4-Fluorobenzaldehyde limonene-based thiosemicarbazone induces apoptosis in PC-3 human prostate cancer cells

Article abstract of DOI:10.1016/j.lfs.2018.04.024

Aims: This study evaluated parameters of toxicity and antiproliferative effects of (+)-N(1)-4-fluorobenzaldehyde-N(4)-{1-methyl-1-[(1R)-4-methylcyclohexene-3-il]-ethyl}-thiossemicarbazone (4-FTSC) in PC-3 adenocarcinoma prostate cells. Main methods: Cytotoxicity of 4-FTSC in PC-3 cells was evaluated using MTT assay. Morphology examination of PC-3 cells treated with 4-FTSC was also performed as well as the cell death mechanisms induced were investigated using flow cytometry. Parameters of toxicity of 4-FTSC was conducted by the investigation of its potential myelotoxicity and lymphotoxicity, hemolytic activity and acute oral toxicity profile. Key findings: 4-FTSC showed promising cytotoxic effects against PC-3 cells (IC₅₀ = 18.46 μM). It also triggered apoptotic morphological changes, phosphatidylserine externalization and a significant increase of DNA fragmentation in PC-3 cells. Moreover, 4-FTSC did not show changes in the PC-3 cell cycle with levels of p21, p27, NF?B and cyclin D1 similar to those found in both control and treated cells. 4-FTSC also promoted an increase of p53 levels associated with mitochondrial impairment through loss of ?Ψm and ROS overproduction. 4-FTSC-induced cell death mechanism in PC-3 cells involved activation of caspase-3/-7 through apoptosis intrinsic pathway via caspase-9. Regarding toxicological profile, 4-FTSC showed in vitro lymphotoxicity, although with low cytotoxicity for bone marrow progenitors and no hemolytic potential. Moreover, it was classified as GHS category 5 (LD₅₀ > 2000–5000 mg/Kg), suggesting it has low acute oral systemic toxicity. Significance: 4-FTSC seems to be a promising candidate to be used as a clinical tool in prostate cancer treatment. Further studies are required to better clarify its toxicopharmacological effects found in this compound.

Full text of DOI:10.1016/j.lfs.2018.04.024

Accepted Manuscript
4-Fluorobenzaldehyde
limonene-based
thiosemicarbazone
induces apoptosis in PC-3 human prostate cancer cells
Bruna dos Santos Rodrigues, Renato Ivan de Ávila, Polyana
Lopes Benfica, Ludmila Pires Bringel, Cecília Maria Alves de
Oliveira, Fábio Vandresen, Cleuza Conceição da Silva, Marize
Campos Valadares
PII:
DOI:
Reference:
S0024-3205(18)30205-4
doi:10.1016/j.lfs.2018.04.024
LFS 15659
To appear in:
Life Sciences
Received date:
Revised date:
Accepted date:
2 February 2018
6 April 2018
16 April 2018
Please cite this article as: Bruna dos Santos Rodrigues, Renato Ivan de Ávila, Polyana
Lopes Benfica, Ludmila Pires Bringel, Cecília Maria Alves de Oliveira, Fábio Vandresen,
Cleuza Conceição da Silva, Marize Campos Valadares , 4-Fluorobenzaldehyde limonene-
based thiosemicarbazone induces apoptosis in PC-3 human prostate cancer cells. The
address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Lfs(2017), doi:10.1016/j.lfs.2018.04.024
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ACCEPTED MANUSCRIPT
4-Fluorobenzaldehyde limonene-based thiosemicarbazone induces apoptosis
in PC-3 human prostate cancer cells
Bruna dos Santos Rodrigues1, Renato Ivan de Ávila1, Polyana Lopes Benfica1, Ludmila Pires
Bringel2, Cecília Maria Alves de Oliveira2, Fábio Vandresen3, Cleuza Conceição da Silva3,
Marize Campos Valadares1,*
1Laboratório de Farmacologia e Toxicologia Celular – FarmaTec, Faculdade de Farmácia,
Universidade Federal de Goiás, Goiânia, GO, Brazil.
2Instituto de Química, Universidade Federal de Goiás, Goiânia, GO, Brazil.
3Departamento de Química, Universidade Tecnológica Federal do Paraná, Londrina, PR,
Brazil.
4Departamento de Química, Universidade Estadual de Maringá, Maringá, PR, Brazil.
*Corresponding author. Address: Faculdade de Farmácia – Universidade Federal de Goiás,
Rua 240 esquina com 5ª Avenida, s/n, Setor Universitário, Goiânia, GO, Brazil. CEP:
74605.170.
E-mail address: marizecv@ufg.br / marizeufg@gmail.com

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Abstract
Aims: This study evaluated parameters of toxicity and antiproliferative effects of (+)-N(1)-4-
fluorobenzaldehyde-N(4)-{1-methyl-1-[(1R)-4-methylcyclohexene-3-il]-ethyl}-
thiossemicarbazone (4-FTSC) in PC-3 adenocarcinoma prostate cells.
Main methods: Cytotoxicity of 4-FTSC in PC-3 cells was evaluated using MTT assay.
Morphology examination of PC-3 cells treated with 4-FTSC was also performed as well as
the cell death mechanisms induced were investigated using flow cytometry. Parameters of
toxicity of 4-FTSC was conducted by the investigation of its potential myelotoxicity and
lymphotoxicity, hemolytic activity and acute oral toxicity profile.
Key findings: 4-FTSC showed promising cytotoxic effects against PC-3 cells (IC50=18.46
µM). It also triggered apoptotic morphological changes, phosphatidylserine externalization
and a significant increase of DNA fragmentation in PC-3 cells. Moreover, 4-FTSC did not
show changes in the PC-3 cell cycle with levels of p21, p27, NFĸB and cyclin D1 similar to
those found in both control and treated cells. 4-FTSC also promoted an increase of p53 levels
associated with mitochondrial impairment through loss of ∆Ψm and ROS overproduction. 4-
FTSC-induced cell death mechanism in PC-3 cells involved activation of caspase-3/-7
through apoptosis intrinsic pathway via caspase-9. Regarding toxicological profile, 4-FTSC
showed in vitro lymphotoxicity, although with low cytotoxicity for bone marrow progenitors
and no hemolytic potential. Moreover, it was classified as GHS category 5 (LD50 > 2000-5000
mg/Kg), suggesting it has low acute oral systemic toxicity.
Significance: 4-FTSC seems to be a promising candidate to be used as a clinical tool in
prostate cancer treatment. Further studies are required to better clarify its
toxicopharmacological effects found in this compound.
Keywords: thiosemicarbazone; limonene; apoptosis; prostate cancer.

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1. Introduction
Plant-derived terpenoids are a large group of bioactive molecules with great
structural and biological diversity [1,2]. They have been used as leads to obtain new bioactive
analogues, especially in anticancer drug discovery [3]. Among terpene class, R-(+)-limonene
[1-methyl-4-(1-methylethenyl) cyclohexane], a dietary monoterpene usually found in citrus
fruits (e.g. orange, lemon, lime, grapefruit) [4], has been widely studied due to its promising
antineoplastic properties [5].
Anticancer properties of this compound as well as limonene-rich natural products
have been related to pathways that modulate inflammation, oxidative stress [6], angiogenesis
and metastasis inhibition [7], autophagy [8] and apoptosis induction [7,9]. However, high
hydrophobicity, volatility and loss of activity due to oxidative degradation restrict the use of
limonene [4,10,11]. Considering molecules with this unstable profile, molecular
modifications have been proposed to overcome these limitations along with improve on
biological properties [12].
Exploring this strategy, Vandresen et al. [13] synthesized novel limonene-based
thiosemicarbazones through retaining of chiral integrity of limonene. Among them, (+)-N(1)-
4-fluorobenzaldehyde-N(4)-{1-methyl-1-[(1R)-4-methylcyclohexene-3-il]-ethyl}-
thiossemicarbazone (4-FTSC) (4) was obtained by synthetic route consisting in the addition of
thiocianic acid (HSCN) to limonene (1) leading to the respective isothiocyanoterpene (2). It
was then treated with hydrazine to result in the thiosemicarbazide (3). 4-FTSC (4) was
obtained by reaction through the condensation of equimolar amounts of thiosemicarbazide (3)
with 4-fluorobenzaldehyde using acidic catalyst and ethanol [13] (Figure 1). Preliminary
studies showed that 4-FTSC (4) has cytotoxic effects on PC-3 adenocarcinoma prostate cells,
which were higher than those found for paclitaxel, standard drug for treating human prostate
cancer [13]. Thus, this compound seems to be promising as a new agent against prostate
cancer. However, the cell death-induced pathways as well as toxicity data of 4-FTSC (4) have
not been elucidated.
Thiosemicarbazones have been inserted in many anticancer agents [14] such as
triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) whose clinical trials have
demonstrated a good efficacy, in a single or in combination therapeutic schedules, in the
treatment of cervical cancer [15,16]. Mammalian ribonucleoside reductase inhibition, an
essential enzyme in DNA biosynthesis, has been described as a possible mechanism of action
promoted by thiosemicarbazones in cancer cells [17]. The presence of bulky groups at the

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N(4) position of thiosemicarbazones, as verified in 4-FTSC (4), has shown improvement in
their antiproliferative effects [18,19].
Given the above, the present study was directed towards an understanding of the
antiproliferative effects of 4-FTSC (4) in PC-3 adenocarcinoma prostate cells. The
investigation focused on the mechanisms of cell death to elucidate the possible pathways
involved. Furthermore, toxicity parameters were also investigated to better understand the
toxicological profile of 4-FTSC (4).

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2. Material and methods
2.1 Chemicals
Dulbecco’s modified Eagle’s medium (DMEM), nutrient mixture HAM F-12,
Roswell Park Memorial Institute (RPMI)-1640 medium, penicillin, L-glutamin, sodium
bicarbonate, rhodamine 123, propidium iodide and granulocyte macrophage colony
stimulating factor (GM-CSF) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
May-Grünwald-Giemsa staining was acquired from Merck (Darmstadt, HE, Germany). Fetal
bovine serum (FBS), Hoechst 33342 dye and 3-(4,5-dimetylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased from Invitrogen (Gibco, NY, USA),
whereas ethanol, methanol, acetic acid and dimethyl sulfoxide (DMSO) were obtained from
Vetec (Rio de Janeiro, RJ, Brazil). Sulfuric acid and Triton X-100 were purchased from Synth
(São Paulo, SP, Brazil). Agar was acquired from Difco (CA, USA), whereas standard
granulated chow for mice, xylazine and ketamine were purchased from Presence Nutrição
Animal (São Paulo, SP, Brazil), Syntec (Cotia, SP, Brazil) and König (Santana de Parnaíba,
SP, Brazil), respectively. The following antibodies were obtained from Santa Cruz
Biotechnology (Paso Robles, CA, USA): p53 (clone DO-7), cyclin D1 (clone H-295), NFκB
(clone E-10), p27 (clone F-8) and p21 (clone F-5).
2.2 Obtaining of 4-FTSC (4)
4-FTSC (4) (molecular weight: 333 g/mol) was obtained and characterized by IR
1
spectra, direct infusion ESI-(+)-FTMS, H, and 13C NMR evaluations as described by
Vandresen et al. [13]. For in vitro assessments, a stock solution (15 mM) was prepared in
DMSO and stored at -20°C. It was diluted in culture medium containing heat-inactivated FBS
(10%, v/v) for a final concentration of DMSO at 0.01% (v/v), which it was previously
established as non-toxic to any cell type used here. For in vivo assays, 4-FTSC (4) doses were
prepared by dilution in sunflower oil (vehicle) before administration to the animals, as
recommended by OECD [20].
2.3 Cell cultures
Balb/c 3T3-A31 fibroblasts were kindly donated by Dr. Mari Cleide Sogayar
(Chemistry Institute, University of São Paulo, SP, Brazil), while PC-3 adenocarcinoma
prostate cells were obtained from the Rio de Janeiro Cell Bank (Rio de Janeiro, RJ, Brazil).
3T3 and PC-3 cells were cultured in DMEM and HAM-F12 medium, respectively, containing

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heat-inactivated FBS (10%, v/v), L-glutamine (2 mM), HEPES (4.5 mM), sodium bicarbonate
(0.17 M), penicillin (100 IU/mL) and streptomycin (100 μg/mL), in a humidified atmosphere
of 5% CO2 in air at 37ºC. As PC-3 and 3T3 cell lines are adherent cells, they were removed
from the culture flasks (25 or 75 cm2) using trypsin (0.025%)/EDTA (0.02%) solution when
the confluence reached 60-80%.
2.4 Animals
Swiss male mice, weighting between 27 and 31 g, were obtained from the Bioterium
at the Federal University of Goiás (UFG) (Goiânia, GO, Brazil). The animals were kept under
constant environmental conditions with light-dark (12:12 h) cycles and controlled temperature
(23 ± 2ºC). Water and food (standard granulated chow) were provided ad libitum. All efforts
were performed to ensure the welfare of animals. In addition, experimental procedures and
protocols were reviewed and approved by the Research Ethics Committee of the Federal
University of Goiás. At the end of each experiment, the animals were previously anesthetized
with a mixture of xylazine (10 mg/kg) and ketamine hydrochloride (100 mg/kg), administered
intraperitoneally, and then euthanized by cervical dislocation [21].
2.5 Cytotoxicity assay
Viability of PC-3 cells and lymphocytes after treatment with 4-FTSC (4) was
determined
by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
reduction assay, adapted from Mosmann [22]. Briefly, 1 x 105 cells were seeded overnight in
96-well plates. After that, they were treated with or without 4-FTSC (4) in eight different
concentrations (1.17-150 µM, 6 wells per concentration) for 48 h. After that, 10 µL/well MTT
(5 mg/mL) were added and the plates were incubated for aditional 3 h. After this period, the
supernatant was removed and DMSO (100 µL/well) was added to solubilize the formazan
formed. The absorbance was measured at 560 nm using a plate spectrophotometer (Thermo
Scientific Multiskan Spectrum, Boston, MA, USA). Cell viability was expressed as a
percentage of the control and IC50 value (concentration that inhibited cell growth by 50%
compared to untreated group) was obtained. For the subsequent assays, IC50 value of 4-FTSC
(4) (18.46 µM) was used.
2.6 Cell morphology examination
Morphological changes induced by 4-FTSC (4) was evaluated using May-Grünwald-
Giemsa dye or DNA binding dye Hoechst 33342 based on protocols described by Ávila et al.

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[23]. Briefly, 1 x 105 PC-3 cells were seeded in polystyrene plate (35 x 10 mm) (Becton
Dickinson, NJ, USA) for overnight. Cells were then treated without or with 4-FTSC (4)
(18.46 µM) for 48 h. After that, cells were washed with PBS and stained with May-Grünwald-
Giemsa or Hoechst 33342 dye, at room temperature for 10 min. Qualitative analysis of the
cells was performed and representative images of three independent assays were taken using
light (Leica DM 2000, Solms, Germany) or fluorescence (DMI 4000 B Leica Microsystems,
Bannockburn, IL, USA) microscope.
2.7 Flow cytometry analysis
PC-3 cells (1 x 105 cells) were seeded in culture flasks (25 cm2) overnight.
Posteriorly, cells were treated without or with 4-FTSC (4) (18.46 µM) for 48 h. Cells were
trypsinized and mechanistic evaluations were carried out to investigate the possible 4-FTSC
(4)-induced cell death pathways in PC-3 cells using BD FACSCanto II flow cytometer (BD
Biosciences, San Jose, CA, USA). For each analysis, 10,000 events were recorded.
2.7.1 Annexin V-FITC/PI double-staining
The assays were carried out using Annexin V Apoptosis Detection Kit FITC
(eBioscience®), in accordance with the manufacturer’s instructions. The following
populations were identified: (1) necrotic cells in the upper-left quadrant (high PI and low
Annexin V-FITC signals, PI+/A-); (2) early apoptotic cells in the lower-right quadrant (low PI
and high Annexin V-FITC signals, PI-/A+); (3) late apoptotic cells in the upper-right quadrant
(high PI and Annexin V-FITC signals, PI+/A+); and (4) viable cells in the lower-left quadrant
(low PI and Annexin V-FITC signals, PI-/A-).
2.7.2 DNA fragmentation
After treatment, cells were washed once with PBS followed by 1 h incubation with
lysis buffer containing sodium citrate (0.1%, w/v), Triton X-100 (0.1%, v/v) and PI (2 µg/mL)
at 4ºC. Subsequently, the red PI fluorescence was excited by a wavelength of 488 nm and
detected at 620-700 nm filter using flow cytometer.
2.7.3 Cell cycle assessment
The untreated and 4-FTSC (4)-treated cells were washed twice with ice-cold PBS,
fixed with 1 mL of ice-cold 70% ethanol and stored overnight at 4ºC. Cells were then washed
once with ice-cold PBS and incubated with 1 mL of PBS containing RNase type I (200

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µg/mL) and PI (50 µg/mL) for 1 h, in the dark at 4°C. After that, cells were analyzed using
flow cytometer.
2.7.4 Cell cycle regulatory proteins evaluations
After 4-FTSC (4) treatment, cells were washed once with PBS and then incubated
with 250 µL of Cytofix/CytopermTM solution (BD Bioscience) for 20 min. After that, cells
were washed twice with PBS-tween 20 (T20) (0.5%, v/v) and incubated with the following
antibodies in PBS-T20, according to the manufacturer’s instructions (Santa Cruz
Biotechnology): p53 (clone DO-7), cyclin D1 (clone H-295), NFκB (clone E-10), p27 (clone
F-8) and p21 (clone F-5). After 15 min of incubation, the cells were washed in PBS-T20,
resuspended in 200 µL PBS and analyzed using flow cytometer.
2.7.5 Mitochondrial membrane potential (ΔΨm) evaluation
ΔΨm evaluation of PC-3 cells treated without or with 4-FTSC (4) was analyzed by
cell incorporation of the fluorescent rhodamine 123 dye. In brief, cells were washed once with
PBS followed incubation with rhodamine 123 (1 µg/mL) at 37ºC. After 30 min incubation,
cells were washed with PBS and resuspended with 200 µL PBS for flow cytometer analysis.
2.7.6 Measurement of reactive oxygen species (ROS) levels
Determination of intracellular ROS levels was carried out using acetate 2,7-
dichlorodihydrofluorescein diacetate (DCFH-DA) dye. In intracellular environment, DCFH-
DA origins the compound 2,7-dichlorodihydrofluorescein (DCFH) through hydrolysis process
by cytosolic esterases. In turn, DCFH can react with intracellular oxidants, resulting the
highly fluorescent 2,7-dichlorofluorescein (DCF) [24]. Relative fluorescence intensity of DCF
is considered proportional to the intracellular ROS levels. After 4-FTSC (4) treatment, cells
were washed once with PBS and incubated with 200 µL of PBS containing DCHF-DA (10
µM) for 30 min at 37ºC. Thereafter, cells were washed again with PBS and 200 µL of the
same buffer were added to each tube for flow cytometer assessment.
2.7.7 Determination of caspase activities
The caspase-3/-7, -8 and -9 activities were evaluated using the CaspaTagTM Caspase
-3/7, -8 and -9 In Situ Assay kit (MilliporeTM), in accordance with the manufacturer’s
instructions.

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2.8 Toxicological assessment of 4-FTSC (4)
2.8.1 Investigation of the potential myelotoxicity
Evaluation of potential myelotoxicity of 4-FTSC (4) in murine hematopoietic
progenitor cells was performed by 3D clonogenic assay of colony forming unit-
granulocyte/macrophage (CFU-GM) with modifications of the protocol described by Pessina
et al. [25]. Briefly, one mouse previously anesthetized was euthanized and both femurs were
aseptically extracted for obtaining bone marrow cells. A cellular suspension (1 x 105
cells/mL) was prepared in solution containing DMEM medium 2x concentrated (30%, v/v),
heat-inactivated FBS (20%, v/v), agar (0.6%, p/v) solution (50%, v/v) and GM-CSF (0.5
ng/mL). Cells (2 x 105 cells) were then seeded in polystyrene plate (35 x 10 mm) without or
with eight different concentrations of 4-FTSC (4) (1.17-150 µM) and incubated for 7 days in
5% CO2 at 37ºC. After that, the colonies were scored using stereoscopic microscope (Leica
MZ6, Solms, Germany) and compared to control group (100% of viability). One mouse was
used for each analysis, in triplicate, through two independent assays.
2.8.2 Evaluation of the potential lymphotoxicity
Human whole blood, randomly and anonymously obtained as residual product from a
laboratory, was used to obtain lymphocytes by density centrifugation with Ficoll gradient.
Lymphocytes were cultured in RPMI-1640 medium containing 10% heat-inactivated FBS, in
5% CO2 at 37ºC. Cells (1 x 105 cells) were then seeded in 96-well plates and treated with or
without 4-FTSC (4) in eight different concentrations (1.17-150 µM, 6 wells per concentration)
for 48 h and cell viability evaluated by MTT assay as previously described (item 2.5).
2.8.3 Hemolytic activity
The hemolytic activity of 4-FTSC (4) was investigated following the modified
method by Gaeti et al. [26]. In brief, 100 µL of human erythrocytes (2%, v/v) in saline (0.85%
NaCl solution containing 10 mM CaCl2) were incubated, in sextuplicate, without or with 100
µL of different concentrations of 4-FTSC (4) (0.58-150 µM). In parallel, blank, negative
(vehicle of 4-FTSC, DMSO at 0.01%) and positive (0.5% Triton X-100 to obtain 100%
hemolysis) controls were performed. After incubation in constant agitation for 1 h at room
temperature, the supernatant was removed and the absorbance of released hemoglobin was
measured at 540 nm. Absorbance values were then used to obtain hemolysis ratio (%) as the
following equation: hemolysis (%) = [(Abs4-FTSC – AbsBlank) / (AbsTriton X-100 – AbsBlank)] x 100.

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2.8.4 Neutral red uptake (NRU) assay
The NRU assay was performed based on the standard protocol of Borenfreund and
Puerner [27] modified by ICCVAM [28]. In brief, 3T3 cells (3 x 103 cells) were seeded in 96-
well flat plate overnight. Cells were then treated without or with different concentrations of 4-
FTSC (4) (2.3-300 µM, 6 wells per concentration) for 48 h. Thereafter, the plate was washed
once with PBS (250 µL/well) followed by addition of neutral red (25 µg/mL) solution (250
µL/well). After additional 3 h incubation, cells were washed again and 100 µL of neutral red
desorb (ethanol, acetic acid and ultrapure water, 50:1:49) solution were added to the plate.
The absorbance was measured at 560 nm. The IC50 value was determined and LD50 was
estimated by the following equation [29]: Log (LD50) = 0.545 x log (IC50) + 0.757.
2.8.5 Acute oral systemic toxicity in mice
Evaluation of acute oral toxicity of 4-FTSC (4) was carried out according to OECD
test guideline Nº 423 [20], which describes an alternative acute toxic class method focused on
reducing animals use without reducing the scientific integrity of data obtained. Swiss male
mice (n = 3) were treated orally (by gavage) with a single dose of 4-FTSC (4) (2000 mg/kg).
Clinical signs of animals (e.g. body weight loss, changes in skin, fur, eyes and mucous
membranes; respiratory, circulatory, autonomic and central nervous systems; somatomotor
activity; and behavior pattern) were then observed during the first 10 min, 30 min, 1 h, 2 h, 4
h, 6 h, 12 h and 24 h and daily until the 14th day after treatment, as recommended by
guideline test. On day 14, the mice were euthanized and necropsy performed. The safety of 4-
FTSC (4) was analyzed by flow chart of OECD test guideline Nº 423 and classified according
to the United Nations Globally Harmonized System of Classification and Labelling of
Chemicals (UN GHS).
2.9 Statistical analysis
Data are expressed as mean ± standard deviation (SD) of two or three independent
experiments. Cytotoxicity data were transformed in percentage of the control and IC50 values
were obtained by non-linear regression. Selectivity index was calculated as follows: IC50
lymphocytes or CFU-GM/IC50 PC-3 cells, being a value > 1.0 was considered satisfactory [30]. The
difference between control and 4-FTSC (4)-treated cells was evaluated by unpaired t test or
one-/two-way analysis of variance (ANOVA) followed by Bonferroni’s test using GraphPad
Prism version 5.01 software for Windows (San Diego, CA, USA). Statistical significance was
established as p<0.05.

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3. Results
3.1 Cytotoxic effects of 4-FTSC (4) on PC-3 cells
Cell viability of PC-3 cells treated without or with 4-FTSC (4) (1.17-150 µM) for 48
h was carried out via mitochondrial metabolic activity evaluation by MTT assay. 4-FTSC (4)
promoted cytotoxicity in a concentration-dependent manner (Figure 2A). The IC50 value
obtained was 18.46 µM. Vehicle control (DMSO at 0.01%) showed cell viability similar to
control (data not shown). Moreover, morphologic analysis of PC-3 cells treated with 4-FTSC
(4) (18.46 µM) was performed using May-Grünwald-Giemsa and DNA binding dye Hoechst
33342 staining. Contrasting with control cells (Figure 2B and D), it was observed that 4-
FTSC (4)-treated cells showed apoptotic cell morphology such as change in the
nucleus/cytoplasm ratio, shrinkage, irregular chromatin and pyknosis (Figure 2C and E).
3.2 Effects of 4-FTSC (4) on phosphatidylserine externalization in PC-3 cells
4-FTSC (4)-treated cells showed an apoptotic profile in phosphatidylserine
externalization evaluation by annexin V-FITC/PI double-staining. As shown in Figure 3A, it
was observed that 4-FTSC (4) promoted a significant reduction of ~15% of viable cells (PI-
/A-) when compared to control. As a consequence, early apoptotic cells (PI-/A+) (24.97 ±
5.16%) showed a significant 2-fold increase (p<0.001) in comparison to control (11.93 ±
3.82%). Control and 4-FTSC (4)-treated cells did not show statistically significant difference
in late apoptosis (PI+/A+) and necrosis (PI+/A-) stages. A representative profile of the
phosphatidylserine externalization evaluation of control and 4-FTSC (4)-treated cells is
reported in Figure 3B.
3.3 Effects of 4-FTSC (4) on DNA fragmentation and cell cycle of PC-3 cells
It was observed that 4-FTSC (4) triggered a significant (p<0.05) increase of DNA
fragmentation in PC-3 cells when compared to untreated cells (39.2 ± 8.51% vs. 10.2 ±
4.33%) (Figure 4A). In addition, PC-3 cells treated with 4-FTSC (4) did not show changes in
cell cycle as compared to control (Figure 4B and C). Corroborating these findings, cell cycle
regulator proteins as p21, p27, NFĸB and cyclin D1 showed similar levels in both control and
treated cells (Figure 4D). 4-FTSC (4) also promoted a 4.2-fold increase in p53 levels (7.0 ±
0.30%) (p<0.05) when compared to control (1.67 ± 0.98%) (Figure 4D).
3.4 Effects of 4-FTSC (4) on ΔΨm and ROS levels in PC-3 cells

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Rhodamine 123 staining showed that 4-FTSC (4) promoted significant reduction of
28% (p<0.01), 16.7% (p<0.05) and 20.4% (p<0.05) in ΔΨm after 6, 24 and 48 h of treatment,
respectively, when compared to control cells (Figure 5A). 4-FTSC (4) also triggered an
increase of 27.2% (p<0.05) in ROS production after 6 h of treatment, whereas at 24 and 48 h
ROS levels were reduced to similar values to control (Figure 5B).
3.5. Effect of 4-FTSC (4) on caspase activities in PC-3 cells
Regarding involvement of 4-FTSC (4) in caspase activities, it was observed that
treated cells showed no significant changes in initiator caspase-8 activity in comparison to
control. On the other hand, 4-FTSC (4) promoted significant 2.5- and 1.5-fold increases
(p<0.05) of executioner caspase-3/-7 and initiator caspase-9, respectively (Figure 6). Thus, it
is suggested that the cell death mechanism promoted by 4-FTSC (4) involves activation of
caspase-3/-7 through apoptosis intrinsic pathway via caspase-9. In parallel, it was also
demonstrated that the autophagy process does not seem to be concomitantly involved in 4-
FTSC (4)-induced cell death, since this compound did not trigger increase of acid autophagic
vesicles as observed by acridine orange staining (data not shown).
3.6 Toxicological assessment of 4-FTSC (4)
The myelosuppressive potential of 4-FTSC (4) (1.17-150 µM) in mouse bone
marrow progenitor cells was investigated by 3D CFU-GM assay and data are reported in
Figure 7A. 4-FTSC (4) was significantly able to inhibit the growth and differentiation of
granulocyte/macrophagic progenitor cells in a dependent-concentration manner, obtaining an
IC50 value of 38 µM. Similar cytotoxic profile was observed for lymphocytes with an IC50 of
9.40 µM (Figure 7B). Considering that value found for PC-3 cells (IC50 = 18.46 µM), the
selectivity index obtained was 2.05 and 0.51 for bone marrow progenitors and lymphocytes,
respectively. Thus, 4-FTSC (4) seems to be lymphotoxic with low cytotoxicity for bone
marrow progenitors.
Moreover, in the hemolytic activity evaluation, 4-FTSC (4) was not able to promote
significant disruption of membrane of human erythrocytes (Figure 7C). 4-FTSC (4)
concentration range (0.58-150 µM) tested only triggered a reduced hemolysis ratio (<5%).
3T3 cells were treated with 4-FTSC (4) (2.3-300 µM) for 48 h and cell viability
evaluated by NRU assay via incorporation and binding of supravital dye neutral red in the
lysosomes of viable cells. 4-FTSC (4) promoted a reduction of cell viability in a
concentration-dependent manner, obtaining an IC50 value of 13.1 µM (Figure 7D). Thus, the

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estimated LD50 was 620.05 mg/kg, which classified 4-FTSC (4) in the UN GHS category 4
(300 < LD50 < 2000 mg/kg). To confirm these results, in vivo acute oral systemic toxicity
hazard was carried out by oral administration of a single dose of 4-FTSC (4) (2000 mg/kg) in
mice. No deaths or clinical sign of toxicity (e.g. body weight loss, changes in skin/fur, and
behavior pattern) were observed during 14 days of observation of the animals. Necropsy also
showed no apparent changes in the organs. Therefore, 4-FTSC (4) was classified in the UN
GHS category 5 (LD50 > 2000-5000 mg/Kg).

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4. Discusssion
The interest for new compounds with enhanced anticancer properties has stimulated
the rational design and synthesis of natural products analogues. Combination of partial active
structures of molecules to produce a more active compound is a widely spread concept [31].
Regarding this,
Vandresen et al.
[13] synthesized
different limonene-based
thiosemicarbazones and performed an antiproliferative activity screening against tumor cell
lines (U251 glioma, UACC-62 melanoma, MCF7 breast, NCI/ADR-RES ovarian resistant,
786-0 kidney, NCI-H460 lung, PC-3, OVCAR-3 ovarian, HT29 colon, K562 leukemia cancer
cells). Comparing the antiproliferative activity of synthesized compounds, 4-FTSC (4)
demonstrated high selectivity and capability to inhibit PC-3 cells growth. Here, it was
demonstrated that 4-FTSC (4)-induced antiproliferative activity seems to be related to
apoptotic cell death mechanisms through induction of different pathways, including DNA
damage, ROS overgeneration, mitochondrial dysfunction and increase of caspase-3 and -9
activities. In addition, our data suggest p53 involvement.
The design of 4-FTSC (4) molecular structure involved the substitution of limonene-
based thiosemicarbazone at the N(4) position with p-fluorobenzaldehyde to obtain 4-FTSC
(4), which was crucial for antiproliferative effect against PC-3 cells. Different studies have
reported the correlation of the enhanced anticancer activity of N(4)-substituted
thiosemicarbazone derivatives against different cancer cell lines to the presence of N(4)
substitution in the derivatives [32,33]. In order to enhance the antiproliferative properties,
fluorine was added in the molecular structure of 4-FTSC (4). Peculiar properties (e.g.
metabolic stability, hardness/low van der Waals interaction due to the low polarizability and
increased lipophilicity) have been attributed to fluoro organic compounds, which may
improve their pharmacological properties [34–37]. In fact, fluorinated antitumor agents are
widely used in cancer treatment, such as 5-fuorouracil (5-FU) and its analogue 5-fuoro-2’-
deoxyuridine (FdUrd) [35]. 4-FTSC (4) cytotoxic profile suggested that the presence of
fluorine in the molecular structure contributed to the better antiproliferative effects observed
in PC-3 cells.
Similarly to fluorinated antitumor agents [38], 4-FTSC (4) also triggered DNA
damage. The significant increase in DNA fragmentation suggested the induction of DNA
damage in PC-3 cells after treatment with 4-FTSC (4). These results were supported by other
cellular evaluation including cell morphology changes, such as nucleus shrinkage, irregular
chromatin and pyknosis. Studies have shown that DNA impairment triggered by some

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anticancer agents may additionally induce ROS overgeneration with consequent apoptotic cell
death through p53-dependent pathway [39–41]. PC-3 cell death induced by 4-FTSC (4) might
follow similar mechanism. The results suggested that 4-FTSC (4) may cause DNA
impairment as well as ROS overproduction and increase of p53 levels.
In this cell oxidative stress context, mitochondria exerts an important role in the
regulation of apoptosis, since increased ROS levels promote mitochondrial dysfunction by
loss of Δψm, culminating in pro-apoptotic factors release (e.g. cytochrome c), which in turn
activates the apoptosis intrinsic pathway through caspase-9 and -3 activation [42–44]. Early
events in 4-FTSC (4)-induced cell death is involved in decrease of Δψm and subsequent
activation of intrinsic pathway of caspase cascade. Further investigation is needed to elucidate
the mitochondrial dysfunction and the pro-apoptotic factors involved in cascade activation.
Moreover, it has been demonstrated that p53, in c-Jun N-terminal kinase (JNK)-dependent
manner, also inhibits several survival oncogenes and p53 inhibitors in the apoptotic process
[41]. Considering this, our data suggest apoptosis via p53 involvement and it seems to be the
main cell death pathway induced by 4-FTSC (4) in PC-3 cells.
As cytotoxic compounds used in cancer treatment have limitations related to high
incidence of severe side effects due to their non-selective actions for tumor cells only,
important parameters of toxicity were investigated for 4-FTSC (4). Although it showed low
cytotoxicity for mouse bone marrow progenitors, 4-FTSC (4) showed a potential lymphotoxic
profile. Doxorubicin, one of the drugs used in chemotherapy for prostate cancer, has
demonstrated to induce depletion of thymus and spleen cells, peripheral blood leukocytes and
lymphocytes; however, this drug does not seem to affect mice bone marrow cell population
[45], similarly to 4-FTSC (4).
Following the toxicological evaluation of 4-FTSC (4), this compound was not able to
promote disruption of the membrane of human erythrocytes. Compounds with this feature
have been considered promising for intravenous use as anticancer drugs [46,47], since
intravenous infusion allows to obtain an adequate pharmacokinetics and reduced
gastrointestinal toxicity, for instance [48]. Furthermore, 4-FTSC (4) showed orally well
tolerated, being classified in the UN GHS category 5 (LD50 > 2000-5000 mg/Kg), which may
consider it as a compound with relative low acute oral systemic toxicity hazard [20]. Overall,
further experimental investigation would be required to elucidate the mechanisms underlying
4-FTSC (4) antiproliferative properties.
5. Conclusions

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Taken together, it was demonstrated that cytotoxic effects triggered by 4-FTSC (4)
involved different routes that lead to apoptosis in PC-3 cells via p53 involvement. Regarding
toxicological profile, 4-FTSC (4) showed moderate hematological toxicity and orally well
tolerated with relative low acute oral systemic toxicity hazard. These features highlight 4-
FTSC (4) as a promising candidate to be used as a clinical tool in prostate cancer treatment.
Further studies are required to better clarify toxicopharmacological effects of 4-FTSC (4)
found in this study.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This study was supported by Brazilian research funding agencies, such as Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e
Pesquisas (FINEP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES).
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