Vol.:(0123456789)
1 3
JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-019-01644-7
ORIGINAL PAPER
6‑Methoxyquinoline complexes aslung carcinoma agents: induction
ofoxidative damage onA549 monolayer andmulticellular spheroid
model
J.F.Cadavid‑Vargas
1,2
· C.Villa‑Pérez
1
· M.C.Ruiz
1,2
· I.E.León
1
· G.C.Valencia‑Uribe
3
· D.B.Soria
1
·
S.B.Etcheverry
1,2
· A.L.DiVirgilio
1,2
Received: 9 November 2018 / Accepted: 17 January 2019
© Society for Biological Inorganic Chemistry (SBIC) 2019
Abstract
The aim of this work was to study the antitumor effects and the mechanisms of toxic action of a series of 6-methoxyquinoline
(6MQ) complexes invitro. The Cu(II) and Zn(II) complexes (Cu6MQ and Zn6MQ) are formulated as M(6MQ)
2
Cl
2
; the
Co(II) and Ag(I) compounds (Co6MQ and Ag6MQ) are ionic with formulae [Ag(6MQ)
2
]
+
NO
3
−
and H(6MQ)
+
[Co(6MQ)
Cl
3
]
−
(where H(6MQ)
+
is the protonated ligand). We found that the copper complex, outperformed the Co(II), Zn(II) and
Ag(I) complexes with a lower IC
50
(57.9µM) in A549 cells exposed for 24h. Cu6MQ decreased cell proliferation and
induced oxidative stress detected with H
2
DCFDA at 40µM, which reduces GSH/GSSG ratio. This redox imbalance induced
oxidative DNA damage revealed by the Micronucleus test and the Comet assay, which turned into a cell cycle arrest at G2/M
phase and induced apoptosis. In multicellular spheroids, the IC
50
values tripled the monolayer model (187.3µM for 24h). At
this concentration, the proportion of live/dead cells diminished, and the spheroids could not proliferate or invade. Although
Zn6MQ also decreased GSH/GSSG ratio from 200µM and the cytotoxicity is related to oxidative stress, the induction of the
hydrogen peroxide levels only doubled the control value. Zn6MQ induced S phase arrest, which relates with the increased
micronucleus frequency and with the induction of necrosis. Finally, our results reveal a synergistic activity with a 1:1 ratio
of both complexes in the monolayer and multicellular spheroids.
Keywords 6-Methoxyquinoline complexes· Lung carcinoma· A549 cells· Multicellular spheroid model· Oxidative
damage
Introduction
The constant worldwide expansion of a disease such as
cancer is challenging the research and development of new
drugs and is pushing scientists to find new and creative ways
to fight against tumor development.
Oxidative damage is considered a potential therapeutic
approach for the development of novel ROS-based anti-
cancer agents. It is very well established that cancer cells
display an altered metabolism with hallmarks such as an
increase in the glucose uptake, increase in lactate synthe-
sis, and an altered redox homeostasis level [1, 2]. In fact,
tumor cells have higher levels of endogenous reactive oxy-
gen species (ROS) than normal cells, and this difference
makes them more vulnerable to ROS-induced injury [3].
Therefore, further oxidative stress induced by exogenous
agents is a strategy to selectively inhibit tumor prolifera-
tion without producing significant toxicity to normal cells
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0077 5-019-01644 -7) contains
supplementary material, which is available to authorized users.
* A. L. Di Virgilio
aldivirgilio@biol.unlp.edu.ar
1
CEQUINOR (CONICET-UNLP), Bv. 120N 1465, LaPlata,
Argentina
2
Facultad de Ciencias Exactas, Universidad Nacional de La
Plata, 47 y 115, 1900LaPlata, Argentina
3
GIAFOT, Departamento de Química, Facultad de Ciencias,
Universidad Nacional de Colombia-Sede Medellín, Medellín,
Colombia
JBIC Journal of Biological Inorganic Chemistry
1 3
[4]. Growing evidence suggests that increased amounts of
ROS can trigger oxidative damage to lipids, proteins, and
DNA. Severe permanent DNA injury leads to a mitotic
catastrophe, which may then be followed by apoptosis or
necrosis [5].
On the other hand, epigenetic control reversibly influ-
ences on the onset and progression of cancer [6]. This
reason led to the development of new drugs that target
histone deacetylases [6]. In fact, it has been reported that
these enzymes may act as oncogenes since they have been
found overexpressed in solid tumors and it is a point of
vulnerability for cancer cells [6]. Indeed, histone deacety-
lases inhibition significantly alters tumor cells, inducing
cell cycle arrest, differentiation, cell death, reduction of
angiogenesis and also can induce an increase in the level
of intracellular oxygen reactive species [7, 8]. Moreover,
it has been highlighted that histone deacetylase inhibitors
provoke genomic instability contributing to the cytotoxic
effects of these drugs [9].
Many quinoline-based drugs that have been used in the
treatment of malaria, arthritis, and lupus, showed to inhibit
histone deacetylase activity [10, 11]. In addition, it has been
demonstrated that quinolines induce DNA damage and apop-
tosis [10] and display antiproliferative activity in invitro and
invivo systems [11, 12] Significant oxidative stress induced
in cells by quinolone derivatives might contribute to the anti-
tumor effect [13, 14].
Previously, it has been reported the synthesis, thermal,
spectral and magnetic studies of metal coordination com-
pounds with 6-methoxyquinoline (6MQ) as ligand [15, 16].
Moreover, the crystal structure of many complexes with
6-methoxyquinoline as ligand and transition metals have
been recently reported by some of us [17]. The synthesis of
these complexes has been undertaken since it is known that
coordination with metals may reinforce therapeutic activity
of the compounds or may allow the acquisition of beneficial
actions. These complexes have shown to improve the anti-
bacterial effect on Gram-positive and Gram-negative bac-
teria after complexation, although nothing is known about
their activity as anticancer drugs [17].
On these bases, we are interested to evaluate if the com-
plexation process of 6-methoxyquinoline with Ag(I), Co(II),
Cu(II) and Zn(II) generates compounds with antitumor activ-
ity for lung carcinoma. Our study was carried out on mon-
olayer and in a multicellular spheroid model of human lung
carcinoma A549 cells, considering cell viability as a starting
point to study, and the mechanisms of action involved in
their antiproliferative effects. We focused our attention on
the role of oxidative stress, and the cytotoxicity and geno-
toxicity actions of Cu(II) and Zn(II) complexes (Cu6MQ and
Zn6MQ) whose formula is M(6MQ)
2
Cl
2
(Fig.1 shows the
crystallographic structure) since these two resulted to be the
most active and to differ from the cation effect.
Materials andmethods
Materials
Tissue culture materials were purchased from Corning
(Princeton, NJ, USA) and APBiotech (Buenos Aires,
Argentina), Dulbecco’s modified Eagle medium (DMEM),
TrypLE™ from Gibco (Gaithersburg, MD, USA), and
fetal bovine serum (FBS) from Internegocios SA (Bue-
nos Aires, Argentina). 2′,7′-Dichlorodihydrofluorescein
diacetate (H
2
DCFDA) and dihydroethidium (DHE) were
obtained from Molecular Probes
®
(Eugene, OR, USA).
Annexin V, Fluorescein isothiocyanate (FITC), propid-
ium iodide (PI) were bought from Invitrogen Corporation
(Buenos Aires, Argentina). Reduced glutathione (GSH),
o-phthalaldehyde (OPT), n-ethylmaleimide (NEM), vita-
min E (α-tocopherol), cytochalasin and the agaroses were
acquired from Sigma Aldrich (St. Louis, MO, USA). Vita-
min C (ascorbic acid) from Merck (Buenos Aires, Argen-
tina). Fluorescein diacetate and Resazurin sodium salt
were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). A549 (CCL-185) and MRC-5 (CCL-175)
cell lines were purchased from ATCC
®
.
Synthesis oftransition metal complexes
of6‑methoxyquinoline andaqueous stability
Four monomeric complexes of Cu(II), Zn(II), Co(II)
and Ag(I) with 6-methoxyquinoline (6MQ) as ligand
have been prepared and identified according to Villa-
Pérez etal. [17]. The Cu(II) and Zn(II) complexes are
formulated as Cu(6MQ)
2
Cl
2
and Zn(6MQ)
2
Cl
2
; the
Co(II) and Ag(I) compounds are ionic with formulae
[Ag(6MQ)
2
]
+
NO
3
−
and H(6MQ)
+
[Co(6MQ)Cl
3
]
−
(where
H(6MQ)
+
is the protonated ligand). Hereafter, the com-
pounds will be referred as Cu6MQ, Zn6MQ, Co6MQ, and
Ag6MQ, respectively.
Aqueous stability in 1000µM solutions of the com-
plexes was measured in phosphate-buffered saline (PBS)
using a Shimadzu UV–Vis spectrophotometer UV-2600
Fig. 1 ORTEP plots of Cu6MQ and Zn6MQ
JBIC Journal of Biological Inorganic Chemistry
1 3
in the range 200–400nm every hour for 24h. To ensure
the stability in biological conditions, 250µM solutions
of Cu6MQ and Zn6MQ were prepared in DMEM and the
spectra were recorded in the range 200–400nm every hour
for 24h as well. The UV spectra were analyzed using the
software SpectraGryph (version 1.2.7, Oberstdorf, Ger-
many), determining the area under the curve in the whole
range, followed by the evaluation of the change of area
compared with the area at time 0.
Cell culture (monolayer andmulticellular spheroids)
The A549 human lung carcinoma (passages 15–35) and
MRC-5 normal lung fibroblasts (passages 5–10) cell lines
were cultured in DMEM supplemented with 10% FBS, 100
U/mL penicillin, and 100µg/mL streptomycin at 37°C in a
humidified atmosphere with 5% of CO
2
. Cells were seeded
in a T75 flask, and when 80–90% of confluence was reached,
cells were subcultured using TrypLE™.
Experiments were carried out in multiwell plates, where
cells were allowed to attach and were washed with DMEM
before each treatment.
A549 carcinoma multicellular spheroids (MCS) were
cultured using the liquid overlay method [18]. Briefly, a 96
wells plate was coated with 50µL 1% (w/v) sterile agarose
in PBS; the gel was allowed to solidify for 20min. 10
4
cells/
mL (150µL) were seeded in each well and incubated at
37°C. Half of the culture medium was replaced with com-
plete fresh medium every other day. On the eighth day, MCS
reached an average diameter between 350 and 400µm and
were suitable to be treated with the complexes [19].
Cell viability assay
Monolayer cell viability was determined using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), which is reduced by mitochondria in viable
cells to a purple formazan dye [20]. Briefly, 2.5 × 10
4
cells
were seeded on 96-well plates and incubated at 37°C. After
24h, cells were exposed to different dilutions of each com-
plex, metallic salt, and ligand for 24, 48 or 72h. Afterward,
the monolayers were washed and incubated with 0.5mg/mL
of MTT in DMEM for 3h. The absorbance of the formazan
extracted with DMSO (100 µL/well) was recorded at a wave-
length of 570nm using a multiplate reader Multiskan FC
(Thermo Scientific). The cell viability is shown graphically
as a percent of the control value (cells treated with DMSO
as vehicle).
To evaluate the role of ROS levels on cell viability, a mix-
ture of 50µM ROS scavengers (vitamin C and E) was simul-
taneously added to the culture medium with the complexes.
After the incubation, the cell viability was determined by the
MTT assay as previously described.
With the goal of achieving a complete outlook of the
harmful effect exerted by the complexes, cell morphology
was also studied. A549 cells were cultured in 6-well plates
(2.5 × 10
5
cells/well), and different concentrations of the
complexes were added for 24h. To observe cell morphologi-
cal changes, the monolayer was fixed with absolute ice-cold
methanol for 5min and stained with Giemsa (1:20 in PBS).
The morphological changes were recorded using an inverted
microscope Olympus BX-51 coupled to a digital camera.
Clonogenic assay
To explore if the compounds affect cell proliferation, a
clonogenic assay was conducted according to [21]. 5 × 10
2
exponential growing A549 cells were plated on 6-well plates
and allowed to attach overnight under standard culture
conditions. The cells were washed twice with sterile PBS
and treated with the complexes for 24h. Next, cells were
washed with PBS twice and incubated with complete culture
medium for 10days. Fixation and the staining process were
conducted with glutaraldehyde 6.0% (v/v) and crystal violet
0.25% (w/v). Colonies formed by more than 50 cells were
recorded for the calculations. The surviving fraction of cells
was plotted versus concentration.
Oxidative stress determination
Reduced (GSH) and oxidized (GSSG) glutathione lev-
els were determined as described by Hissin and Hilf [22].
Confluent A549 monolayer cultured in 24-well plates were
treated with different concentrations of Cu6MQ and Zn6MQ
for 6 and 24h. Then, the monolayer was washed with PBS,
and the cells were lysed with 250µL 0.1% Triton X-100
for 30min at 4°C. For GSH determination, 100µL of the
cellular lysate were added to 1.8mL of ice-cold phosphate
buffer (Na
2
HPO
4
0.1M EDTA 0.005 pH 8.0) and 100µL
o-phthaldialdehyde (OPT) (0.1% in methanol). For the deter-
mination of GSSG, 100µL of the cell lysate were mixed
with 20µL 0.04M of N-ethylmaleimide (NEM) for 20min
at 4°C, then 1.8mL of NaOH 0.1M and 100µL OPT 0.1%
were added. Fluorescence was registered using a fluorometer
Shimadzu RF-6000, the samples were excited at 350nm, and
the emission signal was acquired at 420nm. GSH/GSSG
ratio was calculated as % of the basal for all the experimental
conditions.
Transition metal complexes were tested for reactive oxy-
gen species (ROS) induction as a mechanism of death cell
by flow cytometry. 3 × 10
5
A549 cells were seeded in 12
well plates and incubated overnight. The culture medium
was replaced with different concentrations of the complexes
for 24h. H
2
O
2
0.75mM for 20min was employed as a posi-
tive control. Then, the cellular monolayer was washed with
PBS and detached with Tryple. The cells were centrifuged,
JBIC Journal of Biological Inorganic Chemistry
1 3
and the pellet was incubated with DHE or H
2
DCFDA
(0.8µM) protected from light for 30min. Afterward, cells
were washed twice with PBS, resuspended in 250µl PBS
and transferred to flow cytometry tubes. 2 × 10
4
events were
acquired in FL1 for H
2
DCFDA, or FL2 for DHE using a BD
FACscalibur™ flow cytometer (BD Biosciences, USA) and
further analyses were performed using FlowJo 7.6 software.
Apoptosis
Cells going through different stages of apoptosis were
detected with Annexin V–FITC and propidium iodide (PI)
staining by measuring the externalization of phosphatidyl-
serine (PS) and the cellular membrane integrity, respectively.
Cells exposed to different concentrations of Cu6MQ and
Zn6MQ for 24h were detached using Tryple™ and cen-
trifuged at 2500 RPM for 5min. Afterward, the cellular
pellet was resuspended in 100 µL of binding buffer, and 2
µL of Annexin V-FITC were added, cells were incubated for
20min at room temperature protected from light, and before
de measurement 1µL of PI (50µM) was added. For each
sample, 2 × 10
4
events were analyzed using a BD FACscali-
bur™ flow cytometer (BD Biosciences, USA) and further
analyses were performed using FlowJo 7.6 software.
Cell cycle
DNA content in G1/G0, S, and G2/M phases was analyzed
using flow cytometry. Cells were seeded on 6-well plates and
treated with different concentrations of Cu6MQ and Zn6MQ
for 24h. The harvested cells were washed with PBS, fixed
and permeabilized with 70% ice-cold ethanol for 2h. After-
ward, cells were suspended in 300 µL staining buffer (PBS/
EDTA 2mM, pH 8.0) and 15 µL of RNAse (1mg/mL) and
incubated at 37°C for 15min. Cells were stained with PI (15
µL of a solution 1mg/mL) overnight at 8°C. 10
4
single cells
were analyzed with a BD FACscalibur™ flow cytometer;
histograms depicted the relative DNA distribution within
each sample. The percentage of cells in the G1/G0, S, G2/M
phases and the sub-G1 peak was then calculated using the
cell cycle analysis module in the FlowJo 7.6 software.
Genotoxicity studies
The cytokinesis-block micronucleus (MN) assay was set up
with cultures in the log phase of growth. A549 cells were
seeded onto 6-well plates and incubated at 37°C for 24h.
Then, the cells were treated with different concentrations of
the complexes along with cytochalasin B (4.5μg/mL). After
24h, cells were rinsed and subjected to hypotonic conditions
with 0.075% KCl at 37°C for 5min, fixed with pure metha-
nol at − 20°C for 10min and stained with a 5% Giemsa
solution. For the MN assay, 500 binucleated (BN) cells were
scored at 400× magnification per experimental point from
each experiment. The examination criteria employed were
reported by Fenech [23]. A pulse of 30min of 0.5μg/mL
bleomycin was employed as the positive control.
For detection of DNA damage, the single cell gel elec-
trophoresis assay (Comet assay) was employed based on
the method of Singh etal. [23] with minor modifications.
Briefly, A549 cells were treated with different concentra-
tions of the complexes. After 24h, cells were suspended
in 0.5% low melting point agarose and immediately poured
onto microscope slides precoated with 0.5% normal melting
point agarose. Two slides were prepared for each condition;
one slide was used to observe single-strand DNA breaks and
the other, to obtain information on the presence of oxidized
DNA bases using digestion with the enzyme EndoIII [23].
Slides were immersed in ice-cold lysis solution (2.5M NaCl,
100mM Na2–EDTA, 10mM Trizma–HCl, pH 10 and 1%
Triton X-100, 10% DMSO at 4°C, pH 10) for 1h to lyse the
cells, remove cellular proteins and to allow DNA unfolding.
After that, the slides were washed three times (5min each
time) with enzyme buffer (0.1M KCl, 0.5mM Na
2
–EDTA,
40mM HEPES–KOH, 0.2mg/ml BSA, pH 8.0) and incu-
bated for 45min at 37°C with EndoIII in the enzyme buffer
or with buffer alone. Then, the slides were placed on a hori-
zontal gel electrophoresis tank, and the DNA was allowed
to unwind for 20min in freshly prepared alkaline electro-
phoresis buffer (300mM NaOH and 1mM Na
2
-EDTA, pH
12.7). Electrophoresis was carried out in the same buffer for
30min at 25V (≈ 0.8V/cm across the gels and ≈ 300mA)
in an ice bath condition. Afterward, slides were neutralized
and stained with Syber Green. The analysis was performed
in an Olympus BX50 fluorescence microscope. A total of
150 randomly captured cells per experimental point were
used to determine the tail moment using Comet Score ver-
sion 1.5 software. A pulse of 20min of 10μg/mL bleomycin
just before the cells were harvested was employed as the
positive control.
Multicellular spheroids (MCS) viability assay
The spheroid viability was assessed using the resazurin dye,
which is irreversibly reduced by intracellular oxidoreduc-
tases to a pink-red fluorescent dye known as resorufin [24].
The spheroids were cultured as described and incubated with
different concentrations of the complexes for 24 or 48h.
After the exposure, the medium was replaced with 50µM
resazurin solution in DMEM, and the spheroids were incu-
bated overnight at 37°C. Fluorescence was registered using
a fluorometer Shimadzu RF-6000 (excitation at 570nm,
emission at 585nm). Results were corrected by subtraction
of the fluorescence of resazurin and DMEM alone incubated
under the same conditions. Cell viability was plotted as a
percentage of the basal condition (solvent control).
JBIC Journal of Biological Inorganic Chemistry
1 3
Moreover, morphological changes were studied with
a live–death cell staining. Multicellular spheroids treated
with different concentrations of the complexes were incu-
bated for 24 or 48h and stained with fluorescein diacetate
(8 × 10
−3
mg/mL) and propidium iodide (2 × 10
−2
mg/mL).
The spheroids were incubated in the dark for 5min at room
temperature. The fluorescence was registered using an epi-
fluorescence inverted microscope Nikon Ti Eclipse with
FITC and Texas Red filters. The raw images were processed
using ImageJ
®
software, and composite RGB images were
obtained.
Multicellular spheroids spreading assay
To evaluate if the cells in the spheroids can migrate and
proliferate after the exposure to Cu6MQ and Zn6MQ for
24h, the spheroids were transferred into a 96-well plate
containing 150µL of DMEM supplemented with 10% FBS
and were incubated at 37°C in a humidified atmosphere with
5% of CO
2
. The development of outward cellular projec-
tions from the spheroids into the well surface was registered
through phase contrast microscopy after 24 and 72h.
Synergistic calculations
To determine the existence of a synergistic effect between
Cu6MQ and Zn6MQ on A549 cells, the concentration fixed
ratios 1:1, 1:3 and 1:4 of the complexes were tested. Fol-
lowing the same procedure applied in the cell viability assay
(see “Cell viability assay”), and the data were analyzed using
the Chou–Talalay method through the CompuSyn software.
The results are expressed as the combination index (CI):
synergistic effect (CI < 1), additive effect (CI = 1) and antag-
onism effect (CI > 1) [5].
Statistical analysis
Results are expressed as the mean of three independent
experiments and plotted as mean ± standard error of the
mean (SEM). The total number of repeats (n) is specified in
the legends of the figures. The Tukey test (two way ANOVA)
was employed to compare means in all the experiments
performed.
Results
Stability ofthecomplexes
The stability of the complexes was evaluated using UV–Vis
spectroscopy (Fig.1 from Supplementary Material shows
the electronic absorption spectra of Cu6MQ and Zn6MQ
in DMEM and Co6MQ and Ag6MQ in PBS). After 24h
in PBS, all the complexes kept their spectroscopic charac-
teristics and showed a degradation rate below the 10%. The
stability follows: Cu6MQ = Ag6MQ > Zn6MQ> Co6MQ
(Fig.2A from Supplementary Material). Moreover, in bio-
logical conditions, Cu6MQ and Zn6MQ remain stable for
24 h (Fig.2B from Supplementary Material).
Eect of6‑methoxyquinoline complexes oncell
viability
Results from the MTT assay (Fig.2a) in A549 cell line
show that Co6MQ caused no harmful effect on the tumor
cells, and Ag6MQ was the most active antiproliferative
compound. However, despite the remarkable effect of
the silver compound, it did not show a differential cyto-
toxic effect when compared to the cation Ag
+
in the same
range of concentrations (data not shown). On the other
Fig. 2 a Effect of Cu6MQ, Zn6MQ, Co6MQ and Ag6MQ on A549
cell viability. Cells were incubated alone (control) or with differ-
ent concentrations of the compounds at 37 °C for 24h. The results
are expressed as the percentage of the basal level and represent the
mean ± the standard error of the mean (SEM) (N = 9). Asterisks
represent a statistically significant difference in comparison with
the basal level *(p < 0.05) **(p < 0.001). b Differential behavior of
Cu6MQ and Zn6MQ on A549 and MRC-5 cell viability. The results
are expressed as the percentage of the basal level and represent the
mean ± SEM (N = 9). Asterisks represent a statistically significant dif-
ference in comparison with the basal level *(p < 0.05) **(p < 0.001).
Number sign (#) represents a statistically significant difference when
the same complex concentration is evaluated on A549 and MRC-5
cell lines (p < 0.05)
