Synthetic resveratrol-curcumin hybrid derivative inhibits mitosis progression in estrogen positive MCF-7 breast cancer cells

Article abstract of DOI:10.1016/j.tiv.2018.02.020

Curcumin (1) and resveratrol (2) are bioactive natural compounds that display wide pharmacological properties, including antitumor activity. However, their clinical application has been limited due to their low solubility and bioavailability. Nevertheless

Full text of DOI:10.1016/j.tiv.2018.02.020

Accepted Manuscript
Synthetic resveratrol-curcumin hybrid derivative inhibits mitosis
progression in estrogen positive mcf-7 breast cancer cells
Matheus de Freitas Silva, Letícia Ferreira Coelho, Isadora
Mitestainer Guirelli, Rodrigo Machado Pereira, Guilherme Álvaro
Ferreira da Silva, Graciana Y. Graravelli, Renato de Oliveira
Horvath, Ester Siqueira Caixeta Nogueira, Marisa Ionta, Claudio
Viegas
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DOI:
Reference:
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doi:10.1016/j.tiv.2018.02.020
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27 February 2018
28 February 2018
Please cite this article as: Matheus de Freitas Silva, Letícia Ferreira Coelho, Isadora
Mitestainer Guirelli, Rodrigo Machado Pereira, Guilherme Álvaro Ferreira da Silva,
Graciana Y. Graravelli, Renato de Oliveira Horvath, Ester Siqueira Caixeta Nogueira,
Marisa Ionta, Claudio Viegas , Synthetic resveratrol-curcumin hybrid derivative inhibits
mitosis progression in estrogen positive mcf-7 breast cancer cells. The address for the
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SYNTHETIC RESVERATROL-CURCUMIN HYBRID DERIVATIVE INHIBITS MITOSIS
PROGRESSION IN ESTROGEN POSITIVE MCF-7 BREAST CANCER CELLS
Matheus de Freitas Silvaa, Letícia Ferreira Coelhoa, Isadora Mitestainer Guirellia, Rodrigo Machado Pereirab,
Guilherme Álvaro Ferreira da Silvab, Graciana Y. Graravellib, Renato de Oliveira Horvathb, Ester Siqueira
Caixeta Nogueirab, Marisa Iontab and Claudio Viegas Jr.a
aPeQuiM- Laboratory of Research in Medicinal Chemistry, Federal University of Alfenas, Jovino Fernandes
Sales Avenue, 2600, Alfenas/MG, 37130-000, Brazil.
bInstitute of Biomedical Sciences, Federal University of Alfenas, Gabriel Monteiro da Silva Street, 700,
Alfenas/MG, 37130-000 Brazil.
*Corresponding authors: Claudio Viegas Jr. or Marisa Ionta
Phone: +55 35 37011880
e-mail: cvjviegas@gmail.com or marisaionta@gmail.com
Abstract
Curcumin (1) and resveratrol (2) are bioactive natural compounds that display wide pharmacological properties,
including antitumor activity. However, their clinical application has been limited due to their low solubility and
bioavailability. Nevertheless, independent studies have considered these compounds as interesting prototypes
for developing new chemical structures useful for anticancer therapy. Here in, we report the synthesis of novel
curcumin-like hydrazide analogues (3a and 3b), and a series of curcumin-resveratrol hybrid compounds (4a-f),
and the evaluation of their cytotoxic potential on three tumor cell lines MCF-7 (breast), A549 (lung), and
HepG2 (liver). Cell viability was significantly reduced in all tested cell lines when compounds 4c-4e were used.
The IC50 values for these compounds on MCF-7 cells were lower than those for curcumin, resveratrol, or
curcumin combined with resveratrol. We evidenced that 4c promoted a drastic increase of G2/M population.
The accumulation of cells in mitosis onset in treated cultures was due to, at least in part, the ability of 4c to
modulate nuclear kinase proteins, which orchestrate important events in mitosis progression. We have also
observed significant reduction of the relative RNAm abundance of CCNB1, PLK1, AURKA, AURKB in samples
treated with 4c, with concomitant increase of CDKN1A (p21). Thus, compound 4c is a promising multi-target
antitumor agent that should be considered for further in vivo studies.
Keywords: Breast cancer, Aurora kinases, Curcumin-resveratrol hybrid compounds, cell cycle regulators, multi-
target antitumor activity.
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1. Introduction
Cancer is a complex disease that arises due to multiple genetic and epigenetic alterations (Hanahan and
Weinberg, 2011, 2000; Saracci and Wild, 2015). Breast cancer (BC) is one of the most common female cancers
in the world. It is a heterogeneous disease characterized by a wide spectrum of clinical, pathological, and
molecular features (Perou et al., 2000; Sorlie et al., 2003). According to gene expression profile, five different
intrinsic subtypes have been described: i) luminal A/B, which express estrogen and progesterone receptors (ER+
and PR+); (ii) HER2-enriched (HER+) that display amplification and/or overexpressing of human epidermal
factor growth type 2 gene (HER2/ERBB2); iii) basal-like, also known as triple negative once the majority of
these tumors do not express ER, PR or HER2, but are enriched with markers of epithelial-to-mesenchymal
transition and stem cell-like and/or tumor initiating features; iv) claudin-low, that emerged from basal like
subtype and express claudins 3/4/7; and v) normal breast like (Perou et al., 2000; Sorlie et al., 2003, 2001).
Luminal A/B tumor subtypes are ER-positive, and are the frequent BC diagnosed (Prat et al., 2015). Notably,
although the luminal A subtype of tumors respond well to endocrine therapy, luminal B tumors exhibit a higher
proliferative index, are less responsive, and are more likely to become metastatic(Ellis et al., 2012; Nielsen et
al., 2010). In this context, it is crucial to identify new chemical entities (NCEs) for developing innovative
anticancer agents to improve therapeutic propose for breast cancer. The nature represents an important source
for discovering new substances with pharmacological potential due to the great chemical diversity of secondary
metabolites. Many antineoplastic agents were derived from natural compounds (NCs) including taxol,
vincristine, vimblastine, camptothecin and ephotilone (Newman and Cragg, 2016).
Curcumin (1) (Fig. 1) is a polyphenol metabolite derived from turmeric (Curcuma longa) that is among
the widely studied natural compounds due to its therapeutic activity against various types of cancer with
minimal cytotoxic effects (Strimpakos and Sharma, 2008; Ravindran et al., 2009). (1) exerts pleiotropic effect
once module multiple cell signaling pathways in cancer cells (Kunnumakkara et al., 2017; Ravindran et al.,
2009), and therefore has been considered as a good prototype for multi-targed drug development. Studies of
structure-activity relationship (SAR) indicate that α,β-unsaturated di-ketone system of (1) is important for its
antitumor activity (Chen, 2015). Carbocyclic curcumin-analogue (CUR3d) inhibited cell proliferation of human
hepatocellular carcinoma (HepG2 cells) by downregulating expression of different kinases such as cyclin-
dependent kinases (CDKs) and aurora kinases, which are critical for cell cycle progression (Bhullar et al., 2015).
Resveratrol (2) (Fig. 1) is another polyphenol derivative, very abundant in red grapes, that similarly to
(1) exhibits chemopreventive and antitumor activities (Jang et al., 1997; Pozo-guisado et al., 2002; Chao et al.,
2014; Han et al., 2015). SAR studies have demonstrated that the hydroxyl groups present in the structure of 2
are important for its biological activity (Sinha et al., 2015). However, resveratrol analogues containing hydroxyl
groups replaced by methoxy groups have increased biological activity reinforcing the use of resveratrol as an
important prototype for searching other bioactive compounds (Hsieh et al., 2011).
Molecular hybridization has been widely used by medicinal chemists in both academia and industry for
generating a polyfunctional NCE containing different pharmacophores groups. This strategy represents one of
the most attractive alternative for molecular design allowing to access new molecular scaffolds (Barreiro et al.,
2002). In the present study, we synthetized and characterized two new curcumin-like hydrazide analogues (3a
and 3b), and a series of six curcumin-resveratrol hybrid compounds (4a-f). The structural pattern of 4 was
designed by molecular hybridization between the 3-methoxy-4-hydroxy-cynamoil subunit (Fig. 1A) from
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curcumin, linked to an O-substituted conjugated-phenyl system (Fig. 1B and C) from resveratrol, using a
hydrazone functionality as spacer moiety (D, Fig. 1), once solubility and pharmacokinetics are the main
restrictive factors for therapeutic application of curcumin or resveratrol. Moreover, we expect that the insertion
of the N-acylhydrazone moiety could contribute for increasing solubility and absorption, distribution,
metabolism and excretion (ADME) properties due to its higher polarity, with donor and acceptor hydrogen bond
sites, allowing additional molecular interactions in the biophase (Anand et al., 2007; Bellina et al., 2015; J. Li et
al., 2015; Neves et al., 2012). Curcumin-like hydrazide analogues (3) were also designed to evaluate the
contribution of the hydrazide spacer for antiproliferative activity. Data from the literature suggest that the
insertion of a heteroatom between the carbonyl groups of curcumin could improve the antiproliferative activity,
bioavailability and metabolic stability (Chen, 2015). Herein, cytotoxicity profile of the synthetized compounds
was evaluated against three tumor cell lines derived from human cancers, and molecular mechanism underlying
antitumor activity of 4c on MCF-7 was deeply investigated.
Fig. 1: Design of the new curcumin-like hydrazide analogues (3a and 3b) and a series of curcumin-resveratrol
hybrid compounds (4a-4f).
2. Results and Discussion
2.1. Synthesis
The curcumin analogues 3a and 3b were prepared from 3-methoxy-4-hydroxy-cinnamic acid (5a,
ferulic acid) or 3-hydroxy-4-methoxy cinnamic acid (5b, isoferulic acid), respectively (Scheme 1). The starting
carboxylic acids were converted into their respective acyl-chorides (6), followed by addition of hydrazine
hydrate to furnish the desired compounds 3a and 3b in 10 and 18% yields, respectively (Table 1).
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Scheme 1. Synthetic route for the preparation of curcumin analogues PQM-165 (3a) and PQM-166 (3b).
The synthesis of the target compounds 4a-f was based on the preparation of the key-hydrazide
intermediates 7a and 7b from the 3-methoxy-4-hydroxy-cinnamic acid (5a, ferulic acid) or 3-hydroxy-4-
methoxy cinnamic acid (5b, isoferulic acid), respectively. In a first step, compounds 5a and 5b were converted
into their acyl-chlorides 6a and 6b that were then reacted with hydrazine hydrate to furnish the hydrazides 7a
and 7b. These key-intermediates were then coupled to a series of substituted benzaldehydes (8a-e) leading to the
desired curcumin-resveratrol hybrid compounds 4a-f (Scheme 2) in 11-48% yields (Table 1).
Scheme 2. Synthetic route for the preparation of the curcumin-resveratrol hybrid derivatives 4a-e and 4f.
Taking the hydrazide intermediate 7a on hands, it was coupled with a series of substituted
benzaldehydes to furnish the desired series 4a-f. Among compounds 4a-e there is a diverse structural pattern,
with compounds 4a and 4b keeping the curcumin pattern on the subunit A (Fig. 1), but varying the mono- or di-
hydroxy substitution on ring system B, covering the two possible arrangements as in the resveratrol molecule.
Otherwise, compounds 4c and 4d have hydroxy substituents at the resveratrol derived subunit B/C changed by
methoxy groups aiming to evaluate their pharmacophoric contribution on the antiproliferative activity. On the
compound 4e, the resveratrol derived ring system C was substituted by the 3-methoxy-4-hydroxy substitution
pattern as from curcumin. It is important to note that compounds 3b and 4f were prepared from iso-ferulic acid
in order to investigate the structure-activity influence related to the position of the methoxy and hydroxy
substituents in the curcumin moiety. Compounds 3a and 3b are dimeric hydrazide analogs of curcumin,
differing by the regiochemistry of the hydroxy and methoxy substituents on ring systems A derived from
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curcumin (Fig. 1). Structural characterization of all synthetized compounds was made by NMR, IR and high
resolution mass spectrum (HRMS) techniques.
Table 1. Experimental data for compounds 3a and 3b, and 4a-f.
R1
OH
R2
R3
R4
-
R5
Yield (%) MP (ºC)
OCH3
OH
-
-
-
-
10.3
17.5
59.3
16.8
53.2
47.5
48.1
24.5
200.5
216.0
262.5
232.5
226.0
214.5
157.5
222.5
3a
3b
4a
4b
4c
4d
4e
4f
OCH3
OH
-
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
OH
H
H
OH
OH
H
OH
H
OH
OCH3
H
OH
OCH3
OCH3
H
OCH3
H
OH
OH
OCH3
OCH3
H
2.2 Biological Results
All synthetized compounds (3a and 3b; 4a-f) were tested at 40 µM for 48h using three tumor cell lines
(A549, MCF-7, and HepG2). Compounds 4c, 4d and 4e significantly reduced cell viability in all lines tested,
while compound 4f was active only in A549 and HepG2 cells (Fig. 2). Compounds 3a and 3b are curcumin
analogs, and one of most important biochemical properties of curcumin are its antioxidant and free radical-
scavenging ability, which plays an important role for almost all biological properties exhibited by this natural
product (Deguchi, 2015; Kunchandy and Rao, 1990; Simon et al., 1998). Despite we have not investigated the
correlation between cytotoxicity and antioxidant or radical scavenging properties, in 3a and 3b both Nitrogen
atoms of the diamide subunit can broke this scavenging activity, which could, in turn, be a reasonable
explanation for the lack of biological activity of these compounds. Considering the responsiveness profile of the
MCF-7 cells to compounds 4c, 4d and 4e, their IC50 values (concentration that inhibits 50% of the growth) were
determined. We found higher cytotoxic activity all these three on MCF-7 cells, when compared to curcumin
alone, resveratrol alone or combined curcumin-resveratrol (Table 2). In addition, compounds 4c and 4d
displayed an excellent selectivity index once they were significantly less cytotoxic to normal cells (CCD-
1059Sk) than to breast tumor cells (MCF-7) (Table 2).
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Fig. 2. Cell viability determined from MTS assay. Cell cultures were treated with different
compounds (3a, 3b and 4a-f) at 40 µM for 48 h. *p ˂ 0.05, **p ˂ 0.01, *** p ˂ 0.001 according
to ANOVA followed by Tukey’s post-test. Data represent mean ± SD of 3 independent
experiments.
Table 2. IC50 values (µM) determined from MTS assay after treatment of MCF-7 cells for 48h.
Compounds
MCF-7
CCD-1059Sk
SI*
40.49 ± 1.01
19.09 ± 0.67
42.99 ± 2.07
68.25 ± 2.59
128.85 ± 8.52
61.71 ± 2.42
158.30 ± 5.30
130.10 ± 3.92
74.58 ± 2.41
132.60 ± 3.65
187.50 ± 8.04
83.11 ± 2.38
3.90
6.81
1.73
1.93
1.45
1.35
4c
4d
4e
Curcumin
Resveratrol
(R + C)
(R + C): resveratrol plus curcumin. *Selectivity index: IC50 of normal cells/IC50 of tumor cells.
The reduction in cell viability, previously observed by MTS assay, may be consequent of cell cycle
arrest and/or cell death. Independent studies have showed that antitumor activities of curcumin and reveratrol
are associated to cycle arrest and/or apoptosis induction (Gundewar et al., 2015; Pozo-Guisado et al., 2002).
Thus, we performed cell cycle analysis to verify a possible influence of the compounds 4c, 4d, and 4e on cell
cycle progression of MCF-7 cells. Increased Sub-G1 population was observed in all treated samples, reinforcing
that these compounds display cytotoxic activity on MCF-7 cells. However, drastic alteration in cell cycle
progression was evidenced in samples treated with compound 4c (Fig. 3B, Table 3). There was reduction in both
G0/G1- and S-population, with concomitant increase in G2/M population (66.47 % ± 0.50) when compared to
control samples (29.41 % ± 0.28). It has been reported that curcumin induces cell cycle arrest in G2/M in breast
cancer cells (Banerjee et al., 2010; Simon et al., 1998), and that antitumor activity of curcumin is associated to
multiple biological mechanisms (Banik et al., 2017; Koohpar et al., 2015).
Morphological features of MCF-7 cells were observed immediately before cell cycle analysis (Fig.
3A). Control cells exhibited typical epithelial-like morphology, with cell-to-cell contacts well established. It was
possible to observe drastic alteration in cell morphology in cultures treated with compounds 4d and 4e including
cell shrinkage, picnotic nuclei, and disturbance of the cell-to-cell contacts. Interestingly, in cultures treated with
4c, we evidenced a high frequency of rounded cells, which probably were in mitosis once at the onset of mitosis
a mammalian cell changes from flat to round, condenses its chromosomes, undergoes nuclear envelope
breakdown, and reorganizes its microtubules network into a mitotic spindle(Morgan, 2006). Thus, we performed
a series of methodological approaches to understand better the effects of 4c on MCF-7 cells. We used
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fluorescent cytological preparations, stained for microtubule and microfilament, to determine mitotic index and
to evaluate possible interference of 4c on mitotic spindle dynamic and actin cytoskeleton.
Fig. 3: (A) Illustrative images obtained by phase contrast microscopy showing morphological features non-
treated and treated MCF-7 cells after 24 h of treatment. (B) Representative histograms of 3 independent
obtained by flow cytometry. Cell populations distributed in the different phases of cell cycle, after 24 h of
treatment, are evidenced by different colors (SubG1, brown; G0/G1, pink; S, green; G2/M, blue; and cells with
DNA content ˃ 4C, dark green).
Table 3 Cell cycle analysis after 24 h of treatment with compounds 4c-e at 40 µM.
Sub-G1
G0/G1
S
G2/M
0.72 ± 0.10
49.15 ± 0.84
21.76 ± 1.25***
46.10 ± 1.48
42.94 ± 1.04**
20.72 ± 1.02
8.12 ± 0.29***
12.11 ± 0.84**
10.91 ± 2.60**
29.41 ± 0.28
Control
4c
3.65 ± 0.15***
2.15 ± 0.18**
2.49 ± 0.25**
66.47 ± 0.50***
39.64 ± 0.34***
43.66 ± 0.88***
4d
4e
Data represent mean ± SD from 3 independent experiments. **p<0.01, and ***p<0.001 according to
ANOVA analysis followed by Tukeyˈs post-test.
We demonstrated that increased G2/M population detected previously by flow cytometry was due to
ability of 4c disturbs mitosis progression. Mitotic index was significantly higher in treated cultures than control
cultures (Fig. 4B). The analysis performed by fluorescence microscopy indicated 4c may have interfered in
chromosome alignment dynamic, and consequently in mitosis progression (Fig. 4A). In addition, we observed
diffuse staining for actin cytoskeleton in treated cells when compared to normal cells. In order, we verified by
trypan blue exclusion test a lower frequency of viable cells in treated cultures than control group in both 24 h
and 48 h of treatment with 4c (Fig. 4D). Confocal microscopy analysis evidenced that 4c really disturbed mitotic
spindle dynamic and impaired chromosome migration to metaphase pate (Fig. 4E). Curiously, we detected by
immunoblot only a moderate increase in phospho-histone H3 (Ser10) in treated samples, in comparison to
control group, despite of mitosis arrest had been clearly observed (Fig. 4C). These findings motivated us to
investigate the possible molecular target for 4c in MCF-7 cells considering that G2/M transition and onset steps
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of mitosis involve numerous nuclear protein kinases, which normally are deregulated in cancer cells
(Malumbres and Barbacid, 2005; Diaz-Padilla et al., 2009). Thus, we decided to investigate the expression
profile of CDKN1A (p21), PLK1 (polo-like kinase, PLK1), CCNB1 (cyclin B1), AURKA (aurora A), and
AURKB (aurora B) considering that these genes are critical for G2/M transition and M phase progression (Otto
and Sicinski, 2017).
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Fig. 4: (A) Fluorescent images showing microtubule (green), microfilaments (red) and nuclei (blue). (B)
Quantitative analysis of mitosis frequency. (C) Immunoblot showing expression profile of phospho-histone
(Ser10) H3. -tubulin was used as a loading control. Doxorubicin (DXR) was used as a control of anti-
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neoplastic agent. (D) Growth curve determined by trypan blue exclusion test. (E) Images obtained by confocal
microscopy exhibiting cells in mitosis. Anti-α-tubulin and DAPI were used for evidencing microtubule spindle
(green) and chromosomes were (blue). The quantitative analyses were performed from 3 independent
experiments. ***p<0.001 according to ANOVA analysis followed by Tukeyˈs post-test.
We found significant increase in CDKN1A mRNA abundance in treated samples than control group.
Interestingly, CDKN1A mRNA levels also were higher in samples treated with 4c in comparison to samples
treated with combined use of resveratrol and curcumin at the same concentration suggesting that hybrid
molecule is more potent in promoting p21 upregulation in MCF-7 cells in comparison to combined use of
resveratrol and curcumin. p21 is a member of the Cip/Kip protein family, that promote cell cycle arrest by
binding to and inhibiting cyclin-dependent kinases (CDKs). Despite of p21 can act as tumor suppressor by
different mechanisms, a primary activity of p21 is involved in inhibiting CDK2 and CDK1 (also known as
CDC2) leading to growth arrest at specific stages in the cell cycle in response to variety of cellular stresses. In
addition, p21 can inhibit DNA synthesis by binding to proliferating cell nuclear antigen (PCNA) and interferes
with PCNA-dependent DNA polymerase activity in response to DNA damage preventing genomic
instability(Abbas and Dutta, 2009; Cazzalini et al., 2010). In the present study, we evidenced reduced cell
population in s-phase by cytometry. It is possible that this event has been consequent of p21 upregulation
induced by 4c. Indeed, we observed that 4c reduced mRNA abundance of CCNB1, which encode cyclin B1, a
major regulator of G2/M transition. The efficiency of 4c in reducing cyclin B expression levels was comparable
to use combined of curcumin and resveratrol. The expression levels of cyclin B1 increase during G2 phase, and
its interaction with CDK1 is critical for activating the cyclin B/CDK1 complex at G2/M transition. However, the
CDK1 activity is dynamically regulated during mitosis once it is low in prophase, increase in prometaphase and
reaches its peak at metaphase; after that, it is sharply reduced due to cyclin B degradation, its regulatory subunit.
Our data demonstrate that cell cycle arrest at G2 and early M phase were due to ability of 4c in promoting
upregulation of CDKN1A with concomitant downregulation of CCNB1. Similar results were obtained by Berrak
et al. (2016) who reported cell cycle arrest at G2/M due to downregulation of cyclin B1 and CDK1 activity in
MCF-7 cultures treated with curcumin (Berrak et al., 2016).
We also found significant reduction of mRNA abundance for three mitotic kinases (aurora A, aurora B
and PLK1) in cultures treated with 4c in comparison to control group, however combined use of resveratrol and
curcumin was more effective than compound 4c in negatively modulate the expression of these kinases.
Interestingly, the reduction of mitotic kinases expression levels promoted by 4c was sufficient to promote
mitosis arrest at early stages. The ability of this compound in modulating expression of Aurora A, aurora B and
PLK1 is very important once these kinases orchestrate important steps of mitosis process (Combes et al., 2017;
Tang et al., 2017), and commonly are altered in cancer cells (Tang et al., 2017). Amplification and
overexpression of aurora A has been observed in cancer cells including breast cancer (Cirak et al., 2015;
Ferchichi et al., 2013), which contribute to chromosome instability and aneuploidy (Lapenna and Giordano,
2009). Similarly, elevated aurora B expression has been reported in breast cancer patients contributing to chemo
resistance and predicts poor prognosis (Zhang et al., 2015). In addition, Noh et al. (2015) proposed that auroras
kinases A and B are involved with invasion process in breast cancer. There are evidences indicating that PLK1
contribute to optimal Aurora B activation. Treatment with high doses of PLK1 inhibitors reduced Aurora B
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levels but further reduced its autophosphorylation, and phosphorylation of the Aurora B substrate histone H3
(Ser 10). We evidenced in the present study increased expression of phospho-histone H3 in cultures treated with
4c that reinforce the mitosis arrest previously observed. It has been reported that PLK1 inhibition induces
prolonged mitotic arrest and subsequent onset of apoptosis(Liu et al., 2006; Yeap et al., 2015). Thus, we treated
MCF-7 cells with 4c at 20 and 40 µM for 48h and evaluated its ability in induce apoptosis. We found increased
frequency of positive cells for annexin V in cultures treated with 4c at 20 µM and 40 µM in relation to control
cultures, however significant difference was detect only in the last concentration. The data indicate that
apoptosis may be a subsequent onset of prolonged mitosis arrest induced by 4c treatment (Fig. 6B). Cellular
debris can be observed in phase contrast images obtained from treated cultures immediately before apoptosis
analysis, i.e., after 48h of treatment; reinforcing the hypothesis that 4c induces mitosis arrest and subsequent
apoptosis cell death. In addition, treated cells seem to be larger than control cells evidencing that 4c promoted
drastic morphological alteration.
Fig. 5: Relative mRNA abundance of critical regulators of G2/M transition and M phase determined by real-
time-PCR after 24 h of treatment using ATCB as housekeeping. Compound 4c was used at 40 µM. Combination
of resveratrol and curcumin (R + C) was obtained using final concentration at 40 µM. Data represent mean ± SD
from 4 independent experiments. **p<0.01, and ***p<0.001 according to ANOVA analysis followed by
Tukeyˈs post-test.
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Fig. 6: (A) Illustrative images obtained by phase contrast microscopy showing morphological features non-
treated and treated MCF-7 cells after 48 h of treatment with 4c at 20 and 40 µM. (B) Representative dot plots of
the Annexin V-FITC/7-AAD assay. Viable cells (lower left quadrants); necrotic cells (upper left quadrants);
early apoptosis (lower right quadrants); and late apoptosis (upper right quadrants). (C) Quantitative analysis of
Annexin V-FITC/7-AAD assay considering early apoptosis and late apoptosis). Data represent mean ± SD from
3 independent experiments. *p<0.05 according to ANOVA analysis followed by Tukeyˈs post-test.
3. Conclusion
Considering all data set discussed above, we identified the curcumin-resveratrol acylhydrazone hybrid
4c as a promising antitumor multi-target agent, with an innovative structural scaffold. We demonstrated that this
compound modulates kinase proteins that orchestrate mitosis progression in positive estrogen receptor breast
cancer, leading to cell cycle arrest in mitoses onset and, subsequently, to apoptosis. Further in vivo studies
should be performed for validating anticancer efficacy of this compound as well as its pharmacokinetic
properties, once N-acylhydrazone subunit was used as part of the designed structural scaffold aiming to increase
the coefficient of solubility and absorption.
4. Experimental section
4.1 CHEMISTRY
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4.1.1 Generalities
The spectra in the infrared region (IR) have been generated in an infrared spectrometer Nicolet iSso
(Thermo scientific USA) coupled to Pike Gladi ATR technologies in analysis and characterization of drugs
1
laboratory (LACFar) at the Federal University of Alfenas (UNIFAL-MG). NMR spectra of H and 13C were
1
obtained on a Bruker AC-300 spectrometer operating at 300 MHz for H NMR and 75 MHz for 13C NMR in
Nuclear Magnetic Resonance Laboratory at the UNIFAL-MG. All reagents used in synthesis were purchased
from Sigma-Aldrich without further purifications. Thin layer chromatography experiments were performed on
silica gel sheet 60 F254, Merck and purification by chromatography column was performed on flash silica gel
(220-440 mesh, 0.035 mm - 0.075 mm), Sigma-Aldrich. The visualization of the substances was done in UV
chamber ( = 254 or 365 nm) or with iodochloroplatinate reagent. The solvents dichloromethane, ethanol,
triethylamine and dimethylformamide were distilled and dried according to literature (Perrim and Armarengo,
1988). Melting point were set on Mars equipment (PFM II) with crushed sample and packaged in capillary tube
without correction. All spectra are available in the supplementary material.
4.1.2 Synthesis of curcumin-like derivatives 3a, b
A solution containing 300 mg of trans-ferulic acid (5a) or 3-hydroxy-4-methoxy-trans-cinnamic acid
(5b) (1.546 mmol, 1 eq.), thionyl chloride (10.05 mmol, 6.5 eq) and DMF (37%) in dichloromethane (15 mL)
was stirred for 2 hours at 25°C until consumption of all starting acid. After the reaction medium was
concentrated under low pressure and then directed to a high vacuum line to ensure the removal of thionyl
chloride excess. A solution containing triethylamine (1.86 mmol, 1.2 eq), dichloromethane (20 mL) and
hydrazine hydrate (0.77 mmol, 0.5 eq) was prepared and accommodated in the addition funnel, the flask solid
was solubilized in dichloromethane and placed under stirring, the contents of the funnel addition was then added
to the flask dropwise, the system was stirred for 20 min at 25 ° C. The reaction product was taken to the rotary
evaporator under reduced pressure to remove the solvent and the reaction was purified by CC with flash silica
gel (220-440 mesh, 0.035 mm - 0.075 mm) employing ethyl acetate and hexane, gradient (8-2 to 6-4) as mobile
phase, the obtained products provided the data described below.
4.1.2.1 (E)-3-(4-hydroxy-3-methoxyphenyl)-N'-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl) acrylohydrazide
(3a, PQM165)
Yield 10.3%, white solid, m.p. 200.5 ºC. IR (ATR)  3430.74, 3016.12, 2968.39, 2840.15, 1688.37,
1
1590.02, 1430.92, 1376.44 and 1033.66 cm-1. H NMR (300 MHz, CD3OD) 7.59 (d, J = 15.89 Hz, 2H,
HC=CH), 7.17 (d, J = 1.93 Hz, 2H, Ar•H), 7.06 (dd, J = 1.91, 8.24, 2H, Ar•H), 6.81 (d, J = 8.17, 2H, Ar•H),
6.31 (d, J = 15.88 Hz, 2H, HC=CH) and 3.89 (s, 6H, CH3). 13C NMR (75 MHz, CD3OD)  171.0, 150.5, 149.4,
146.9, 127.8, 124.0, 116.5, 115.9, 111.7 and 56.4.
4.1.2.2 (E)-3-(3-hydroxy-4-methoxyphenyl)-N'-((E)-3-(3-hydroxy-4-methoxyphenyl)acryloyl) acrylohydrazide
(3b, PQM166)
Yield 17.5%, white solid, m.p. 216 ºC. IR (ATR)  3396.03, 2941.88, 2844.49, 2585.59, 1668.61,
1
1611.72, 1441.53, 1357.16 and 1023.53 cm-1. H NMR (300 MHz, DMSO-d6) 7.42 (d, J = 15.91 Hz, 2H,
HC=CH), 7.07 (m, 5H, Ar•H), 6.92 (d, J = 8.61, 2H, Ar•H), 6.22 (d, J = 15.93 Hz, 2H, HC=CH) and 3.78 (s,
6H, CH3). 13C (75 MHz, DMSO-d6)  168.3, 150.3, 147.1, 144.6, 127.6, 121.5, 116.8, 114.5, 112.42 and 56.07.
13

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4.1.3 Synthesis of hydrazide intermediate (7a and 7b)
To a suspension of the corresponding cinnamic acid (5a or 5b) (1.55 mmol, 1.0 eq.) in 20 mL CH3CN
at room temperature, was added HOBt (1.86 mmol, 1.2 eq.) in a single portion, followed by EDC (1.86 mmol,
1.2 equiv). The reaction was stirred at 25°C for 1.5 h. The resulting mixture was then slowly added to a solution
of hydrazine hydrate (15.50 mmol, 10.0 eq.) in 5 mL of CH3CN, kept between 0 and 10ºC. The reaction was
usually completed upon the end of addition. Them the solvent was removed under low pressure. The solid was
resuspended in saturated NaHCO3 solution and the insoluble solid was separated by filtration and washed with
cold saturated NaHCO3 solution, resulting in the desired corresponding hydrazide.
4.1.3.1 (E)-3-(4-hydroxy-3-methylphenyl)acrylohydrazide (7a)
Yield 95%, white solid. IR (ATR): 3404.76, 3306.89, 3260.61, 1656.58, 1579.92, 1514.84, 1440.59
1
and 1028.37 cm-1. H NMR (300 MHz, CD3OD)  9.18 (s, 1H, Ar-OH), 7.35 (d, J = 15.75 Hz, 1H, HC=CH),
7.11 (d, J = 1.82 Hz, 1H, Ar•H), 6.99 (dd, J = 1.82, 8.19 Hz, 1H, Ar•H), 6.79 (d, J = 8.12 Hz, 1H, Ar•H), 6.36
(d, J = 15.75 Hz, 1H, HC=CH), 4.39 (s, 2H, CONHNH2) and 3.80 (s, 3H, CH3). 13C NMR (75 MHz, CD3OD) 
165.2, 148.3, 147.9, 138.71, 126.5, 121.4, 116.9, 115.7, 110.9 and 55.6.
4.1.3.2 (E)-3-(3-hydroxy-4-methylphenyl)acrylohydrazide (7b)
Yield 89%, yellow solid. IR (ATR): 3253.38, 3287.33, 3147.31, 2938.55, 2838.27, 1651.76,
1
1586.19, 1510.98, 1424.20 and 1027.41 cm-1. H NMR (300 MHz, CD3OD)  7.47 (d, J = 15.73 Hz, 1H,
HC=CH), 7.12 (d, J = 1.51 Hz, 1H, Ar•H), 7.03 (dd, J = 1.57, 8.08 Hz, 1H, Ar•H), 6.80 (d, J = 8.14 Hz, 1H,
Ar•H), 6.38 (d, J = 15.77 Hz, 1H, HC=CH), and 3.88 (s, 3H, CH3). 13C NMR (75 MHz, CD3OD)  168.6, 162.5,
149.9, 149.3, 142.1, 128.2, 123.2, 116.5, 111.5 and 56.4.
4.1.4 Coupling reaction of hydrazide (7) with aldehydes for the preparation of 4a-f.
To a solution of the corresponding hydrazide derivative (7a or 7b) (0.961 mmol) in dry ethanol (30
mL) containing a catalytic amount of 40% aq. hydrochloric acid was added 1.153 mmol of the desired
benzaldehyde. The mixture was stirred at 25ºC for 12 hours. After confirmation of completion of the reaction
(TLC), the solvent was removed under reduced pressure and the product purified from chromatography column
(CC) with flash silica gel (220-440 mesh, 0.035 mm - 0.075 mm) and suitable eluent, providing good yields.
The final products provided the following data.
4.1.4.1 (E)-3-(4-hydroxy-3-methoxyphenyl)-N'-(4-hydroxybenzylidene)acrylohydrazide (4a, PQM 160)
Yield 59.3%, brown solid, m.p. 262.5 ºC. IR (ATR): 3075.42, 3014.19, 2921.63, 2828.10, 1678.73,
1
1606.41, 1580.38, 1455.99, 1350.89 and 1102.60. cm-1. H NMR (300 MHz, CD3OD) 8.12 and 7.95 (s, 1H,
N=CH), 7.69 (d, J= 8.63 Hz, 2H, Ar-H) 7.51 (d, J=15.11 Hz, 1H, HC=CH), 6.84 (m, 6H, Ar-H), 7 6.72 (d,
J=2.17 Hz, 2H, Ar-H), 6.51 (d, J=15.64 Hz, 1H, HC=CH) and 3,82 (s, 3H, CH3). 13C NMR (75 MHz, CD3OD)
166.3, 161.8, 160.4, 147.9, 146.5, 143.2, 130.2, 128.9, 128.7, 126.3, 125.2, 121.9, 115.8, 111.0 and 55.6. ESI-
MS m/z: 313.1 [M + H]+; HRMS: Calcd for C17H17N2O4 [M + H]+ 313.1188, found 313.1187. Calcd for
C17H16N2NaO4 [M+Na]+ 335.1008, found 335.1009.
4.1.4.2 (E)-N'-(3,5-dihydroxybenzylidene)-3-(4-hydroxy-3-methoxyphenyl)acrylohydrazide (4b, PQM 161)
Yield 16.8%, light yellow solid, m.p. 232.5 ºC. IR (ATR): 3374.39, 3230.23, 3047.03, 2962.17,
1
1622.83, 1585.23, 1467.11, 1343.68 and 1018.73 cm-1. H NMR (300 MHz, CD3OD)  7.96 and 7.82 (s, 1H,
N=CH), 7.66 (d, J=15.41 Hz, 1H, HC=CH), 7.16 (d, J=1.53 Hz, 1H, Ar-H), 7.09 (dd, J=1.31, 8.07 Hz, 1H, Ar-
14

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H), 6.82 (d, J=8.14 Hz, 1H, Ar-H), 6.72 (d, J=2.17 Hz, 2H, Ar-H), 6.47 (d, J=15.62 Hz, 1H, HC=CH), 6.32 (s,
1H, Ar-H), 3,90 (s, 3H, CH3). 13C NMR (75 MHz, CD3OD) 165.8, 160.0, 149.6, 149.4, 146.0 (Z), 145.1 (E),
144.3, 137.3, 128.3, 123.6, 123.4, 116.6, 111.8, 107.1 and 55.5. ESI-MS m/z: 329.1 [M + H]+; HRMS: Calcd for
C17H17N2O5 [M + H]+ 329.1137, found 329.1141. Calcd for C17H16N2NaO5 [M+Na]+ 351.0957, found 351.0957.
4.1.4.3 (E)-3-(4-hydroxy-3-methoxyphenyl)-N'-(4-methoxybenzylidene)acrylohydrazide (4c, PQM 162)
Yield 53.1%, light yellow solid, m.p. 226.0 ºC. IR (ATR)  3116.40, 3046.50, 2928.86, 2833.40,
1651.25, 1605.63, 1507.58, 1420.80, 1398.14 and 1031.25 cm-1. 1H NMR (300 MHz, CD3OD) 8.08 and 7.95
(s, 1H, N=CH), 7.75 (d, J = 8.83 Hz, 2H, Ar-H), 7.66 (d, J = 15.61 Hz, 1H, HC=CH), 7.17 (d, J = 1.79 Hz, 1H,
Ar-H), 7.10 (dd, J = 1.81, 8.22 Hz, 1H, Ar-H), 6.97 (d, J = 8.86 Hz, 2H, Ar-H), 6.83 (d, J = 8.13 Hz, 1H, Ar-H),
6.48 (d, J = 15.62 Hz, 1H, HC=CH), 3.91 (s, 3H, CH3) and 3.83 (s, 3H, CH3). 13C NMR (75 MHz, CD3OD)
165.7, 163.2, 150.4, 149.3, 145.0, 144.0, 130.5, 129.8, 128.1, 123.5, 116.6, 116.4, 115.3, 111.8, 56.4 and
55.9MS m/z: 327.1 [M + H]+; HRMS: Calcd for C18H19N2O4 [M + H]+ 327.1345, found 327.1343. Calcd for
C18H18N2NaO4 [M+Na]+ 349.1164, found 349.1163.
4.1.4.4 (E)-N'-(3,5-dimethoxybenzylidene)-3-(4-hydroxy-3-methoxyphenyl)acrylohydrazide (4d, PQM163)
Yield 47.5%, beige solid, m.p. 214.5 ºC. IR (ATR)  3153.53, 3055.17, 2934.16, 2833.88, 1660.89,
1
1628.59, 1587.13, 1423.69, 1363.43, 1054.87 cm-1. H NMR (300 MHz, CD3OD) 8.10 and 7.93 (s, 1H,
N=CH), 7.50 (d, 1H, J=15.82 MHz, HC=CH), 7.31 (s, 1H, Ar-H), 7.13 (m, 3H, Ar-H), 6.83 (s, 1H, Ar-H), 6.81
(d, J=2.26 MHz, 1H, Ar-H), 6.51 (d, J=15.67 MHz, HC=CH), 3.85 (s, 3H, CH3 (Z)), 3.84 (s, 3H, CH3), 3.83 (s,
3H, CH3 (E)) and 3.82 (s, 3H, CH3). 13C NMR (75 MHz, CD3OD) 162.0, 160.7, 149.0, 148.8, 146.1 (Z), 142.4
(E), 141.2, 136.5, 126.2, 122.8, 122.0, 116.7 (Z), 115.7, 111.0, 104.8, 102.3 (E), 55.5, 55.4 (E), 55.2 and 55.0
(Z). ESI-MS m/z: 357.1 [M + H]+; HRMS: Calcd for C19H21N2O5 [M + H]+ 357.1450, found 357.1453.
4.1.4.5 (E)-N'-(4-hydroxy-3-methoxybenzylidene)-3-(4-hydroxy-3-methoxyphenyl)acrylohydrazide (4e,
PQM164)
Yield 48.1%, yellow solid, m.p. 157.5 ºC. IR (ATR)  3212.34, 3059.03, 2965.02, 2840.15, 1651.73,
1
1585.20, 1427.55, 1373.55 and 1025.46 cm-1. H NMR (300 MHz, DMSO-d6)  8.13 and 7.95 (s, 1H, N=CH),
7.51 (d, J = 15.74 Hz, 1H, HC=CH), 7.31 (s, 1H, Ar•H), 7.15 (m, 5H, Ar•H), 6.84 (dd, J = 8.13, 1.51 Hz, 4H,
Ar•H), 6.56 (d, J = 15.65 Hz, 1H, HC=CH), 3.85 (s, 6H, CH3 (E)), 3.84 (s, 3H, CH3 (Z)) and 3.82 (s, 3H, CH3
(Z)). 13C (75 MHz, DMSO-d6) 166.3 (E), 161.9 (Z) 149.0, 148.8, 148.1, 147.9, 146.8 (E), 143.1 (Z), 140.7,
126.3, 125.8, 122.1, 121.9, 117.1, 115.8, 115.5, 111.0, 109.2 and 55.6. ESI-MS m/z: 343.1 [M + H]+; HRMS:
Calcd for C18H19N2O5 [M + H]+ 343.1288, found 343.1299.
4.1.4.6 (E)-3-(3-hydroxy-4-methoxyphenyl)-N'-(4-methoxybenzylidene)acrylohydrazide (4f, PQM167)
Yield 24.5%, light yellow solid, m.p. 222.5 ºC. IR (ATR) 3331.91, 3196.43, 2929.34, 2835.33,
1
1652.21, 1599.66, 1574.59, 1442.98 and 1015.34 cm-1. H NMR (300 MHz, CD3OD)  8.14 and 7.98 (s, 1H,
N=CH), 7.66 (d, J = 8.52 Hz, 4H, Ar-H (E e Z)), 7.46 (d, J = 16.11 Hz, 1H, HC=CH, (E)), 7.34 (d, J = 16.00
Hz, 1H, HC=CH, (Z)), 7.15-6.94 (m, 7H, Ar-H), 6.45 (d, J = 15.75 Hz, 1H, HC=CH) and 3.97 (s, 6H, CH3). 13
C
NMR (75 MHz, CD3OD)  163.8, 162.4, 151.0, 147.9, 144.3, 142.2, 130.2, 129.8, 128.4, 122.2, 118.8, 115.7,
114.9, 113.4, 56.63 and 57.0. MS m/z: 327.1 [M + H]+; HRMS: Calcd for C18H19N2O4 [M + H]+ 327.1345,
found 327.1352. Calcd for C18H18N2NaO4 [M+Na]+ 349.1164, found 349.1172.
15

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4.2 BIOLOGICAL ASSAYS
4.2.1 Cell lines and treatment schedule
Cell lines derived from human cancers: A549 (lung adenocarcinoma), MCF7 (breast adenocarcinoma),
and HepG2 (hepatocellular carcinoma); and fibroblasts derived from normal skin (CCD-1059Sk) were used in
this study, which were purchased from Rio de Janeiro Cell Bank. The cell cultures were maintained in DMEM
(Dulbecco’s Modified Eagle’s Medium, Sigma, CA, USA) supplemented with 10% fetal bovine serum
(Vitrocell, Campinas, Brazil). Cells were grown in a 37° C humidified incubator containing 5% CO2.
Synthetic compounds were solubilized in dimethyl sulfoxide (DMSO) at 20 mM, and stock solution
was stored at -20o C until use. Cells were seeded into plates containing 96, 24, or 6 wells depending of the
experimental approach. After attachment (24h), the cells were treated for 24 or 48h.
4.2.2 Cell viability assay
Cell viability was measured by MTS (dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium)
assay using CellTiter 96® Aqueous Non-Radiative Cell Proliferation assay (Promega) according to
manufacturer’s instructions. 1 x 104 cells were seeded into 96 well/plate and synthetic compounds were
screened at 40 µM for 48 h. After that dose-response curves were performed using a concentration range (12.5 –
100 µM) for 48 h. Formazan, the reduced form of tetrazolium, absorbs light at 490 nm and viable cells rate is
directly proportional to the amount of produced formazan by dehydrogenase enzymes. Experiments were
conducted in triplicate wells. Data are presented as the mean ± standard deviation (SD) of 3 independent
experiments. The IC50 value was determined from non-linear regression using GraphPad Prism® (GraphPad
Software, Inc., San Diego, CA, USA).
4.2.3 Cell cycle analysis
Cells were seeded onto 24 well/plates at 2 x 105 density. Treatment was performed for 24 h or 48 h
with 4c and 4d at 20 µM and 40 µM concentrations. The cells were fixed with 75% ethanol at 4 C overnight,
rinsed twice with cold phosphate-buffered saline (PBS). Following, cells were homogenized in dye solution
[PBS containing 30 μg/mL propidium iodide (PI) and 3 mg/mL RNAase]. DNA was quantified 1h after
staining. The analysis was performed by flow cytometry (Guava easyCyte 8HT, Hayward, CA, USA). Results
are presented as means ± standard deviations (SD) of 3 independent experiments.
4.2.4 Transcript level evaluation
Total RNA from the treated and untreated MCF-7 cells was extracted using the RNeasy® Micro kit
(Qiagen, Mississauga, ON, Canada), according to the manufacturer's instructions, and eluted in 30 μL of
RNAse-free water. RNA concentrations were measured by spectrophotometer using a NanoDrop® ND 1000
(Thermo Scientific, Wilmington, DE, USA) and 1 µg of total RNA was incubated with DNase I (1 U/µg;
Invitrogen, São Paulo, SP, Brazil) to eliminate possible contamination with genomic DNA, and then subjected
to reverse transcription using random primers and the High Capacity cDNA Reverse Transcription Kit®
(Applied Biosystems, São Paulo, SP, Brazil), according to the manufacturer's instructions. The reagents were
incubated at 25°C for 10 min, 37°C for 120 min, and finally 85°C for 5 min to inactivate the enzyme.
Expression of the target genes (CDKN1A, PLK1, CCNB1, AURKA, and AURKB) was investigated by
real-time PCR with an ABI 7500 thermocycler using Power Sybr Green PCR Master Mix (Applied Biosystems)
using the primers as stated in Table 1. Reactions were performed in a final volume of 25 μL and the genes were
16

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amplified using the following conditions: 95°C for 10 min (1 cycle), denaturation at 95°C for 10 s, and
annealing and extension at 60°C for 1 min (40 cycles). To select the most stable housekeeping gene, β-actin
(ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18srRNA)
amplification profiles were compared using the geNorm applet for Microsoft Excel (medgen.ugent.be/genorm;
Vandesompele et al. (2002); the most stable housekeeping gene was ATCB. The relative abundance of each
target gene was calculated using the ΔΔCt method with efficiency correction and a control sample for
calibration (Pfaffl, 2001). The average efficiency values for each gene were calculated through the amplification
profile of each sample using the LinRegPCR program (Ramakers et al., 2003). Each sample was run in triplicate
and non-template control was included. The data are presented as mean ± standard error of the mean (SEM)
from 4 independent experiments.
4.2.5 Immunoblot
Cells were homogenized in RIPA lysis buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate,
0.1% SDS and 50 mM Tris pH 8.0) containing both protease and phosphatase inhibitors (Sigma). Lysates were
centrifuged (10,000 × g) for 10 min at 4 °C. Supernatants were recovered, total proteins were quantified (BCA
kit, Pierce Biotechnology Inc., Rockford, IL, USA) and resuspended in Laemmli sample buffer containing 62.5
mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue. An
aliquot of 50 μg protein was separated by SDS–PAGE (12%) and transferred (100 V, 250 mA for 2h) on to a
PVDF membrane (Amersham Bioscience), blocked for 1h at 4 °C with blocking solution [5% non-fat milk in
Tris-buffered saline (TBS) + 0.1% (v/v) Tween-20] to prevent nonspecific protein binding. The membrane was
probed with primary antibodies: phospho(ser10)-histone H3 (Santa Cruz ‒1:200) or α-tubulin (Sigma – 1:1000)
overnight at 4 °C. After washing with TBS-tween (0.1%), the membrane was incubated with a secondary
antibody (anti-rabbit or anti-mouse peroxidase conjugated) for 2h at room temperature. Immunoreactive bands
were visualized with the ECL Western Blotting Detection Kit (Amersham Pharmacia). A reprobing protocol
was followed for detecting immunoreactive bands for different antibodies. The quantification of
immunoreactive bands was performed using a public program (Image J) from 3 independent experiments.
4.2.6 Apoptosis detection by Annexin V/7-AAD
Kit Guava Nexin® (Merk Millipore, Massachusetts, EUA) was used according to manufacturer’s
instructions. Briefly, cells were collected by enzymatic digestion (Trypsin/EDTA, Sigma), centrifuged at 1,000
rpm for 5 min at 4 °C, washed with ice-cold PBS, and then 2 × 104 cells were resuspended in 100 μL of DMEM.
In the next step, it was added 100 μL of mix solution containing buffered Annexin V-PE and 7-AAD. The
samples were read after 20 min of incubation at room temperature in a dark chamber. The analysis was
performed by flow cytometry using GuavaSoft 2.7 software. The experiments were conducted in triplicate and
repeated twice. Data are presented as mean ± SD.
4.2.7 Mitotic index
Mitotic cells were counted from fluorescent cytological preparations containing DAPI stained nuclei
and α-tubulin immunostaining. It was counted 1000 cells per sample. Data are shown as mean ± SD of three
independent experiments.
4.2.8 Trypan blue exclusion test
Cells were seeded into 35mm plates at a density of 2 x 105. Cells were harvested with trypsin/EDTA at
24 h and 48 h after treatment. Trypan blue solution (0.4%) was added to cellular suspension (1:1), and
17

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subsequently, cells were counted using hemocytometer chamber and light microscope. viable cells (unstained)
were quantified from three independent experiments. The results are presented as mean ± SD.
4.2.9 Statistical Analysis
Significant differences were determined using one-way analysis of variance (ANOVA) followed by
Tukey’s post-test. The values were expressed as mean ± SD or mean ± SEM from of at least three independent
experiments. p<0.05 was considered as statistically significant.
Acknowledgements
The authors are grateful to the Brazilian Agencies CAPES, FAPEMIG (# APQ-02810-16), CNPq
(#454088/2014-0, #310082/2016-1) and FINEP for financial support and fellowships. We also thank
CAPES (M.F.S., G.A.F.S.), FAPEMIG (R.O.H), and PIBIC-CNPq-UNIFAL (G.Y.G) for the research
fellowships. The authors are grateful to Dr. J.S.Garcia and Dr. M. Trevisan for the help in the use of the
UPLC equipment.
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Graphical abstract
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Highligths
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Resveratrol-curcumin hybrid 4c inhibits mitosis progression of MCF-7 cells
4c induces accumulation of cells in mitosis onset and apoptosis in MCF-7 cells
4c promotes strong increase of CDKN1A expression in MCF-7 cells
Concomitantly, compound 4c reduces level expression of AURKA, AURKB, PLK-1 & CCNB1
4c represents an innovative multi-target directed antitumor drug candidate prototype
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