in G0/G1 indicates that complex 2
can lead to the restriction of the cell cycle in the G0/G1
phase, where important parameters are established for the
advancement of the cell cycle, related to nutritional suf-
ꢁciency, disposition of growth factors, size, DNA damage
The interaction of both complexes with DNA was stud-
ied by UV–Vis spectrophotometric titration. DNA-binding
3
3
−1
constants, Kb, found (5.2×10 and 1.5×10 M for com-
plexes 1 and 2, respectively) suggest a moderate interac-
tion of the complexes with the ctDNA, such as non-covalent
ones including hydrogen bonds or hydrophobic interactions
[
46, 47]. In the literature, high binding constants at approxi-
6
−1
mately 10 M occur for classical intercalator agents, such
as for ethidium bromide [48].
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JBIC Journal of Biological Inorganic Chemistry
and other essential elements [54, 55]. However, DNA dam-
age is the most possible mechanism, considering results
with other Ru complexes that showed the same cell cycle
behavior [40, 81]. Other studies with Ru complexes dem-
onstrate that part of its mechanisms of action refers to
direct and indirect DNA-targets [9, 56].
For the morphological characteristic tests, we used triple
dyes. HO dye is a blue, ꢂuorescent dye that binds speciꢁ-
cally and quantitatively to DNA. The more the chromatin is
condensed, the greater the ꢂuorescence observed; while HO
is captured by living cells, PI is internalized only by dead
cells. The PI dye also binds to nucleic acid and hence is
commonly used as DNA staining, emitting red ꢂuorescence
[64]. A disturbance in membrane phospholipid symmetry
carries to the phosphatidylserine externalization (PS) from
the inner membrane layer, which can be labeled by Annexin
V-FITC in green ꢂuorescence [64]. The morphologic tech-
nique using triple dyes was relevant to allow a visual deter-
mination of key cellular characteristics and discrimination
between early and late apoptosis (secondary necrosis) [30].
This method has been described for the ꢁrst time using Ru
complexes.
The comet assay is a common test used to measure
DNA damage and repair [57]. According to the results, the
progression of DNA damage seems to be related to time
and compound concentration. An increase in DNA dam-
age assessed by DI induced by complex 2 was observed in
Fig. 2d for both times and concentrations utilized; these data
demonstrate that complex 2 can interact with DNA, leading
to chromatin fragmentation as shown in Fig. 2.
According to some authors, a comet population can be
distinguished by the shape of the comet and by quantitative
parameters such as tail and head sizes. Comets originated
from apoptotic cells usually presenting large tails and small
heads [34, 35].
The results for the cell death proꢁle developed after treat-
ments with complex 2, found in the morphological assay
(Fig. 5), conꢁrm the results of Annexin V-FITC staining
by ꢂow cytometry, showing a regulated cell death proꢁle
consistent with the late apoptosis aspects for both treatment
times.
The low level of intracellular ROS was measured in breast
cancer cells after exposure to complex 2 (Fig. 3). Although
the relationship between the ROS and XO levels is not clear
here, these results suggest that low ROS levels seem to be
related to the ability of the Ru/allo complexes to selectively
inhibit XO, as previously reported [15]. Although many
studies have revealed that ROS are implicated in the under-
lying mechanisms of DNA damage, currently new ꢁndings
have revealed that ROS in a dose-dependent manner contrib-
utes to inhibiting distinct signaling pathways associated with
cancer pathogenesis [58] and chemoresistance. In fact, while
there is some evidence showing that high ROS levels are
associated with resistance to chemotherapy [59], preclinical
studies have indicated that antioxidant drugs when combined
with chemotherapic agents may act synergistically, decreas-
ing tumor progression compared to either drug individually
Previous reports indicate that apoptosis is the cell death
type most described for Ru(II) compounds [65, 66]. The
apoptosis is triggered by two major pathways: extrin-
sic (death receptors) and intrinsic (mitochondrial content
release). Through both signaling pathways, it is well known
that the caspase family plays the main role in apoptosis exe-
cution. However, other relevant proteins are also involved in
the apoptotic cascade [32].
Our ꢁndings indicate that complex 2 triggers apoptosis
by multiple pathways. The overexpression of Beclin-1 and
Bim becomes a new feature in apoptotic Ru (II) compounds
(Fig. 6). Members of the BH3-only protein family, Bim,
and Beclin-1, may play a role in inhibiting autophagy and
promoting the ampliꢁcation of apoptogenic signals through
caspase cleavage and outer mitochondrial membrane per-
meabilization [67–69]. For some authors, Beclin-1 has
been considered a mediator agent between apoptosis and
autophagy [68–71]. Overexpression of p53 is generally pri-
marily correlated with DNA damage, mediating the mito-
chondrial pathway apoptotic chain and cell cycle arrest [72,
73]. In some way, the upregulation of p53 is directly or indi-
rectly linked to Bim, Beclin-1 and caspases-3 and 7 induc-
tion [74–76]. On the other hand, these caspases promote
Beclin-1 cleavage and this generated C-terminal fragments,
possibly accumulated in the mitochondria, promoting the
release of cytochrome c and allowing the formation of apop-
tosomes. All these factors together possibly generate positive
feedback and ampliꢁcation of apoptotic events.
[
60, 61].
Supporting this idea, a study in vitro with cancer cells
showed a consistent argument in the thiopurine-induced
cytotoxicity by co-administration with allopurinol [62]. In
agreement with our ꢁndings, a previous report in human
breast cancer cells demonstrated that allopurinol signiꢁ-
cantly reduced the H O levels XO-generated, but it did not
2
2
prevent either DNA fragmentation or contribute to a pro-
tective eꢀect against cell death [14]. Thus, these results are
consistent, conꢁrming our ꢁndings reported here and sup-
porting the hypothesis that allopurinol might, at least in part,
contribute to Ru-induced apoptotic eꢀects.
Analyzing the flow cytometry results (Fig. 4), we
observed a rapid induction of late apoptosis, before 24 h,
which may be related to the time of action of complex 2 and
the type of cell treated, because the time of onset from early
apoptosis to induction until its ꢁnal stage (late apoptosis)
may depend on several factors [63].
In addition, several studies disclosed that Beclin-1 acts
as a tumor suppressor and is reduced in some human breast
carcinomas [77–79]. In addition, the role of Bim in the
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JBIC Journal of Biological Inorganic Chemistry
for complex 2. X-ray data showed that phosphorus atoms
from the diphosphine are trans positioned to the nitrogen
atoms of allo ligand, while the chlorido is trans to chlorido.
The compounds had their cytotoxicity evaluated in vitro
against breast tumor cells, showing good activity. Complex
2
showed a more potent antiproliferative response in breast
cancer, probably by its interaction with DNA, promoting
DNA damage, cell cycle arrest in the early stage of repli-
cation, and morphological and biochemical breast cancer
cell changes compatible with apoptosis features. Although
the apoptotic mechanisms involving Bim and Beclin-1 have
not yet been fully elucidated, according to the results, we
suggest that complex 2 may act as a target drug for BH3-
only proteins, signaling intrinsic pathway events. The over-
expression of p53 after treatment with complex 2 was not
unexpected due to its capacity to promote DNA damage.
Moreover, upregulation of p53 can be linked to low ROS
levels, Bim, Beclin-1, and caspases-3 and -7 induction. All
these factors together possibly generate a kind of positive
feedback and ampliꢁcation of apoptotic events (Fig. 7).
Thus, the characteristics attributed to complex 2 may have
promising results in antitumor therapy, in which the activa-
tion and increase of pro-apoptotic factors are involved in the
tumorigenesis inhibition process.
Fig. 7 Complex 2 inducing apoptosis by multiple targets. The tumor
cell interaction with complex 2 triggers DNA damage with subse-
quent upregulation of p53. The p53 promotes inhibition of cell rep-
lication with cycle arrest in G0/G1 and leads directly or indirectly to
Bim, Beclin-1 and caspases-3 and 7 overexpression. Once activated,
caspases-3 and 7 cleave Beclin-1 protein. The Beclin-1 C-fragment
(
Beclin-1C) accumulates in mitochondria producing in collaboration
with Bim the release of cytochrome c (Cyt c) and apoptosome forma-
tion. The apoptosome activates the executor caspases (caspases-3 and
7
) expanding the apoptogenic signals
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s00775-021-01862-y.
pro-apoptotic response of breast cancer to chemotherapeutic
agents is well understood through the study of the mecha-
nism of action of Paclitaxel, Geꢁtinib and Erlotinib in Bim
upregulation [80]. Therefore, activating the Bim and Bec-
lin-1 pathways, cooperating together, may provide a new
generation of target anticancer agents.
Acknowledgements This work was supported by the Brazilian
National Counsel of Technological and Scientiꢁc Development (CNPq)
(Grant numbers 403588/2016-2 and 308370/2017-1) and the Founda-
tion for Research Support of the State of Minas Gerais (FAPEMIG)
(
Grant number APQ-01674-18).
Authors contributions IOT: conceptualization, methodology, formal
analysis, investigation, writing, and visualization; FM-A: conceptual-
ization, methodology, investigation, writing, and visualization; RPC:
investigation and formal analysis; WCP: investigation and formal anal-
ysis; PFFdS: investigation and formal analysis; RSC: conceptualiza-
tion, methodology, formal analysis, investigation, writing, and funding
acquisition; TT: methodology, formal analysis, investigation; AM-O:
conceptualization, methodology, formal analysis, and writing; AAB:
conceptualization, methodology, writing, and resources; EdPS-L: con-
ceptualization, project administration, and resources.
In our study, we observed changes in the DNA mole-
cule caused by complex 2 that led to DNA fragmentation,
observed by apoptotic characteristics, cell cycles arrested in
the G0/G1 phase (Fig. 20S) and by the comet assay (Fig. 2).
Altogether our ꢁndings suggest p53-mediated apoptosis due
to cell cycle arrest, DNA damage (Fig. 2), and pro-apoptotic
protein induction (Fig. 6 and Table 4S). Although the anti-
tumor mechanisms of the Ru/allo complexes are still under
investigation, our results suggest that complex 2 is a prom-
ising candidate for preclinical antitumor and antimetastatic
studies, due to its action on multiple intracellular targets.
Declaration
Conflict of interest The authors declare that they have no known com-
peting ꢁnancial interests or personal relationships that could have ap-
peared to inꢂuence the work reported in this paper.
Conclusions
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Authors and Aꢀliations
Ingrid O. Travassos1 · Francyelli Mello‑Andrade
1,2
· Raíssa P. Caldeira · Wanessa C. Pires ·
1
1
1
3
3
4
5
Paula F. F. da Silva · Rodrigo S. Correa · Tamara Teixeira · Alisson Martins‑Oliveira · Alzir. A. Batista ·
Elisângela P. de Silveira‑Lacerda1
3
*
Elisângela P. de Silveira-Lacerda
silveiralacerda@gmail.com
Department of Chemistry, Federal University of Ouro
Preto—UFOP, Ouro Preto, MG 35400-000, Brazil
4
5
UniEvangelica University Center, Anapolis, GO 75083-515,
Brazil
1
Laboratório de Genética Molecular E Citogenética Humana,
sala 213, Departamento de Genética, Instituto de Ciências
Biológicas I, Campus Samambaia, Universidade Federal
de Goiás, Avenida Esperança, s/n, Cx Postal: 131, Goiânia,
Goiás CEP 74690-900, Brazil
Department of Chemistry, Federal University of Sao
Carlos—UFSCar, Sao Carlos, SP 13565-905, Brazil
2
Department of Chemistry, Federal Institute of Education,
Science and Technology of Goiás, Goiânia,
Goiás 74055-110, Brazil
1
3