Involvement of reactive oxygen species in the oleoylethanolamide effects and its pyrazonilic analogue in melanoma cells

Article abstract of DOI:10.1007/s00044-017-1971-1

The search for more substances that effectively fight melanoma is extremely important, because of its aggressive nature. In this sense, the molecular hybridization is a promising strategy. The aim of this work is to evaluate whether the antiproliferative effect of the endocannabinoid oleoylethanolamide can be improved with the addition of a trifluoromethylated pyrazolinic nucleus on its structure in B16F10 cell line. The pyrazolinic analog was named oleoyl pyrazoline. We also compared the effects of oleoylethanolamide and that of the classic endocannabinoid anandamide (AEA). The cell viability was evaluated by MTT assay, the intracellular reactive oxygen species generation by fluorimetry, and apoptosis/necrosis by fluorescent microscopy. Also, α-tocopherol antioxidant was used to evaluate the involvement of reactive oxygen species in the cellular response. Although the effects of AEA occur in smaller concentrations, the results show that the effects of AEA and oleoylethanolamide were similar. The results showed a decrease in cell viability, induction of apoptosis and necrosis, and increased generation of reactive oxygen species by the oleoylethanolamide, while the oleoyl pyrazoline increased cell proliferation and decreased reactive oxygen species. Additionally, the effects of oleoylethanolamide in cell viability were decreased by inhibiting the generation of reactive oxygen species by α-tocopherol. Therefore, it is possible to suggest the involvement of reactive oxygen species in the effect of oleoylethanolamide in the B16F10 cells. Considering the great need to find substances that can fight melanoma and the lack of greater elucidation in the action mechanisms of cannabinoids and their analogs, this work provides important new information that could serve as reference to other studies.

Full text of DOI:10.1007/s00044-017-1971-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1971-1
ORIGINAL RESEARCH
Involvement of reactive oxygen species in the oleoylethanolamide
effects and its pyrazonilic analogue in melanoma cells
Priscila Antiqueira-Santos1,2 Daiane S. dos Santos3 Carolina R. L. Hack3
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Alex Fabiani C. Flores4 Marcelo G. Montes D’Oca3 Luciana A. Piovesan3,5
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Luiz Eduardo M. Nery1,2 Ana Paula S. Votto1,2
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Received: 29 December 2016 / Accepted: 21 June 2017
© Springer Science+Business Media, LLC 2017
Abstract The search for more substances that effectively
fight melanoma is extremely important, because of its
aggressive nature. In this sense, the molecular hybridization
is a promising strategy. The aim of this work is to evaluate
whether the antiproliferative effect of the endocannabinoid
oleoylethanolamide can be improved with the addition of a
trifluoromethylated pyrazolinic nucleus on its structure in
B16F10 cell line. The pyrazolinic analog was named oleoyl
pyrazoline. We also compared the effects of oleoylethano-
lamide and that of the classic endocannabinoid anandamide
(AEA). The cell viability was evaluated by MTT assay, the
intracellular reactive oxygen species generation by fluori-
metry, and apoptosis/necrosis by fluorescent microscopy.
Also, α-tocopherol antioxidant was used to evaluate the
involvement of reactive oxygen species in the cellular
response. Although the effects of AEA occur in smaller
concentrations, the results show that the effects of AEA and
oleoylethanolamide were similar. The results showed a
decrease in cell viability, induction of apoptosis and
necrosis, and increased generation of reactive oxygen spe-
cies by the oleoylethanolamide, while the oleoyl pyrazoline
increased cell proliferation and decreased reactive oxygen
species. Additionally, the effects of oleoylethanolamide in
cell viability were decreased by inhibiting the generation of
reactive oxygen species by α-tocopherol. Therefore, it is
possible to suggest the involvement of reactive oxygen
species in the effect of oleoylethanolamide in the B16F10
cells. Considering the great need to find substances that can
fight melanoma and the lack of greater elucidation in the
action mechanisms of cannabinoids and their analogs, this
work provides important new information that could serve
as reference to other studies.
●
●
●
Keywords Cell viability Apoptosis B16F10 cell line
●
Oleoylpyrazoline Oxidative stress
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1971-1) contains supplementary material,
which is available to authorized users.
* Ana Paula S. Votto
anavotto@yahoo.com.br
Introduction
1
Programa de Pós-Graduação em Ciências Fisiológicas, Instituto de
Ciências Biológicas, Universidade Federal do Rio Grande–FURG,
The search for more effective substances for treatments that
prevent or reduce the proliferation of melanoma cells, even
in advanced stage, is extremely important due to the
aggressive nature of this pathology. It was estimated that
76,380 new cases of melanoma would be diagnosed in 2016
(American Cancer Society 2016). Even though melanoma
accounts for only 1% of all skin cancer cases worldwide, it
is responsible for the vast majority of skin cancer related
deaths (American Cancer Society 2016).
Rio Grande, RS, Brazil
2
Laboratório de Cultura Celular, Instituto de Ciências Biológicas,
FURG, Rio Grande, RS, Brazil
3
Laboratório Kolbe de Síntese Orgânica, Escola de Química e
Alimentos, FURG, Rio Grande, RS, Brazil
4
Escola de Química e Alimentos, FURG, Rio Grande, RS, Brazil
5
Nanobusiness Informação e Inovação Ltda, Incubadora de
Projetos, Instituto Nacional de Metrologia, Qualidade e
Tecnologia–INMETRO, Duque de Caxias, Rio de Janeiro, Brazil

Med Chem Res
Cannabinoid substances are highlighted among the sev-
eral substances studied for use as chemotherapeutic
(Velasco et al. 2012). These are lipid chemical substances
naturally occurring in the plant Cannabis sativa, being Δ(9)-
tetrahydrocannabinol, considered the most representative of
the group (Pertwee 2008). As well as the natural cannabi-
noids, the synthetic cannabinoids such as WIN-55,512-2,
JWH-133, and (R)-methanandamide, also bind to CB1 and
CB2 cannabinoid receptors to exert their biological effects.
In addition, according to Nikan et al., CB receptors are
over-expressed in some tumor cells, and cannabinoid
receptor ligands affect these tumoral cells selectively (Nikan
et al. 2016). The discovery of two cannabinoid substances
endogenously produced, N-arachidonoylethanolamide
(anandamide, AEA) and sn-2-arachidonoyl glycerol (2-
AG), led to new scientific research that contributed to
identifying another endogenous substance derived from
fatty acid as the oleoylethanolamide (OEA), an N-acyl
ethanolamide (Pertwee 2010; Mechoulam et al. 1995;
Mechoulam et al. 2002). However the effects of OEA
against melanoma have not yet been studied.
The possibility that certain cannabinoid substances can
be used as antitumoral drugs has been investigated for a
long time (White et al. 1976). Since the identification of
these substances in the 1960s and 70 s, several similar
substances have been synthesized and used as therapeutic
drugs with various medicinal effects (Honório et al. 2006).
According to Bifulco et al. (2006) and Sarfaraz et al.
(2008), cannabinoid substances have a potential appli-
cation as antitumor drugs that can cause a decrease
in cell survival and proliferation. It has also recently been
suggested that increased levels of endocannabinoids
and the blocking of its degradation leads to a decreased
viability of tumor cells (Hamtiaux et al. 2012). According to
Cudaback et al. (2010), the increased synthesis of endo-
genous cannabinoid, the catabolism of several ligands
inside cells and tissues, and the increased expression of
different receptors appear to be important in signaling cas-
cades by different kinases. These signaling cascades result
in pro-autophagic, proapoptotic, antiproliferative or anti-
apoptotic effects and such effects have been observed in
some tissues and cancer cells. According to Blázquez et al.
(2006), melanoma cells actively express cannabinoid
receptors (CB1 and CB2) and the activation of these
receptors inhibits the growth of melanoma cells in vivo and
in vitro.
consequently increased oxidative stress. Carracedo et al.
(2006) showed that treatment on pancreatic tumor cells
using a specific cannabinoid increased the activation of
ATF-4 and TRB3 genes, which are related to oxidative
stress (among other pathways) leading to apoptosis. Fur-
thermore, several studies have shown that cannabinoids can
realize its antiproliferative effects and induce cell death
dependent of ROS. For instance, anandamide leads to an
increased ROS generation in tumor cells (Massi et al. 2003;
Sarker et al. 2003). Jacobsson et al. (2001) demonstrated
that anandamide and 2-AG antiproliferative effects in
glioma cells can be completely inhibited by the antioxidant
α-tocopherol. However, according to Ambrosini et al.
(2006), OEA also presents antioxidant properties resulting
in beneficial effects on in vitro capacitation of human
sperm. Thus the role of cannabinoids on regulation of
oxidative stress remains controversial. Although the cell
death induced by ROS seems to be supported by cannabi-
noids, a protective effect by cannabinoids against induced
cell death by ROS can also be observed. The role of CB
receptors on prooxidant and antioxidant effects is unclear
and both receptor-dependent and receptor-independent
effects were related, varying according to different canna-
binoids (Zolese et al. 2005).
Previous studies have shown that trihalomethylated
pyrazolines may have remarkable biological effects, and
because they present a wide range of applications in the
Medicinal Chemistry, they may be considered privileged
structures. According to Hassan et al. (2011), pyrazole
compounds may induce oxidative stress and sometimes the
antitumor activity may be followed by a considerable
increase in superoxide dismutase activity, decrease in cat-
alase and glutathione peroxidase activity, in addition to
decreased levels of glutathione, high production of H2O2,
NO and free radicals, leading to death of tumor cells.
These compounds were reported to have a wide range of
biological applications, by its antimicrobial, antinocice-
ptive, analgesic, intestinal stimulatory secretion, anti-
depressant, anticonvulsivant, antipyretic, anti-inflammatory
and hypophagic properties (Nenajdenko and Balenkova
2011; Sauzem et al. 2008). According to Kumar et al.
(2013), molecular modeling and docking studies have
been discussed for the development of novel anticancer
agents bearing pyrazole moiety. Pyrazole analogs have
been found to inhibit the active site of kinase family
enzymes and various growth factors like vascular endo-
thelial growth factor, fibroblast growth factor, tumor
necrosis factor, tumor growth factor (TGF-α and TGF-β)
and BRAF gene.
The relation between cannabinoid receptors and oxida-
tive stress has already been reported. Bifulco et al. (2008)
observed that the cannabinoid interaction with transient
receptor potential vanilloid promotes the activation of more
than one cell mechanism, such as mitochondrial apoptosis
pathway activation, that occurs at the same time as the
reactive oxygen species (ROS) generation increase, and
The development of synthetic biologically active com-
pounds is of great interest to the biochemistry and phar-
maceutical communities; many heterocyclic compounds
present a great structural diversity and biological activity,

Med Chem Res
which justify the interest in the development of new com-
pounds with therapeutic potential. Among the molecular
design strategies, the molecular hybridization is a very
promising strategy working from the union of two distinct
structural features of bioactive molecules in a new mole-
cule. This strategy can generate new molecules that exhibit
the combination of both activities of the original molecules
in a single molecule, change in selectivity, and reduction in
undesirable side effects (Junior et al. 2007).
With this in mind, and coupled with the excellent results
that we have obtained with regard to the cannabinoid ana-
logs fatty amides (Santos et al. 2015) and fatty heterocycles
as antiproliferative compounds (Treptow et al. 2015; Mor-
aes et al. 2017) – including activity against melanoma–this
study proposes a structural modification of the OEA
by coupling it with an heterocyclic nucleus–oleoyl tri-
fluoromethylated pyrazoline (OPZ) (Fig. 1)–in order to
verify if this association is capable to improve the anti-
proliferative effect of endocannabinoids, AEA and OEA
(Fig. 1). In addition, we verified the involvement of ROS
production in the effects of the endocannabinoid OEA and
its analog OPZ in melanoma cells.
Materials and methods
Cell culture
The murine melanoma cell line, B16F10, was obtained from
the Rio de Janeiro Cell Bank. B16F10 cell line was main-
tained in DMEM medium, supplemented with sodium
bicarbonate (0.2 g/L), L-glutamine (0.3 g/L), Hepes (3 g/L),
10% fetal bovine serum, 1% antibiotic and antimycotic, in
cell culture flasks at 37 °C.
Synthesis of compounds
OEA e OPZ were obtained by the synthetic pathway shown
in Fig. 2 from the same precursor, the methyl oleate,
obtained from the oleic acid. OEA was synthesized
according to a published method (D’Oca et al. 2010; Santos
et al. 2015), from aminolysis reaction of methyl oleate (0.3
mmol) in the presence of ethanolamine (1.8 mmol) in
acetonitrile for 24 h (Fig. 2). The progress of the reactions
was monitored by silica gel thin-layer chromatography.
The raw products were purified via column chromatography
Fig. 1 Evaluated compounds:
O
anandamide (AEA),
oleoylethanolamide (OEA),
oleoyl pyrazoline (OPZ)
OH
( )4
( )3
N
H
AEA
R
O
HO
N
OH
F3C
N
N
H
OEA
( )7
O
( )7
OPZ
Fig. 2 Synthesis of oleoyl ethanolamide (OEA) and oleoyl pyrazoline (OPZ) from oleic acid

Med Chem Res
on silica gel (n-hexane/ethyl acetate, 7:3) and analyzed by
1H NMR, and the data are in agreement with the literature.
The synthesis of OPZ was performed according to Beck
et al., (Beck et al. 2012). In a first step, the methyl oleate
hydrazinolysis was realized in the presence of the hydrazine
to obtain the oleic hydrazide, which was then subjected to
reaction cyclocondensation with (Z)-1,1,1-trifluoro-4-meth-
oxy-8-metilnone-3,7-dien-2-one (enone). After the reaction
was complete, the precipitate that formed was filtered and
dried under vacuum to obtain the new analog OPZ, (Fig. 2).
The compound was analyzed by 1H NMR, and GC/MS, and
the data are in agreement with the literature. Anandamide
(AEA) was synthesized according to a published method
(D’Oca et al. 2010; Santos et al. 2015), by reacting the
respective arachidonic acid (0.3 mmol) with ethanolamine
(0.3 mmol), triethylamine (0.3 mmol), and a catalytic
amount of (dimethylamino) pyridine, and dicyclohex-
ylcarbodiimide (0.3 mmol) in CH2Cl2 was added dropwise
to the above reaction mixture, which was then stirred at
room temperature for 24 h. The solid dicyclohexylurea
formed was removed by filtration and the solvent was
evaporated under reduced pressure (Fig. 3). AEA was
Anandamide
N-arachidonoylethanolamine. C22H37NO2. MW 347.53
g·mol−1. Colorless oil. H NMR (300 MHz, CDCl3) δ 5.90
1
(s, 1H), 5.36 (m, 8H), 3.72 (s, 2H), 3.42 (m, 2H), 2.82 (m,
6H), 2.62 (s, 1H), 2.21 (m, 2H), 2.12 (q, J 6.8 Hz, 2H), 2.05
(q, J 6.9 Hz, 2H), 1.73 (m, 2H), 1.32 (m, 6H), 0.89 (t, J 6.9
Hz, 3H).
Cells and treatments with the compounds
The B16F10 cells were centrifuged, suspense in DMEM
medium (2 × 105 cells/mL) and incubated for 24 h to adhere
in cell culture plates at 37 °C. The compounds oleoyl
ethanolamide (OEA) and oleoyl pyrazoline (OPZ) were
diluted in ethanol and Tween 20 (Synth) and added in the
cells in the maximum volume of 0.05% (0.04% ethanol
and 0.01% Tween) which was not cytotoxic (Fig. 4a).
The anandamide (AEA) was diluted in DMSO and added in
the cells in the maximum volume of 1% which also was
not cytotoxic (Fig. 4b). The control cells received the
same volume of solvent of the highest concentrations of
compounds. The concentrations of OEA or OPZ tested were
50, 125, 250, 375, and 500 µM until 72 h. Already the
concentrations of AEA were 0,1, 1, 10, 50 and 100 µM
until 72 h.
1
analyzed by H NMR, and the data are in agreement with
the literature.
Oleoylethanolamide
N-(2-hydroxyethyl) oleamide: C20H39NO2. MW 325.53 g
mol−1. Yellow solid. H NMR (300 MHz, CDCl3) δ 6.10
(sl, 1H), 5.34 (m, 2H), 3.72 (m, 2H), 3.41 (m, 3H), 2.23 (t,
J = 7,2 Hz, 2H), 2.01 (m, 4H), 1.63 (m, 2H), 1.30 (m, 20H),
0,88 (t, J = 6,9 Hz, 3H).
1
Cellular viability
The viability of B16F10 cells exposed to AEA, OEA and
OPZ was measured by method of MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) 0, 24, 48,
and 72 h after incubation at 37 °C, according to Trindade
et al. (Trindade et al. 1999). Briefly, the cells after incu-
bation were washed with PBS and 200 μl DMEM medium
β-mercaptoethanol free and 20 μl of MTT (5 mg/ml) was
added to each well. The plates were incubated for 3 h at
37 °C. The medium was removed and formazan crystals
were dissolved in 200 µl of dimethylsulfoxide (DMSO,
Sigma) with gentle shaking. The absorbance values at 490
nm were determined on a multiwell plate reader (ELX 800
Universal Microplate Reader, Bio-TEK).
Oleoyl pyrazoline
(Z)-1-(5-hydroxy-3-(4-methylpent-3-enyl)-5-(trifluoromethyl)-
4,5-dihydropyrazol-1-yl)octadec-9-en-1-one. C28H47F3N2O2.
MW 500.68 g mol−1. Yellow solid. H NMR (400 MHz,
1
CDCl3) 6.36 (s, 2H, 2H–C=C), 5.12 (d, 1H, H–C=C
(CH3)2, 3.44 (d, 1H, J 18.5 Hz, H4a), 3.12 (d, 1H, J 18.5
Hz, H4b), 2.67 (m, 6H, 3CH2–CH=C), 2.40 (m, 2H, CH2),
2.31 (m, 2H, CH2), 1.71 (s, 3H, CH3), 1.65 (s, 3H, CH3),
1.56 (m, 2H, 2CH2), 1.27 (m, 20H, 12CH2), 0.90 (t, 3H,
CH3). Obtained Mass: [M+]: 500.0.
Fig. 3 Synthesis of anandamide (AEA) from arachdonic acid

Med Chem Res
Fig. 4 Cells B16F10 treated
with a ethanol and tween; b
DMSO; and c ethanol and
tween, DMSO or both solvents
at the same concentration. Data
are shown as mean standart
error. Asterisk indicates
significant difference from the
respective control at each
exposure time (p < 0.05)
Quantitative analysis of apoptosis and necrosis
Assessment of intracellular ROS formation
The evaluation of apoptosis and necrosis was realized
according to Ribble et al. modified, with the addition of 2
µL of work solution composed by PBS, with 100 µg/mL of
acridine orange and 100 µg/mL of ethidium bromide (Rib-
ble et al. 2005). The analysis was performed from areas
captured from the well plate (20X) with epifluorescence
microscope (Olympus IX81). All cells were analyzed and
the data were expressed in percentage related to total cells
number visualized on the captured area. The cells were
classified according Kosmider et al. modified, as following:
those evincing green or yellow-orange fluorescence cyto-
plasm and green nucleus were considered viable cells; those
presenting orange nucleus with fragmented chromatin were
considered apoptotic. Those cells with uniformly orange-
stained nucleus were considered necrotic (Kosmider et al.
2004).
The B16F10 cells were centrifuged, suspense in DMEM
medium (2 × 105 cells/mL) and incubated for 24 h to adhere
in cell culture plates with DMEM medium at 37 °C. After
this, they were treated in medium with 50, 125, and 250 µM
of OEA or OPZ and incubated at 37 °C during 24 h. Then
the cells were washed with PBS (two times) and incubated
for 30 min at 37 °C with the fluorogenic compound 2′,7′-
dichlorofluorescin diacetate (H2DCF-DA) at a final con-
centration of 40 μM. After the loading with H2DCF-DA, the
cells were washed with PBS two times and then suspended
in fresh PBS. Aliquots of 160 μl of each sample (five
replicates) were placed into an ELISA plate and the fluor-
escence intensity was determined during 90 min at 37 °C,
using a fluorometer (Victor 2, Perkin Elmer), with an
excitation and emission wavelength of 485 and 520 nm,
respectively. ROS levels were expressed in terms of fluor-
escence area, after fitting fluorescence data to a second

Med Chem Res
order polynomial and integrating between 0 and 90 min in
order to obtain its area.
different concentrations. However 72 h after treatment it
was observed a significant increase in the percentage of
viable cells treated with 50, 125, and 375 µM of OPZ
(Fig. 5c).
α-tocopherol
To verify the involvement of ROS generation in the effects
of OEA was used an antioxidant (α-tocopherol-Vitamin E)
at the concentration of de 25 µg/ml dissolved with DMSO.
The cells were treated with α-tocoferol, 250 µM of OEA,
and α-tocoferol and 250 µM of OEA during 24 h. The
control cells received the same volume of solvent used in
each condition (Ethanol and Tween or DMSO, or DMSO
plus Ethanol and Tween) (Figs. 4a–c, respectively). It was
made assessment of intracellular ROS formation and cell
viability assay by MTT. In this case, the data are presented
as percentage of viable cells in relation to respective
controls.
ROS
After 24 h of treatment with OEA, B16F10 cells showed an
increase in ROS generation in concentration of 250 µM
compared to control (Fig. 6a). For OPZ was observed a
reduction in the generation of ROS in concentration of 50
µM, suggesting an antioxidant effect of this molecule
(Fig. 6b).
Apoptosis and necrosis
It was observed a decrease in cell viability while the con-
centration of OEA increased. In the concentrations of 125
and 250 µM, there was a decrease in number of viable cells
and an increase in cell apoptosis. At highest concentrations
(375 and 500 µM) all cells suffered necrosis (Fig. 7a–c).
Statistical analysis
Each experiment was repeated on three independent times
with, at minimum triplicate samples. The results were
expressed as the means SEs. Analysis of variance
(ANOVA) was used to determine significant differences
among groups. Tukey’s significant difference post hoc test
was used for pair wise comparisons after analysis of var-
iance. Statistical significance was accepted at p < 0.05.
α-tocoferol
In the treatment of OEA with α–tocopherol, the ROS gen-
eration was significantly lower than treatment with only
OEA and statistically equal to the treatment with only α-
tocopherol (Fig. 8a). Compared to its control, only OEA
showed significant difference in the percentage of viable
cells (Fig. 8b).
Results
AEA
Discussion
When exposed to AEA the B16F10 cell line showed lower
percentage of viable cells from 24 h after treatment with
concentrations of 50 and 100 µM when compared to the
control (Fig. 5a).
According to Bifulco et al. (2006), the potential application
of cannabinoid substances as antitumor drugs is supported
by the evidence of decreased cell proliferation and survival,
among others. In this study, two endocannabinoid sub-
stances were used: a classic endocannabinoid, the ananda-
mide (AEA), and the OEA, that was proposed as a member
of the endocannabinoid family later (Galve-Roperh et al.
2013). According to Adinolfi et al. (2013), AEA induces
cytotoxicity against human melanoma cells (A375) in
micromolar concentrations through a complex mechanism
which includes, among other mechanisms, the CB1
activation.
Although the effects of AEA occur in smaller con-
centrations, showing that the classic endocannabinoid
(AEA) has a more potent effect than OEA, the results of our
experiment show that the effects of the two cannabinoids
(AEA and OEA) are similar.
OEA
When exposed to OEA the B16F10 cell line showed lower
percentage of viable cells 24 h after treatment with con-
centrations of 250 and 375 µM compared to control. After
48 h, it was observed a lower percentage of viable cells
compared to control in cells treated with a concentration of
375 µM of OEA. At 72 h, this effect was no longer
observed, except for the concentration of 500 µM, which
was cytotoxic from 24 h exposure (Fig. 5b).
OPZ
On OPZ exposure, there were not any significant differ-
ences immediately, 24 and 48 h after treatment with
In the present study, there was a decrease in viable cell
number and an increase in apoptotic cells in concentrations

Med Chem Res
Fig. 5 a Percentage of viable
B16F10 cells treated with
different concentrations of AEA,
b OEA and c OPZ immediately,
24, 48, and 72 h after exposure.
Data shown are the mean
standard error. Asterisk indicates
significant difference from the
respective control at each
exposure time (p < 0.05)
of 125 and 250 µM of OEA, after 24 h. Higher concentra-
tions (375 and 500 µM) led to cell death by necrosis.
According to these results, we suppose that the intermediate
concentrations might allow an initiation to a cellular
response, leading cells to apoptosis, whereas higher con-
centrations do not allow any attempt to cell response.
Supporting that view, Giuliano et al. (2009) demonstrated
that the utilization of the synthetic cannabinoid WIN

Med Chem Res
55,212-2 in liver cancer cells was associated with the
upregulation of proapoptotic factors such as bax, bid, and
bcl-xs, and the corresponding down-regulation of anti-
apoptotic, cell protective factors such as bcl-2.
However, according to Ma et al. (2015) previous
treatment with OEA on human umbilical vein endothelial
cells shows a protective effect against cytotoxic H2O2-
induced effects, decreases the intracellular ROS level and
caspase-3 activation, and reverses H2O2-induced lipid per-
oxidation and cellular antioxidant potential descent on the
cell line.
Nevertheless, when the endocannabinoid was tested in its
original form (OEA), a considerable effect of decrease in
cell viability was observed. This effect on cell viability was
accompanied by an increase of ROS generation in the initial
24 h, for the concentration of 250 µM of OEA. Moreover,
when an antioxidant was used in the same exposure con-
dition, it demonstrated a decrease in ROS generation
accompanied by a decrease in OEA effect on cell viability.
Additionally, according to the results obtained for OPZ, it is
possible to demonstrate that the structural change in OEA
led to loss of oxidant capacity, as well as the loss of ability
to reduce cell viability. Thus, these results suggest the
involvement of ROS generation in the effect of OEA in the
cell line B16F10.
After 72 h of exposure to modified structure (OPZ), the
opposite effect was observed, with an increase in cell pro-
liferation. Previous studies also suggest that different pyr-
azole isoforms can generate different effects on organisms.
Balbi et al. (2011) synthetized thirty-six pyrazole-derived
isoforms and tested its antiproliferative effects on three
tumoral cell lines. Just four of those presented good anti-
proliferative effects and had capacity to induce apoptosis on
Fig. 6 a ROS generation in B16F10 cells treated with 50, 125, and
250 µM of OEA and b OPZ for 24 h. Data shown are the mean
standart error. Asterisk indicates significant difference from the
respective control at each exposure time (p < 0.05)
Fig. 7 a Visual field of B16F10
cells in the control group
demonstrating viable cells and b
cells treated with 125 µM of
OEA showing viable cells, in
apoptosis or necrosis captured
by fluorescence microscopy. c
Percentage of viable, apoptotic
and necrotic B16F10 cells 24 h
after treatment with 50, 125,
250, 375, and 500 µM of OEA
from the visual field captured by
a fluorescence microscope. Data
shown are the mean standard
error. Asterisks (*), (**), and
(***) indicate the significant
difference in relation to control
on viable, apoptotic, and
necrotic cells number,
respectively (p < 0.05)

Med Chem Res
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Molecular mechanisms of OEA action are not yet com-
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Acknowledgements This work was supported by the Programa de
Pós-Graduação em Ciências Fisiológicas (FURG). P.A.S. received a
graduate fellowship from Brazilian CAPES. We are thankful to Anahy
Fazio, Milene Medeiros and Fernanda Lopes for help on the work, and
for financial support from Fundação de Amparo à Pesquisa do Estado
do Rio Grande do Sul (FAPERGS/PRONEM) and Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq).
Compliance with ethical standards
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Conflicts of interest The authors declare that they have no com-
peting interests.

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