Synergistic effect of pyrazoles derivatives and doxorubicin in claudin-low breast cancer subtype

Article abstract of DOI:10.1016/j.biopha.2017.12.062

Background Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the prognosis is poorer and the treatment is still a challenge. Pyrazoles are an important class of heterocyclic compounds and are promising anticancer age

Full text of DOI:10.1016/j.biopha.2017.12.062

Biomedicine&Pharmacotherapy98(2018)390–398
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Synergistic effect of pyrazoles derivatives and doxorubicin in claudin-low
breast cancer subtype
Silvia Saueressiga,b, Josiane Tessmanna,b, Rosiane Mastelaria,b, Liziane Pereira da Silvaa,b
,
Julieti Bussb, Natalia Vieira Segattob, Karine Rech Begninia,b, Bruna Pachecob,
⁎
Cláudio Martin Pereira de Pereirac, Tiago Collaresa,b,c, Fabiana Kömmling Seixasa,b,
a
Programa de Pós-Graduação Em Biotecnologia (PPGB), Biotecnologia/Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil
b
Grupo de Pesquisa Em Oncologia Celular E Molecular (GPO), Laboratório de Biotecnologia Do Câncer, Centro de Desenvolvimento Tecnológico, Universidade Federal de
Pelotas, Pelotas, RS, Brazil
c
Programa de Pós-Graduação Em Bioquímica E Bioprospecção, UFPel, Pelotas, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Triple negative
Claudin-low
Pyrazole
Bromine
Chlorine
Background: Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the
prognosis is poorer and the treatment is still a challenge. Pyrazoles are an important class of heterocyclic
compounds and are promising anticancer agents based on their chemical properties. The present study was
aimed not only at testing pyrazoles previously prepared by our research group in two breast cancer cell lines
characterized by intermediated response to conventional chemotherapy but also at analyzing the possible sy-
nergistic effect of these pyrazoles associated with doxorubicin.
Methods: Four 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles were tested for the first time in MCF-7 and
MDA-MB-231 culture cells. The pyrazoles with best results in cytotoxicity were used in combination with
doxorubicin and compared with this drug alone as standard. The synergic effect was analyzed using Combination
Index method. In addition, cell death and apoptosis assays were carried out.
Results: Two pyrazoles with cytotoxic effect in MCF-7 and especially in MDA-MB-231 were identified. This
activity was markedly higher in pyrazoles containing bromine and chlorine substituents. The combination of
these pyrazoles with doxorubicin had a significant synergic effect in both cells tested and mainly in MDA-MB-
231. These data were confirmed with apoptosis and cell death analysis.
Conclusions: The synergic effect observed with combination of these pyrazoles and doxorubicin deserves special
attention in Claudin-low breast cancer subtype. This should be explored in order to improve treatment results
and minimize side effects.
1. Introduction
are found into the bigger subset of triple negative tumors which lack the
expression of estrogen, progesterone receptor (ER/PR) and do not ex-
Breast cancer remains a disease of high prevalence and is the most
frequently diagnosed cancer worldwide. In some countries, this is the
leading cause of women cancer-related death [1,2]. This entity re-
presents a heterogeneous disease, classified in distinct subsets by gene
expression signature with important implications for treatment, re-
sponses and outcome. The choice of appropriate therapy and prognosis
depends on a number of factors including the molecular subtype of the
pathology, which is important for the identification of predictive factors
for response to a given treatment [3–5].
hibit amplification of the human epidermal growth factor receptor 2
(HER2) gene. MDA-MB 231 cell line is an example of Claudin low
subtype with a low expression of Ki67, E-cadherin, claudin-3, claudinin-
4 and claudinin-7, while MCF-7 represents the luminal breast cancer
cell line, with expression of ER, HER2 negative, PR expression or not
and low Ki67 [9,10]. Despite the therapeutic and molecular advances of
recent years, there is no validated target to be blocked in the triple
negative subset and chemotherapy is the only available systemic
therapy [11]. Currently, taxane and anthracycline-based combination
chemotherapy remains the standard treatment approach for triple ne-
gative subtype, and this approach has not changed much in the last
decade [[17],12,13]. Doxorubicin is an anthracycline antibiotic
Basal-like and Claudin-low subtypes, represent about 19% of all
breast cancers including those with worst prognosis due to its ag-
gressive and metastatic nature and high rates of relapse [6–8]. These
⁎
Corresponding author at: Universidade Federal de Pelotas, Campus Universitário S/N, Capão Do Leão, RS, Cep: 96010-900, Brazil.
E-mail address: seixas.fk@gmail.com (F.K. Seixas).
https://doi.org/10.1016/j.biopha.2017.12.062
Received 30 September 2017; Received in revised form 20 November 2017; Accepted 14 December 2017
0753-3322/©2017ElsevierMassonSAS.Allrightsreserved.

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Fig. 1. A:Synthesis of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles 2a–d; 1B: Effect of the pyrazoles, (2a), (2b), (2c), (2d), on cell viability in human breast cancer MDA-MB-
231(B,D,F,H) and MCF-7 (C,E,G,I) cells, using the MTT assay. Cells were treated with concentrations of 5, 10, 20, 40, and 80 μM for 24, 48 and 72 h. Values are presented as mean SEM
of three independents experiments performed in triplicate. Uppercase letters represent concentration and lowercase letters represent time. Different letters indicate significant differences
among means. Significance was considered at p < .05.
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Fig. 2. Treatment combination with Dox and pyrazoles 2c and 2d for 48 h showed synergistic effect in reducing cell viability. Growth inhibition in MDA-MB-231 (A, C) and MCF-7 (E, G)
were measured by MTT assay. The combination index (CI) in MDA-MB-231 (B, D) and MCF-7 (F, H) were calculed using method described by Chou et al 2010, where CI < 1 indicated
synergistic effect. Data presented mean
p < .05. Dox = doxorubicin.
SEM from three independent experiments. Different letters indicate significant differences among means. Significance was considered at
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Fig. 3. Combination of Dox and pyrazoles incresead MDA-MB-231 human breast cancer cells death. Cells were treated with Dox (0.5 μM); Pz 2c (10 μM); Pz 2d (10 μM) or combination of
Dox and pyrazoles (0.5 + 10 μM) for 48 h. Analysis of cell death was estimated by LIVE/DEAD assay (A). Percentage of dead cells are indicated in (B). Values are presented as
mean
SEM from three independents experiments. Different letters indicate significant differences among means. Significance was considered at p < .05. Pz = pyrazole and
Dox = doxorubicin.
routinely used in the treatment of breast and other types of cancer and
it is, so far, generally used as a standard drug in breast cancer studies
[14–16]. Due to the molecular characteristics of triple negative tumors,
the treatment of this entity is still provocative and the identification of
more active therapies is mandatory to increase the therapeutic arsenal
of this disease subset [17].
it, especially in MDA-MB-231 whose alternatives for systemic treatment
are so restricted. To our knowledge, there are no reports of the com-
bination of pyrazoles and doxorubicin in these cells.
2. Materials and methods
Pyrazoles are an important class of heterocyclic compounds and are
promising scaffolds in medicinal chemistry. They are a five-membered
ring containing a pyridine-like and a pyrrole-like N-atom in adjacent
positions (1,2-diazole). Many pyrazole and pyrazole derivatives have
been extensively cited in recent scientific literature due to their im-
portant biological activities such as antitumor, anti-inflammatory, an-
timicrobial, anti-depressant, anticonvulsant and anti-oxidant [18–26].
Structural variations and modifications of pyrazoles derivatives led to
different cytotoxic activities. The pyrazole activity has been studied in
MCF -7 cells [14,22,23,25], but little is known about its effect on MDA-
MB-231 cells [22,27]. Several recent reports suggest pyrazole deriva-
tives as promising anticancer agents, indicating their use in the devel-
opment of new anticancer agents [14,27–30].
2.1. Cell culture
The human breast cancer MCF-7 and MDA-MB-231 cell lines were
obtained from Rio de Janeiro Cell Bank (PABCAM, Federal University of
Rio de Janeiro, Brazil). MDA-MB-231 cells were cultured in Leibovitz L-
15 medium supplemented with 10% fetal bovine serum (FBS) and
0.2 mg/mL sodium bicarbonate. MCF-7 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
FBS. The mediums and FBS were purchased from Vitrocell Embriolife,
Campinas, Brazil. Cells were grown at 37 °C in an atmosphere of 95%
humidified air and while MCF-7 cells were grown at 5% CO2 the MDA-
MB-231 cells were grown without any CO2. All experiments were per-
formed in triplicate.
The present study is based on the hypothesis that pyrazoles syn-
thesized with specific substituents could have good cytotoxic effect and
may increase the effect of the standard drug doxorubicin when added to
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Fig. 4. Combination of Dox and pyrazoles incresead MCF-7 human breast cancer cells death. Cells were treated with Dox (0.5 μM); Pz 2c (10 μM); Pz 2d (10 μM) or combination of Dox
and pyrazoles (0.5 + 10 μM) for 48 h. Analysis of cell death was estimated by LIVE/DEAD (A). Percentage of dead cells are indicated in (B). Values are presented as mean SEM from
three independents experiments. Different letters indicate significant differences among means. Significance was considered at p < .05. Pz = pyrazole and Dox = doxorubicin.
2.2. Synthesis of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles
2a–d
2c and 2d were used in concentrations of 10, 20 and 40 μM for 48 h.
Cell growth inhibition was measured by MTT assay. To evaluate the
possible synergistic effect between the combination of pyrazoles and
doxorubicin the Combination Index (CI) was calculated using the
method described by Chou et al [32] where CI < 1 indicated sy-
nergistic effect.
The molecules 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyr-
azoles 2a–d were synthesized based on the methodology reported by
Pizzuti et al with minor modifications [20]. The reaction scheme is
shown in Fig. 1A.
The yield of the compounds was similar to the literature. The pyr-
azoles were isolated in high purity > 97%, conform Gas
Chromatograph Mass Spectrometer analysis - Shimadzu QP 2010 SE.
2.5. Live/dead assay
Cell death analysis of MDA-MB-231 and MCF-7 cells was estimated
by Live/Dead viability assay (Invitrogen, Carlsbad, CA, USA) following
the manufacture’s instructions. Cells were treated with doxorubicin
(0.5 μM); pyrazole 2c (10 μM); pyrazole 2d (10 μM) or combination of
doxorubicin and pyrazoles (0.5 + 10 μM) for 48 h. The assay was ana-
lyzed with a fluorescence microscope Olympus IX71 (Olympus Optical
Co., Tokyo, Japan) by multicolor imaging. The recorded images were
analyzed using Cell^F software (Cell^F, New York, USA).
2.3. Pyrazole citotoxicity
Four pyrazoles were tested (2a–2d) in the MCF-7 and MDA-MB-231
cells with five different concentrations (5, 10, 20, 40, 80 μM) and in-
cubated for 24, 48 and 72 h. Cell viability was determined by MTT
assay as previously described [30,31]. The experiments carried out with
cells in logarithmic growth phase. Fig. 1B.
2.6. DAPI assay
2.4. Combination index
Analysis of apoptotic MDA-MB-231 and MCF-7 cells was evaluated
by DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) staining
(Invitrogen, Carlsbad, CA, USA). Cells were treated with doxorubicin
(0.5 μM); pyrazole 2c (10 μM); pyrazole 2d (10 μM) or a combination of
The pyrazoles with great growth inhibition of both cell lines were
used in combination with different doxorubicin concentrations (0.5; 1;
2 μM) and compared with doxorubicin alone as the standard. Pyrazoles
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Fig. 5. Combination of Dox and pyrazoles incresead apoptosis in MDA-MB-231 human breast cancer cells. Cells were treated with Dox (0.5 μM); Pz 2c (10 μM); Pz 2d (10 μM) or
combination of Dox and pyrazoles (0.5 + 10 μM) for 48 h. Analysis of apoptotic cells was estimated by DAPI staining (A). Percentage of apoptotic cells are indicated in (B). Values are
presented as mean
SEM from three independents experiments. Different letters indicate significant differences among means. Significance was considered at p < .05. Pz = pyrazole
and Dox = doxorubicin.
doxorubicin and pyrazoles (0.5 + 10 μM) for 48 h. The assay was ana-
lyzed with a fluorescence microscope Olympus IX71 (Olympus Optical
Co., Tokyo, Japan) by multicolor imaging. The recorded images were
analyzed using Cell^F software (Cell^F, New York, USA).
inhibition growth rate was near 100% for higher concentrations and
exposition times of pyrazoles 2b, 2c and 2d.
3.2. Combination index
2.7. Statistical analysis
Analyzing data on drug combination, the rate of cell growth in-
hibition in MDA-MB-231 cells was significantly higher with association
than with isolated doxorubicin in all doses of combination with both
pyrazoles. Growth inhibition rates in these cells were 80% or higher at
various concentrations of drug combinations with either pyrazol 2c and
2d. The CI favours synergism (< 1) in almost all association doses ex-
cept for one high dose combination. Best synergism rates were observed
with doxorubicin at low dose (0.5 μM) combined with pyrazole 2c at
Statistical analysis was performed by two-way analysis of variance
(ANOVA), followed by Tukey test for multiple comparisons. All values
were presented as mean
EM and the differences were considered
significant when p < .05.
3. Results
the dose of 10 μM (CI = 0.348
0.09) and in the same doses for
3.1. Pyrazole citotoxicity
pyrazol 2d (CI = 0.282 0.03). Fig. 2 (panel A–D).
In MDA-MB-231 the cell growth inhibition effect of the combination
of doxorubicin and pyrazoles 2c and 2d was even greater than that
observed in the other group of cells studied. Fig. 2 (panel A, C, E, G). In
MCF-7 cells, the only combinations doses that were better than dox-
orubicin or pyrazole 2c alone were doxorubicin 0,5 μM and pyrazole 20
or 40 μM. Synergism was observed in almost all combinations with this
pyrazole doses except for one. Fig. 2 (panel E, F). In this group of cells,
the combination with pyrazole 2d was significantly different from
The growth inhibition rate was dose and time dependent for all
pyrazoles tested. Overall the triple negative cells were more sensitive to
the tested compounds - Fig. 1B (panel B, D, F, H). Those with chlorine
and bromine radicals (2c and 2d) showed better results in both cell
lines – Fig. 1B (panel F–I). The IC50 for pyrazoles 2c and 2d in MDA-
MB-231 cells were 19.86 μM and 31.05 μM and for MCF-7 were
54.12 μM and 77.16 μM respectively in 48 h. In triple negative cells, the
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Fig. 6. Combination of Dox and pyrazoles incresead apoptosis in MCF-7 human breast cancer cells. Cells were treated with Dox (0.5 μM); Pz 2c (10 μM); Pz 2d (10 μM) or combination of
Dox and pyrazoles (0.5 + 10 μM) for 48 h. Analysis of apoptotic cells was estimated by DAPI staining (A). Percentage of apoptotic cells are indicated in (B). Values are presented as
mean
SEM from three independents experiments. Different letters indicate significant differences among means. Significance was considered at p < .05. Pz = pyrazole and
Dox = doxorubicin.
doxorubicin or the pyrazole alone just with 0,5 μM of doxorubicin and
10 μM of pyrazole 2d. There was no difference between pyrazole 2d
doses. The CI was lower with doxorubicin 0,5 μM and pyrazol 2d 10 μM
and synergism was observed with all combinations. Doxorubicin dose
did not influence inhibition in all combination analyzes. Fig. 2 (panel G,
H)
MDA-MB-231 human breast cancer cells when compared to doxorubicin
or the pyrazoles alone. There was no difference between the pyrazoles
used. Fig. 5 (panel A, B). In the MCF7 cells, the findings were similar
but the rates of apoptosis with the combination were significantly less
pronounced than in the other cell group tested, although statistically
superior than drugs alone. Fig. 6 (panel A, B).
3.3. Live/dead assay
4. Discussion
Combination of doxorubicin in 0,5 μM and pyrazoles in 10 μM in-
creased MDA-MB-231 cells death significantly when compared to dox-
orubicin or the pyrazoles alone. Both pyrazoles showed equivalent re-
sults when used in combination with the chemotherapeutic agent. Fig. 3
(panel A, B). In MCF7 cells the findings were similar but the proportion
of cell death was significantly lower than in MDA-MB-231 with all the
drugs tested, including the combination. However, the association of
doxorubicin and the pyrazoles tested was statistically superior to ex-
posure to drugs alone. There was no difference between the pyrazoles
tested in combinations. Fig. 4 (panel A, B).
Breast cancer is a disease with substantial genotypic and phenotypic
diversity [33–35]. Each subtype has different prognosis and treatment
response in dependence of molecular features of tumor differentiation,
grade and targets which can be blocked by therapeutic drugs, influen-
cing tumor progression and metastatic spread.
It is well known that cytotoxic-based therapies are more effective in
highly proliferative than in quiescent, low proliferative tumors [36,37].
The cell lines used in this study are characterized by intermediated
response to chemotherapy, both with low proliferation index and many
differences in molecular characteristics. MCF-7 is a hormone responsive
cell, representing luminal breast cancer cell line and MDA-MB-231 cells
were shown to be in the Claudin low subgroup. The latter one has
unique characteristics such as downregulation of claudin-3 and clau-
dinin-4, low expression of Ki67, enrichment for markers associated with
3.4. DAPI assay
Combination of tested drugs increased apoptosis significantly in
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the epithelial–mesenchymal transition and expression of features as-
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Despite these properties, the pyrazoles 2c and 2d showed con-
siderable growth inhibition rates in these cells, especially in MDA-MB-
231. Not surprisingly, the pyrazoles with the highest cytotoxicity were
the halogen-containing chlorine and bromine mono-substituents re-
spectively. Among the anticancer pyrazoles previously studied in MCF-
7, those with substituted halogens (fluorine, chlorine, and bromine)
exhibited more potent activities than compounds with methoxy and
methyl substitutions, respectively [23,28,40,41]. This effect is probably
related to higher electronegativity of these substituents and the re-
lationship of the chemical structure and the cytotoxicity of these
compounds needs to be better studied.
The pyrazole 2c has previously been tested in the normal cell line
MRC-5. The compound showed 30.01% of growth inhibition in the
concentration of 70 μM at 48 h [30], demonstrating that the compound
had a considerable lower cytotoxicity in the normal cells when com-
pared to the results obtained in the tumor cell lines MDA-MB-231 and
MCF-7.
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This work was supported by CAPES, CNPq and FAPERGS.
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