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Characterization of the in vitro cytotoxic eects of brachydins isolated from
Fridericia platyphylla in a prostate cancer cell line
Higor Lopes Nunes
a
, Katiuska Tuttis
a
, Juliana Mara Serpeloni
a
, Jessyane Rodrigues do Nascimento
b
,
Claudia Quintino da Rocha
b
, Viviane Aline Oliveira Silva
c
, André van Helvoort Lengert
c
, Rui Manuel Reis
c,d,e
,
and Ilce Mara de Syllos Cólus
a
a
Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina, Paraná, Brasil;
b
Departamento
De Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal do Maranhão, São Luís, Maranhão, Brasil;
c
Centro de Pesquisa em
Oncologia Molecular, Hospital de Câncer de Barretos, Barretos, São Paulo, Brasil;
d
Life and Health Sciences Research Institute (ICVS), School of
Medicine, University of Minho, Braga, Portugal;
e
ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal
ABSTRACT
Brachydins (Br) A, B, and C are avonoids extracted from Fridericia platyphylla (Cham.) L.G. Lohmann
roots (synonym Arrabidaea brachypoda), whose extract previously exhibited cytotoxic and anti-
tumor activity. In vitro cell culture of human prostate tumor cell line (PC-3) was used to determine
cell viability as evidenced by MTT, neutral red, and LDH release using nine concentrations (0.24 to
30.72 µM) of each brachydin. A triple-uorescent staining assay assessed the mechanism resulting
in cell death. Genomic instability and protein expression were evaluated using comet assay and
western blot analysis, respectively. The pro-oxidant status was analyzed using the5-(and-6)-
chloromethyl-2′,7′-dichlorodihydrouorescein diacetate (CM-H
2
DCFDA) probe. The IC
50
values for
brachydins BrA, BrB, and BrC were 23.41, 4.28, and 4.44 µM, respectively, and all compounds
induced apoptosis and necrosis. BrB and BrC increased p21 levels indicating a possible G1 cell
cycle arrest. BrA (6 µM) and BrB (3.84 µM) decreased phospho-AKT (AKT serine/threonine kinase)
expression, which also inuenced cell cycle and proliferation. BrA, BrB, and BrC elevated cleaved
PARP (poly (ADP-ribose) polymerase), a protein related to DNA repair and induction of apoptotic
processes. Therefore, this study determined the IC
50
values of brachydins in the PC-3 cell line as well
as the inuence on cell proliferation and cell death processes, such as apoptosis and necrosis,
indicating the proteins involved in these processes.
Abbreviations: ANOVA: Analysis of Variance; BrA: Brachydin A; BrB: Brachydin B; BrC: Brachydin C;
CGEN: Genetic Heritage Management Council; CID: Compound identication number; CM-H
2
DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrouorescein diacetate, acetyl ester; CO
2
:
Carbon dioxide; DMSO: Dimethyl sulfoxide; DNA: Deoxyribonucleic acid; DTT: Dithiothreitol; DXR:
Doxorubicin; ECL: Chemiluminescence; EDTA: Ethylenediaminetetraacetic acid; FBS: Fetal bovine
serum; H
2
O
2
: Hydrogen peroxide; HRMS: High-Resolution Mass Spectrometry; IC50: Half maximal
inhibitory concentration; LDH: Lactate dehydrogenase; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphe-
nyl tetrazolium bromide; Na3VO4: Sodium Orthovanadate; NaOH: Sodium hydroxide; NCBI: National
Center for Biotechnology Information; NMR: Nuclear Magnetic Resonance; PBS: Phosphate buer
saline; PCR: Polymerase chain reaction; PSMF: Phenylmethylsulfonyl uoride; RPMI: Roswell Park
Memorial Institute Medium; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis;
STR: Short tandem repeat; TBS-T: Tris-buered saline and Polysorbate 20; UPHLC: Ultra-Performance
Liquid Chromatography
KEYWORDS
Apoptosis; cytotoxicity;
flavonoids
Introduction
Cancer is one of the diseases responsible globally
for high morbidity and mortality rates. This disease
has been treated with several therapies, including
cytotoxic chemotherapy, using synthetic or natural
cytotoxic chemotherapeutics (Huang, Cai, and
Zhang 2009; Siegel, Miller, and Jemal 2016).
Prostate cancer is the type of non-cutaneous cancer
frequently diagnosed in men associated with a high
rate of morbidity and mortality (Pezaro, Woo, and
Davis 2014; Woodrum et al. 2017). Treatments for
this condition include surgery, chemotherapy,
immunotherapy, and radiotherapy. Although
these options are effective for different types of
cancers, there are limitations as adverse effects
occur in patients (Bonam et al. 2018). Considering
CONTACT Juliana Mara Serpeloni julianaserpeloni@yahoo.com.br Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade
Estadual de Londrina, Londrina, Paraná CEP 86057-970, Brasil
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
https://doi.org/10.1080/15287394.2020.1784339
© 2020 Taylor & Francis
factors such as (1) increased incidence of cancer,
(2) aging population, and (3) need to develop thera-
pies with fewer adverse effects, the search for new
therapeutic approaches becomes necessary.
Plant-derived natural compounds have become
indispensable for modern pharmacotherapy
(Corveloni et al. 2020; Gontijo et al. 2018; Rocha
et al. 2019a; Yesil-Celiktas et al. 2010). Medicinal
plants have historically been a valuable drug source
and are still explored regarding their modulation of
several molecular targets for the treatment of
chronic diseases, including cancer (Atanasov et al.
2015; González-Vallinas, Reglero, and Ramírez de
Molina 2015; Majolo et al. 2020).
The products of plant secondary metabolism are
the focus of several new research investigations.
These products need to be extracted, isolated, and
structurally verified before the evaluation of poten-
tial biological and pharmacological activities using
in vitro and in vivo models (Atanasov et al. 2015;
Maistro et al. 2019; Russo et al. 2010).
Brachydin A (BrA), B (BrB), and C (BrC) are
unusual previously described dimeric flavonoids
isolated from the dichloromethane fraction of
a hydroalcoholic extract of the roots of the plant
species Fridericia platyphylla (Cham.) L.G.
Lohmann, identified as Arrabidaea brachypoda
(DC) Bureau (da Rocha et al. 2014). In a recent
study, Serpeloni et al. (2020) demonstrated cyto-
toxic and anti-tumor activity in tumor gastric cells
incubated with Fridericia platyphylla extract mod-
ulating apoptosis- and cell cycle-related genes.
The traditional use of teas prepared from the
roots of this plant is already known for the treat-
ment of kidney stone and arthritis (Rodrigues and
Carlini 2005). De Sousa Andrade et al. (2020)
reported that brachydins BrA and BrB displayed
antimicrobial actions against Staphyloccocus aur-
eus, Escherichia coli, and Candida albicans.
Recently investigators focused predominantly on
the cytotoxic activity of isolated brachydins, mainly
as anti-parasites. da Rocha et al. (2014) examined
the toxicity of these three compounds against the
extracellular trypomastigotes of Trypanosoma
cruzi. In the same study, BrB and BrC exhibited
cytotoxicity against mammalian cells and intracel-
lular amastigotes of T. cruzi and were capable of
inhibiting parasite invasion. Further, in vivo tests
using female BALB/c mice demonstrated that BrB
was able to reduce blood parasitemia and mortality
when compared to controls. Antileishmanial activ-
ity was also observed for BrB, which was toxic to
intracellular amastigotes of Leishmania amazonen-
sis but not to host mammalian cells (Rocha et al.
2019b). It is emphasized that in the Ames test, no
mutagenicity of BrA, BrB, and BrC was noted
(Resende et al. 2017).
The dichloromethane fraction of the hydroetha-
nolic root extract, which contains a mixture of the
three compounds, was also examined using in vivo
and in vitro assays. This extract did not induce
estrogenic activity (Resende et al. 2017); however,
gastroprotective (da Rocha et al. 2017), antinoci-
ceptive, and anti-inflammatory effects (da Rocha
et al. 2011), as well as alleviation of acute pain in
mice, were reported (Rodrigues et al. 2017).
Considering the positive cytotoxic and antitu-
moral activity observations obtained for the crude
extract of Fridericia platyphylla in cancer cells
(Serpeloni et al. 2020), this study aimed to assess
in vitro the possible bioactivities of the three bra-
chydins, the major constituents of the extract, using
the human prostate cancer-derived cell line, PC-3
and determine the molecular mechanisms under-
lying potential medicinal uses, providing insights to
new cancer therapies.
Materials and methods
Plant material and compound obtainment
Fridericia platyphylla root samples were collected in
April 2016 at Sant’Ana da Serra farm João Pinheiro,
Minas Gerais, Brazil (Location: 17º44ʹ45”S,
46º10ʹ44”W). A voucher specimen (n°17935) was
deposited at the Herbarium of the Federal
University of Ouro Preto, Brazil. This procedure
was approved by CGEN – Genetic Heritage
Management Council (A451DE4). Previously the
nomenclature Arrabidaea brachypoda (DC.)
Bureau (da Rocha et al. 2011) proposed by Prof.
Dr. Ana Maria Cristina Braga Messias from Federal
University of Ouro Preto was used, but its taxonomy
was later modified to Fridericia platyphylla (Cham.)
L.G. Lohmann by Kaehler (2020). The plant name
was verified with http://www.theplantlist.org.
All isolation processes and structure elucidation of
the three dimeric flavonoids identified as brachydin
2H. L. NUNES ET AL.
A (BrA), brachydin B (BrB), and brachydin C (BrC)
are described by da Rocha et al. (2014). The structure
elucidation was determined using nuclear magnetic
resonance (NMR) and high-resolution mass spectro-
metry (HRMS) techniques. There is only a single radi-
cal difference between them: a hydroxyl group is
found in BrA, a methoxyl group is present in BrB,
and a single hydrogen occurs in BrC (Figure 1). The
purities of the three compounds were determined to
employ ultra-performance liquid chromatography
(UHPLC- HMRS) analysis and were greater than 98%.
BrA, BrB, and BrC were diluted in dimethyl
sulfoxide (DMSO) purchased from Labsynth,
Diadema, SP, Brazil, and then phosphate buffer
saline (PBS) was added in equal quantity to obtain
a final solution of 50% DMSO, resulting in 0.5% in
cell culture.
Cell line and cell culture
The prostate tumor cell line PC-3 (American Type
Culture Collection – Manassas, VA, USA), which is
derived from a bone metastatic site, was cultivated
using Roswell Park Memorial Institute (RPMI –
Gibco, Grand Island, NY, USA) 1640 culture medium
supplemented with 10% fetal bovine serum (FBS –
Gibco, Grand Island, NY, USA) and 1% antibiotic-
antimycotic solution (Gibco, Grand Island, NY, USA)
at 37°C in an atmosphere containing 5% CO
2
and
95% relative humidity. The assays were performed
using cells cultured between the third and tenth pas-
sages. In addition to the absence of mycoplasma
determined by PCR, the authentication of PC3 cancer
cell line was confirmed at Molecular Diagnostic
Center of Barretos Cancer Hospital by short tandem
repeat (STR) DNA typing and performed according
to the International Reference Standard for
Authentication of Human Cell Lines using a panel
of eight (D5S818, D13S317, D7S820, D16S539, vWA,
TH01, TPOX, and CSF1P0) STR loci and gender
determination amelogenin (AMEL) (Dirks et al.
2005).
Cell viability assays
To perform the MTT, neutral red, and LDH activ-
ity release viability assays, 1 × 10
4
cells were seeded
per well in 96-well culture plates and to adhere for
24 hr. Subsequently, the medium was removed,
and cells washed in PBS. Then, negative control
(PBS), solvent control (DMSO 0.5%), positive
control (Docetaxel 10 µM), and treatments were
added to the medium without FBS for 24 hr. Nine
concentrations of BrA, BrB, and BrC (0.24, 0.75,
0.96, 1.5, 3.84, 6, 15.36, 24, or 30.72 µM), were
selected based upon a previous study (da Rocha
et al. 2014). Three biological replicates and four
technical replicates were performed for each cell
viability assay. The neutral red and MTT results
were expressed as cell viability %, considering the
negative control as 100%. LDH activity release
findings were expressed in absorbance relative to
LDH activity.
MTT assay
The MTT assay was performed as described by
Mosmann (1983) using 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyl tetrazolium bromide (CAS:
298–93-1, Sigma-Aldrich, St. Louis, MO, USA).
After treatments, cells were washed in PBS and
incubated in MTT solution (0.5 mg/ml) for 4 hr at
37°C. Then, the MTT solution was removed, and
Figure 1. The chemical structure of the brachydins.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 3
200 µl DMSO was added to each well. The absor-
bance at 570 nm was read with a spectrophotometer
(Biotek Eon, Winooski, VT, USA).
Neutral red assay
The neutral red assay was conducted following the
protocol of Repetto, Del Peso, and Zurita (2008)
using neutral red (CAS: 553–24-2, Sigma-Aldrich,
St. Louis, MO, USA). After the treatments, cells
were washed with PBS and incubated in neutral
red solution (40 µg/ml) for 3 hr at 37°C. Then, the
neutral red solution was removed, and the cells
were washed with PBS. To enable the reading,
0.1 ml destaining solution (50% ethanol, 49% deio-
nized water, and 1% glacial acetic acid) was added
to each well. The absorbance was read with a Biotek
Eon spectrophotometer at 540 nm.
LDH activity release assay
The Pierce™ LDH Cytotoxicity Assay Kit (Thermo
Fisher Scientific, Waltham, MA, USA) was used to
measure membrane integrity according to the man-
ufacturer’s instructions. An aliquot of 50 µl cell
culture medium was withdrawn to a new plate
after treatment, and kit solutions added into the
new plate. The absorbance was measured with
a Biotek Eon spectrophotometer at 490 nm and
680 nm.
Cell death assay
To perform the cell death assay, 1.5 × 10
5
cells were
seeded per well in 12-well culture plates, stabilized
in culture medium for 24 hr and then treated in
triplicate with three concentrations of BrA, BrB, or
BrC (1.5, 3.84, or 6 µM) selected according to the
results of previous viability assays. A positive (dox-
orubicin – DXR at 0.345 µM) and negative (PBS)
controls were also used. After treatment, cells were
washed with PBS and trypsinized. To assess cell
death morphology, a triple-staining assay using
propidium iodide (1 µg/ml), Hoechst 33342 (4 µg/
ml) and fluorescein diacetate (7.5 µg/ml) was per-
formed (Martínez-López et al. 2010; Proietti De
Santis et al. 2003; Ryhänen et al. 2009). Two hun-
dred cells per treatment were counted using an
Olympus BX 43 fluorescence microscope
(Olympus Microscopy, Europe), recording the
number of viable, necrotic, and apoptotic cells.
Pro-oxidant evaluation
To quantify intracellular reactive species, 1 × 10
4
cells were seeded in 96-well black plates and
allowed to stabilize for 24 hr. Two biological repli-
cates and four technical replicates were performed
for each treatment. Subsequently, treatment in
quadruplicate with 4 concentrations of BrA, BrB,
or BrC was performed (0.96, 1.5, 3.84, or 6 µM) for
4 different treatment periods (1, 3, 12, or 24 hr).
Hydrogen peroxide (H
2
O
2
) 1 mM was used as
a positive control 20 min before the treatment end
point and PBS as a negative control. To measure
reactive species, the probe CM-H
2
DCFDA (Life
Technologies, Eugene, OR, USA) was employed at
5 µM, following the manufacturer’s instructions
and lab protocols (Serpeloni et al. 2015; Tuttis
et al. 2018). The obtained values represent the
fluorescence absorbance of the probe.
Comet assay
The comet assay was performed as described by
Tice et al. (2000) and Singh et al. (1988). Initially,
2 × 10
5
cells were seeded in 24-well plates and
stabilized for 24 hr. The treatments were performed
in two biological and three technical replicates with
three concentrations of BrA, BrB, or BrC (1.5, 3.84,
or 6 µM), PBS as negative control and DXR
(0.345 µM) as positive control. The treatment
time was 4 hr, preventing DNA repair (OECD TG
489, 2016; Sasaki, Nakamura, and Kawaguchi 2007;
Singh et al. 1988). Cell viability was verified (≥90%)
with trypan blue exclusion using the Countess®
Automated Cell Counter (Life Technologies,
Eugene, OR, USA). Then, cells were mixed in low
melting point agarose (0.5%) and placed on slides
coated with normal melting pointing agarose
(1.5%). After drying, the coverslips were removed,
and slides submerged in lysis solution (2.5 M NaCl,
100 mM EDTA and 10 mM Tris, pH 10) for 1.5 hr
at 4°C. For electrophoresis, the slides were allowed
to rest in alkaline solution (300 mM NaOH, 1 mM
EDTA, pH >13) for 20 min at 4°C. Then, electro-
phoresis was carried out for 20 min, 25 V, and
300 mA. After, the slides were submerged in neu-
tralization buffer (0.4 M Tris, pH 7.5) for 15 min,
then fixed in absolute ethanol (99%) for 5 min and
left to air dry. For analysis, slides were stained with
4H. L. NUNES ET AL.
GelRed™ Nucleic Acid Gel Stain (Biotium,
Fremont, CA, USA) and analyzed using fluores-
cence microscopy. A total of 300 cells were counted
for each treatment using the software Comet
Imager v 2.2 (MetaSystems, Germany), and tail
length was determined.
Western blot analysis
To assess cell protein expression, 1 × 10
6
PC-3 cells
were seeded in 6-well plates and allowed to stabilize
for 24 hr. The treatments were performed with two
biological and three technical replicates at two con-
centrations of BrA (3.84 or 6 µM) and one concen-
tration of BrB, and BrC (3.84 µM). The
concentration of 3.84 µM was chosen because it is
close to the IC
50
value. For BrA that presented
higher IC
50
values, the concentration of 6 µM was
also added. After 24 hr incubation, the cells were
harvested and lysed with lysis buffer containing
Tris buffer (50 mM, pH 7.6 ~ 8), sodium chloride –
NaCl (150 mM), EDTA (5 mM), sodium orthova-
nadate – Na
3
VO
4
(1 mM), sodium fluoride – NaF
(10 mM), Na pyrophosphate (10 mM), and NP-40
(1%), supplemented with inhibitor cocktail con-
taining protease inhibitor DTT (1 mM), leupeptin
(1 μg/ml), aprotinin (1 μg/ml), phenylmethylsulfo-
nyl fluoride – PSMF (1 mM) and EDTA (1 mM) for
1 hr. After centrifugation at 12,000 g for 15 min at
4°C, the soluble proteins were quantified by
a Bradford assay kit (Sigma-Aldrich, St. Louis,
MO, USA) following the manufacturer’s instruc-
tions. Proteins (20 µg) were separated through
SDS-PAGE (12% and 15%) and transferred into
a nitrocellulose membrane (Hybond-C,
Amersham Biosciences, Little Chalfont, UK) with
a mini Trans-blot® Turbo Transfer System (Bio-
Rad, CA, USA). The membranes were blocked
with milk (5%) in Tris-buffered saline (TBS) and
0.1% Tween 20 (TBS-T) for 1 hr. Then, membranes
were incubated with the corresponding primary
antibodies overnight. The antibodies used were
anti-total PARP (Poly(ADP-ribose) polymerase),
anti-phospho AKT (AKT serine/threonine kinase),
anti-p21 (Cyclin-dependent kinase inhibitor 1A)
and anti-p27 (Cyclin-Dependent Kinase Inhibitor
1B) (Cell Signaling Technology, MA, USA) diluted
1:1000. α-Tubulin (Cell Signaling) was utilized as
a loading control. The membranes were washed
with TBS-T and incubated with peroxidase-
conjugated secondary antibody (Cell Signaling
Technology, MA, USA). The detection was per-
formed through chemiluminescence (ECL) western
blot detection reagents (RPN2109, GE Healthcare,
IL, USA), and the signal was detected and imaged
using ImageQuant™ LAS 4000 mini (GE
Healthcare). The results were quantified into den-
sitometry data using the software ImageJ v. 1.41.
Comparison of similar molecules
The search for molecules with ≥90% structural simi-
larity to brachydins (compound identification num-
ber – CID: – BrA −102339051; BrB – 102339053;
BrC – 102339052) was conducted using a PubChem
Chemical Structure Search.
Statistical analysis
Normality of all results was obtained using the
Shapiro–Wilk test. With the null hypothesis
accepted, analysis of variance (ANOVA) was applied
followed by a Dunnett’s test or Tukey’s test. The
comet assay results were analyzed with a non-
parametric Kruskal–Wallis test followed by Dunn’s
test. All statistical analyses were performed using the
free software RStudio© v. 1.1.442 (RStudio, Inc.).
Results
Cell viability
The MTT test illustrated that BrA, BrB, and BrC
produced cytotoxicity in the PC-3 prostate tumor
cell line (Figure 2). BrA, BrB, and BrC decreased
cell viability starting at 15.36 µM, 3.84 µM, and
3.84 µM, with IC
50
values of 23.41 µM, 4.28 µM,
and 4.44 µM, respectively.
Figure 3 shows results obtained in the neutral red
assay for the PC-3 cell line. BrA, BrB and BrC
induced cytotoxicity starting at 15.36 µM, 6 and
and 3.84 µM, respectively.
Further, the LDH activity release assay showed
that BrA, BrB, and BrC induced the death of PC-3
cells at concentrations of 15.36 µM, 3.84 µM, and
6 µM, respectively (Figure 4). In order to assess cell
death induced by brachydins, a triple-staining cell
death assay was performed which demonstrated
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 5
that BrA (6 µM) induced necrosis, BrC (1.5, 3.84, or
6 µM) produced apoptosis and BrB (6 µM) initiated
both necrosis and apoptosis (Table 1).
Pro-oxidant eects
Due to the cell death observed in the triple-staining
assay, reactive intracellular species were quantified
to verify if the results observed were related to pro-
oxidant effects in PC-3 cells treated with brachy-
dins. Only one concentration of BrA (6 µM) at one
time point (24 hr) initiated a significant increase in
reactive intracellular species (Figure 5). Both BrB
and BrC, which were cytotoxic at lower concentra-
tions, failed to markedly affect reactive species
generation.
Figure 2. Cell viability (%) of the PC-3 cell line evaluated by MTT assay after 24 hr of treatment with different concentrations of BrA, BrB
and BrC. NC – negative control (PBS: phosphate buffer saline). SV – solvent control (dimethyl sulfoxide 0.5%). PC – positive control
(Docetaxel 10 µM). Data are presented as the mean ± SD. *Statistically different from negative control (p < .05) by ANOVA followed by
Dunnett’s test.
Figure 3. Cell viability (%) of the PC-3 cell line evaluated by neutral red assay after 24 hr of treatment with different concentrations of
BrA, BrB and BrC. NC – negative control (PBS: Phosphate buffer saline). SV – solvent control (dimethyl sulfoxide 0.5%). PC – positive
control (Docetaxel 10 µM). Data are presented as the mean ± SD. *Statistically different from negative control (p < .05) by ANOVA
followed by Dunnett’s test.
Figure 4. LDH assay of PC-3 cells treated for 24 hr with different concentrations of BrA, BrB and BrC. NC – negative control (PBS:
Phosphate buffer saline). SV – solvent control (dimethyl sulfoxide 0.5%). PC – positive control (cells lysed by manufacturer’s reagent).
Data are presented as the mean ± SD. *Statistically different from negative control (p < .05) by ANOVA followed by Tukey’s test.
6H. L. NUNES ET AL.
Table 1. Percentage of PC-3 cells that were viable, apoptotic, and necrotic after treatment with three
different concentrations of BrA, BrB, and BrC and their respective controls.
Cells (%)
Treatment Viable Necrotic Apoptotic
NC (PBS) 96.00 ± 2.00 1.67 ± 1.53 2.33 ± 1.53
PC (DXR −0.345 µM) 80.33 ± 1.53* 9.00 ± 2.65* 10.67 ± 2.08*
SV (DMSO 0.5%) 91.67 ± 2.52 5.00 ± 1.73 2.00 ± 1.00
Brachydin A (µM)
1.5 96.00 ± 2.65 3.67 ± 2.08 0.33 ± 0.58
3.84 94.67 ± 0.58 4.33 ± 1.53 1.00 ± 1.00
6.00 91.67 ± 2.08 7.00 ± 2.65* 1.33 ± 0.58
Brachydin B (µM)
1.5 81.67 ± 8.98 14.83 ± 9.36 3.50 ± 0.50
3.84 85.50 ± 3.04 8.17 ± 2.02 6.33 ± 1.53
6.00 64.50 ± 3.50* 17.00 ± 2.18* 18.50 ± 3.90*
Brachydin C (µM)
1.5 90.17 ± 3.21 1.67 ± 0.29 8.17 ± 3.33*
3.84 89.67 ± 2.25 4.33 ± 2.02 6.00 ± 1.50*
6.00 90.00 ± 1.32 2.33 ± 0.58 7.67 ± 1.89*
NC – negative control (PBS: Phosphate buffer saline). SV – solvent control (DMSO 0.5%). PC – positive control (DXR
0.345 µM). Data are presented as the percentage (%) of cell morphology type found with triple staining.
*Statistically different from the negative control by ANOVA followed by Tukey’s test (p < 0.05).
Figure 5. Quantification assay of intracellular reactive species using the CM-H
2
DCFDA probe. NC – negative control (PBS: Phosphate
buffer saline). SV – solvent control (dimethyl sulfoxide 0.5%). PC – positive control (H
2
O
2
1 mM). Data are presented as the mean ± SD.
*Statistically different from negative control (p < .05) by ANOVA followed by Tukey’s test.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 7
Genotoxic eects
None of the concentrations evaluated of brachydins
were genotoxic to PC-3 cells after 4 hr incubation
(data not shown).
Protein expression
The results obtained from western blot analysis are
presented in Figure 6. The protein p21 promotes
cell cycle arrest in response to a variety of stimuli
and was overexpressed in PC-3 cells treated with
3.84 µM of BrB or BrC but not BrA. However, the
expression of the protein p27, which also regulates
the cell cycle, was not markedly altered by any of
the treatments. pAKT expression was reduced in
cells treated with BrA at 6 µM and BrB at 3.84 µM,
indicating dysregulation of cellular survival pro-
cesses. The expression of cleaved PARP was higher
in all treatments examined, suggesting cell death by
apoptosis.
Similar molecules have similar eects
The similarity of brachydins with other molecules
was investigated using the public database PubChem
from the National Center for Biotechnology
Information (NCBI). Among 1330 molecules with
similarity (≥90%) to brachydins, 101 were already
evaluated in bioassays, and 44 of them were active.
Some similar molecules exhibited cytotoxicity in
several tumor cell lines (HepG2 – liver; MCF-7 and
T-47D – mammary epithelium; A549 – lungs;
SV480 – colon) but did not induce mutagenic or
genotoxic effects (Table 2).
Discussion
The three classical prostate cancer cell lines,
namely, DU145, PC-3, and LNCaP, are most widely
used in prostate cancer research (Namekawa et al.
2019). Further, the PC-3 cell line was used in the
present study as an in vitro model to assess the
effects of brachydins.
Different methodologies may be employed for
analysis of cytotoxicity of compounds and the com-
bination between these is an important factor
(Specian et al. 2016). The biological effects observed
after the combination of tests may enable inference
regarding the mechanisms of action of the com-
pounds, which may result in the identification of
target molecules and/or organelles to determine
their biological activities (Atanasov et al. 2015). In
the present study, different assays were performed
to evaluate the effects of brachydins to different
cellular targets such as respiratory chain activity
(MTT), lysosomal activity (Neutral Red), and
membrane integrity (LDH activity release). It is
noteworthy that similar results were detected with
the brachydins.
Table 2. PubChem public database study summary and compar-
ison of brachydins and similar molecules.
Biological activities
Similar mole-
cules
(PubChem)
Brachydins
Reference
Cytotoxicity ✔ ✔ Present study
Activity against pathogens ✔ ✔ da Rocha et al.
(2014)
Genotoxicity ✘ ✘ Present study
Mutagenicity ✘ ✘ Resende et al.
(2017)
Induction of reactive intracellular
species
✘ ✘ Present study
✔ presence of effect, ✘ absence of effect.
Figure 6. Densitometry quantification of p21, p27, pAKT and
cleaved PARP of western blot with PC-3 cell lines treated with
different concentrations of BrA, BrB, and BrC after 24 hr. NC –
negative control (PBS: Phosphate buffer saline). PC – positive
control (DXR 0.345 µM). A – Brachydin A, B – Brachydin B, C –
Brachydin C. Data are presented as the mean ± SD. *Statistically
different from negative control (p < .05) by ANOVA followed by
Tukey’s test.
8H. L. NUNES ET AL.
The cytotoxic concentration of each brachydin was
determined to utilize the PC-3 cell line. The IC
50
values found were 23.41, 4.28, and 4.44 µM for BrA,
BrB, and BrC, respectively. These values are also
similar to those obtained in PC-3 cells in vitro with
the chemotherapy drug cisplatin (3.3 µM), employed
in the treatment of prostate cancer (Altaf et al. 2019).
Da Rocha et al. (2014) estimated the IC
50
values of
brachydins BrB and BrC in Trypanosoma cruzi (5.3
and 6.6 µM, respectively), intracellular amastigotes
(6 µM and 6.8 µM, respectively) and in macrophages
(15.6 µM and 17.3 µM), while BrA was inactive.
In addition to inducing cytotoxicity, chemother-
apeutic compounds might influence cell cycle pro-
gression and cell death mechanisms, which are
mechanisms that limit cancer progression and are
important parameters for the evaluation of potential
chemotherapy drugs (Sikes 2007). In order to under-
stand the influence of brachydins on cellular path-
ways, some of the intracellular pathways following
short-term exposure to these plant extracts were
examined in prostate cancer cells. The AKT protein,
which is involved in the PI3K/AKT/mTOR meta-
bolic pathway, regulates quiescence, proliferation,
and cell longevity (King, Yeomanson, and Bryant
2015) and its activity was significantly reduced in
PC-3 cells after treatment with BrA and BrB.
Further, BrB and BrC also decreased cell cycle pro-
gression through induction of p21 expression. Since
p27 expression is not markedly changed by brachy-
dins, p21 may have prevented cyclin-CDK4/6 com-
plex formation, inducing cell cycle arrest at G1
phase. This finding might be confirmed in further
studies with flow cytometry. These data indicate
a possible interference of dimeric flavonoids in the
cell cycle as well as in cell growth and metabolic
processes, which may favor and explain the observed
apoptotic cell death (Nitulescu et al. 2018).
With respect to cell death, LDH activity release
and cleaved PARP expression support the hypoth-
esis that brachydins are cytotoxic for human pros-
tate tumor cell line. The LDH assay data are
consistent with observations for cell death. The
PARP protein is involved in DNA repair and pro-
grammed cell death, and the cleaved version of
PARP, which occurs in apoptosis cell death, is fre-
quently used as a marker of this type of cell death
(Morales et al. 2014). The increase in cleaved PARP
expression after cell treatment with brachydins
suggests the induction of apoptosis processes by
these compounds.
The exact type of cell death induced could not be
identified with the assays performed because triple
staining demonstrated both apoptotic and necrotic
morphology. Nevertheless, overexpression of PARP
signaled apoptosis, and the results of the LDH
activity release assay suggested cell death by mem-
brane disruption such as necrosis. It is noteworthy
that western blot analysis revealed brachydins
influenced the expression of cell cycle and cell
death proteins, corroborating the viability assays,
MTT and neutral red, which measure death and
proliferation.
Oxidative stress induced by unbalanced control
between reactive species and antioxidants might be
related to necrotic cell death (Choi et al. 2009).
However, no marked alteration in intracellular reac-
tive species was noted after brachydin treatment
except for 6 µM of BrA, which elevated the reactive
species level with 24 hr treatment. This concentration
also induced necrosis in the triple-staining assay.
The identification of genotoxicity and mutagenicity
is crucial for the evaluation of new compounds, since
these processes are related to the capability of
a compound to damage genetic material (Eastmond
et al. 2009). Resende et al. (2017) used the Ames test to
demonstrate that the extracts obtained from leaves,
roots, and stalks of F. platyphylla extract were not
mutagenic. F. platyphylla root extract increased the
frequency of nuclear buds in tumor gastric cells
(Serpeloni et al. 2020). No genotoxicity and mutageni-
city were detected for brachydins isolated from
F. platyphylla in the present study (comet assay), nor
by Resende et al. (2017) in Ames test, indicating
noDNA strand breaks or mutations in genetic
material.
To compare brachydin A with other molecules,
a computational method was used based upon
Tanimoto’s coefficient (Maggiora et al. 2014) in
PubChem. This method is employed in chemical
informatics and drug discovery and enabled us to
verify our results in relation to similar molecules
from the literature. In the search for similar mole-
cules, some active molecules, such as licoricidin
(CID: 480865) and glyasperin D (CID: 480860),
were cytotoxic to HepG2, MCF-7, A459, and
SV480 tumor cells. In proliferation assays, two
molecules showed divergent activity: erycibenin
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 9
E (CID: 16093658) inhibiting proliferation in
tumor lymphocytes, and vestitol (CID: 182259) sti-
mulating proliferation in T-47D tumor cells. These
data demonstrated similarities to the cytotoxic pro-
files of brachydins in tumor cells, as well as activity
against pathogens (de Sousa Andrade et al. 2020;
Rocha et al. 2019b), and absence of genotoxicity
and mutagenicity (Resende et al. 2017).
Due to the cytotoxicity of BrB at lower doses than
others (reducing the necessity of raw material), BrB is
a more promising compound for further investiga-
tion. With only one radical difference in the three
brachydins evaluated in this study (BrA – hydroxyl,
BrB – methoxyl, and BrC – hydrogen), it is still not
possible to conclude if these differences confer distinct
biological activities of these phytochemicals. Thus,
additional studies need to be performed in order to
understand the influence of brachydins on different
cellular pathways.
Conclusions
The viability/cytotoxicity assays performed indicate
that the flavonoids brachydins A, B, and C,
obtained from the root extract of Fridericia platy-
phylla (Cham.) L.G. Lohmann, induce cytotoxicity
in the human prostate tumor cell line PC-3.
Although the present study examined the effect of
brachydins only in PC-3 cells, it was possible to
define minimum toxicity thresholds, which might
be used in subsequent studies to assess efficacy
without adverse consequences. The results showed
that the cytotoxicity and lower proliferation rate are
sustained by the responses observed in western blot
assays with p21, pAKT, and PARP proteins and by
computational comparison between similar mole-
cules in a public database. These compounds need
to be further investigated to (1) assess whether
effects are selective on normal cells and (2) better
understand the underlying mechanisms of action
and molecular targets to be potentially considered
as phytopharmaceuticals for disease treatment,
especially for prostate cancer.
Authors’ contributions
Conceptualization and design: Serpeloni, J.M.; Cólus, I.M.S.;
Nunes, H.L. Provision of study materials and funding acquisi-
tion: Serpeloni, J.M.; Rocha, C.Q.; Reis, R.M.; Cólus, I.M.S.
Collection and assembly of data: Nunes, H.L.; Tuttis, K.;
Lengert, A.H.; Nascimento, J.R.; Silva; V.A.O. Data analysis
and interpretation: Nunes, H.L.; Serpeloni, J.M.; Cólus, I.M.S.
Writing – original draft and editing: Nunes, H.L.; Cólus, I.
M.S.; Serpeloni, J.M.;
Supervision and project administration: Cólus, I.M.S.;
Serpeloni, J.M.
Conicts of interest
No potential conflict of interest was reported by the authors.
Funding
The authors are thankful to CNPq (No. 401516/2016-4),
FAPEMA (No. 00849/18), CAPES (Financial code 001), and
the State University of Londrina and Barretos Cancer Hospital
(HCB), which made the development of this study possible.
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