Biomedicine & Pharmacotherapy 103 (2018) 18–28

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Phosphoethanolamine induces caspase-independent cell death by reducing T

the expression of C-RAF and inhibits tumor growth in human melanoma
model
⁎
Lisley I. Mambellia, , Sarah F. Teixeirab, Salomão D. Jorgea,c, Bárbara Kawamuraa,d,
⁎
Renato Menegueloe, José A.M. Barbutoa, Ricardo A. de Azevedoa,c, Adilson K. Ferreiraa,c,d,
a
Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil
b
Department of Pharmacology, Institute of Biomedical Science, University of Sao Paulo, Sao Paulo, Brazil
c
Alchemy, Innovation, Research & Development, Department of Oncology, CIETEC/IPEN, University of Sao Paulo, Sao Paulo, Brazil
d
Medical Science, University of Sao Paulo Medical School, Sao Paulo, Brazil
e
Instituto Tecnológico da Aeronáutica, Sao Jose dos Campos, Sao Paulo, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Phosphoethanolamine (PEA) is a fundamental precursor during the biosynthesis of cell membranes phospholi-
Phosphoethanolamine pids. In the past few years, it has been described as a potential antitumor agent. In previous studies, we de-
Caspase-independent cell death monstrated that PEA showed antitumor properties in vitro and in vivo in a wide range of tumor cell lines. Herein,
Antitumor properties we showed that PEA possesses cytotoxic properties and notably revealed to induce caspase-independent cell
Melanoma
death. Of interest, we provided evidence that PEA inhibits melanoma cells proliferation through the reduction of
C-RAF. Molecular docking of PEA evidenced that this compound indeed fits satisfactory in the binding site
located between the dimers of C-RAF protein with 107,01 Å and score of −29,62. Also, PEA arrested A2058 cells
at G2/M phase in the cell cycle. Moreover, cell proliferation, migration and adhesion capacities of A2058 cells
were also inhibited by PEA. Most importantly, PEA inhibited tumor growth of melanoma tumors and prolonged
survival rate of mice. Also, PEA induced a significant immune response in a syngeneic metastatic melanoma
model. Taken together, these data indicate that PEA is a promising candidate for future developments in cancer
field.

1. Introduction
The primary amine phosphoethanolamine is the central precursor of
ethanolamine branch in the Kennedy pathway to produce phosphati-
dylethanolamine-phospholipids, which besides many functions, are also
related to the turnover of cell membranes [1,2]. These phospholipids
take part in the lipid signaling pathways and when hydrolyzed result in
the formation of the second messengers [3]. Consequently, these mo-
lecules can regulate the cell proliferation and survival through lipids
signalling [4]. Recently, the therapeutic potential of Synthetic Phos-
phoethanolamine (Pho-s) was demonstrated in the acute promyelocytic
leukemia (APL) using a transgenic mouse model harboring the PML/
RARα fusion [5]. Pho-s showed anti-proliferative effects in APL by
blocking the dissemination of malignant clones and reducing the
number of CD117+ cells and Gr-1+ immature myeloid cells in the bone
marrow, spleen and liver. Importantly, Pho-s attenuated the expansion
of malignant clones CD34+/CD117+, as well as CD34+ and Gr-1+ cells
in the bone marrow. Accordantly, Pho-s induced in vivo cell death of
these immature cells in the spleen and liver, which is a notable effect.
Recently, we demonstrated that Pho-s has a putative anti-metastatic
effect which was validated in a renal carcinoma model. Potentially,
Pho-s inhibited lung metastasis in nude mice and blocked endothelial
cell proliferation, migration and tube formation, besides provoked cell
cycle arrest at the G2/M phase [6].
Metastatic malignant melanoma (MM) is a form of skin cancer
which is highly aggressive. According to American Cancer Society,
clinical treatments for this tumor type are based on systemic che-
motherapy by using chemotherapeutic drugs such as dacarbazine, pa-
clitaxel and cisplatin as standard therapy. Such strategies showed a
poor prognosis for patients with a response rate of only 10–20% [7,8].
More recently, strategies based on melanoma biology understanding
have been focused on blocking the often-activating mutation in the
RAS-RAF-MEK-ERK signaling as a possible target for therapy [9–11].
The use of chemotherapy agents for the MM treatment and either their

⁎
Corresponding authors at: Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
E-mail addresses: lis_mambelli@yahoo.com.br (L.I. Mambelli), ferreira-kleber@usp.br (A.K. Ferreira).

https://doi.org/10.1016/j.biopha.2018.03.135
Received 28 November 2017; Received in revised form 22 March 2018; Accepted 22 March 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
L.I. Mambelli et al. Biomedicine & Pharmacotherapy 103 (2018) 18–28

Fig. 1. Cytotoxic effect of PEA in A2058 cell line. (A) Chemical structure of PEA. MTT assay demonstrated that PEA is cytotoxic to A2058 cell line with IC50 value of
160 mM. (B) Colony forming units assay was used to analyze the capacity of PEA on reducing cell proliferation and diminish the formation of new colonies. In
accordance to MTT assay, IC50 values of PEA significantly reduced the capacity of A2058 cells to proliferate and form colonies. Paclitaxel, used as positive control,
showed to be more efficient on reducing the viability and the capacity of these cells to proliferate. (C) Panel of apoptotic and anti-apoptotic proteins analyzed by
western blotting. We demonstrated that PEA did not induce cleavage of caspases 3 and 8 and also PARP. Although, it is notable the alteration in the expression of
other important proteins, such as cytochrome c, BAD, and BCL-XL. (* p < 0.05; ** p < 0.001; *** p < 0.0001, relative to control). β-actin was used as total protein
loading control. Proteins quantification graph representing the values of each band normalized by the corresponding β-actin expression.

combination with radiotherapy has not demonstrated an impressed 2. Results

impact on patient’s survival. Thus, the development and the optimiza-
tion of new compounds, which could be new candidates for MM
treatment are urgently required. Therefore, the main purpose of the
present work was to investigate the potential anti-melanoma effects of
O-Phosphorylethanolamine (PEA) in a human melanoma model.
2.1. PEA induces cytotoxic effect and caspase-independent cell death in
human melanoma tumor cells
The cytotoxicity of PEA (Fig. 1A) was evaluated at different con-
centrations in comparison to paclitaxel, used as positive control. MTT

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L.I. Mambelli et al.
assay was performed to evaluate such cytotoxic effect. Human mela-
noma cell line (A2058 cells) was incubated for 24 h with increasing
concentrations of PEA and paclitaxel. MTT reduction shows that PEA is
cytotoxic to A2058 (Fig. 1A) with IC50 value of 160 mM. In accordance
to MTT assay, we observed that PEA at IC50 values, reduced the ca-
pacity of A2058 cells to form colonies, when compared to paclitaxel
used as control (Fig. 1B; ** p < 0.001). As expected, paclitaxel was
cytotoxic to A2058 cells with IC50 value of 10μM and also inhibited the
capacity of A2058 cells of proliferating and forming new colonies
(Fig. 1B; *** p < 0.0001). Of note, for colony-forming units experi-
ment, we observed a slight increase of new colonies formed using
40 mM of PEA (Fig. 1B; * p < 0.05), when compared to paclitaxel.
To further evaluate the molecular mechanisms underlying cell death
by PEA, we investigated proteins related to apoptosis by western blot-
ting. A2058 cells were treated for 24 h with 10μM of paclitaxel (positive
control) and different concentrations of PEA (40, 80 and 160 mM). Our
results showed that PEA did not induce the cleavage of caspases 3 and
8. In accordance, PEA also did not cleavage the Poly (ADP-ribose)
polymerase (PARP), a molecular sensor of DNA repair and programmed
cell death (Fig. 1C). Based on these findings, we propose that the cy-
totoxic effects of PEA are based on a caspase-independent manner. In-
terestingly, PEA at the concentration of 80 mM down-regulated BCL-XL,
an antiapoptotic protein of BCL-2 family. In agreement to the reduction
of BCL-XL, PEA upregulated the expression of BAD, an apoptotic
member of BCL-2 family. BAD is not capable of promoting apoptosis by
itself, so it needs to be dimerized with different BCL-2 family members
into the conserved BH1 and BH2 domains. Thus, our data suggests that
the cell death induced by PEA in A2058 cells can be dependent on the
depletion of BCL-XL expression (Fig. 1C).
2.2. Cell cycle arrest of A2058 cells in response to PEA treatment
To further investigate whether PEA exerts anti-proliferative effects
on A2058 cells flow cytometry analysis was performed. Initially, A2058
cells were synchronized by deprivation of serum for 12 h. To reenter in
the cell cycle, serum was added and at this moment cells were treated
with 10μM of paclitaxel (positive control) or IC50 value (160 mM) of
PEA for 24 h. Cell cycle results revealed that the treatment with PEA at
the concentration of 160 mM reduced the population of A2058 cells in
G0/G1 phase and increased the population of cells in G2/M phase,
when compared to cells treated with IC50 value of paclitaxel
(Fig. 2A,B). Our data indicates that PEA induces cell cycle arrest in G2/
M phase in A2058 cell line. The population of cells at Sub-G0/G1 phase
was not significant for this analysis. Such data was confirmed by wes-
tern blotting when analyzed proteins associated to cell cycle. It was
observed a slight decrease in the expression of proteins associated to
cell proliferation and expansion, when A2058 cell line was treated for
24 h with IC50 value of paclitaxel (10μM; positive control) and PEA
(160 mM). Proteins as p.RB, which besides many properties is re-
sponsible for G1 checkpoint, blocking S-phase entry and consequently
cell growth; and p.CDC2, mainly responsible for the entry of eukaryotic
cells into mitosis were also downregulated when compared to control
(non-treated cells). It was noted a minimal increase in the expression of
p.P38, which can be in response to stress stimuli. Importantly, we ob-
served a significant reduction in the expression of cyclin proteins, such
as cyclin D1 and D3 and cyclin-dependent kinases, CDK 4 and 6
(Fig. 2C). Taken together, our data indicates that PEA impair A2058 cell
division and proliferation by arresting cells at G2/M phase.
2.3. PEA reduces A2058 cell mobility and fits in silico into the C-RAF
binding site
In order to evaluate the effect of PEA on interfering in A2058 cells
capacity of adhering to plastic, or to a matrix or interfering in cell-cell
interaction, adhesion assay was performed. Indeed, PEA demonstrated
similar effects of paclitaxel (positive control) on adhesion ability of
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Biomedicine & Pharmacotherapy 103 (2018) 18–28
A2058 cells. It was possible to note that when cultures were treated
with IC50 values of PEA (160 mM) or paclitaxel (10μM; positive con-
trol) for 24 h, the capacity of cells of adhering to the matrix and to the
plastic decreased significantly, when compared to control (non-treated
cells; Fig. 3A; *** p < 0.0001 and ** p < 0.001, respectively). In
contrast, when we used the same conditions previously mentioned,
both compounds significantly increased cell-cell interaction ability of
A2058 cells (Fig. 3A). Notably, PEA was capable of significantly in-
crease cell-cell interaction of A2058, when compared to paclitaxel
(positive control; Fig. 3A; *** p < 0.0001).
The inhibitory effect of PEA on tumor cell migration, another crucial
step of melanoma skin cancer progression, was examined by the wound-
healing assay. Approximately 80% of decreasing of A2058 cell migra-
tion was observed after 24 h of tumor cells incubation with IC50 value
of PEA, when compared to control (non-treated cells; Fig. 3B; *
p < 0.05). The treatment with 10μM of paclitaxel for 24 h showed
similar result as observed for PEA (Fig. 3B; ** p < 0.001). However,
after 48 h of treatment with PEA, the “scratch” was completely closed
(Fig. 3B), while in the treatment with paclitaxel (positive control) the
“scratch” remained opened.
Of interest, we observed a significant decrease in the expression of
p.C-RAF and p.ERK when cells were treated with IC50 value of PEA
(Fig. 3C). C-RAF is a well-known proto-oncogene that in combination
with ERK forms a complex signaling pathway (RAF-MEK-ERK) re-
sponsible for regulating proliferative, differentiation, and survival
pathways of many tumors including melanoma. Also, it was possible to
note an increase in the expression of A-RAF, when compared to control
(non-treated cells; Fig. 3C). We did not observe differences in the ex-
pression of p.B-RAF as well as non-phosphorylated C-RAF in A2058 cell
line treated with different concentrations of PEA and with IC50 value of
paclitaxel (Fig. 3C).
Having observed a significant decrease in the expression of C-RAF
protein by western blotting, we investigated whether PEA could sa-
tisfactory fits into C-RAF protein. Docking simulations were used to
investigate the possible binding site of PEA in C-RAF kinase (Figure 5D,
5E), the orientation and accommodation of such compound in the
mapped binding sites, as well as to calculate the affinity of PEA for each
one (Gibbs interaction free energy, ΔG) with the formation of the en-
zyme-ligand complex. The intermolecular interactions established
among PEA and amino acids of the interaction site were determined in
the best model of dock simulation according to the ΔG (Fig. 5F).
The simulations showed that PEA fits well in the binding site located
between the dimers, in a binding site with 107,01 Å and score of
−29,62. The scores values varied from −7,57 to −29,62. PEA estab-
lished interactions with the amino acid residues of both dimers into the
binding pocket through four hydrogen bonding interactions, via three
oxygen atoms to the PEA side chain of the catalytic Arg401(a) residue
and Trp342 residue of both dimers (a and b), and via protonated ni-
trogen of amine on the oxygen of Arg398(a) residue.
2.4. Up-regulation of p.STAT-3 induced by PEA leads to the decrease of
MEK1/2 expression
We demonstrated that PEA did not induce caspase-dependent cell
death in A2058 cell and fits well into C-RAF binding site. Then, we
investigated if PEA could induce cell death trough RAS/RAF/MEK/ERK
signaling/pathway. It is important to note that this pathway plays a role
in melanoma initiation, maintenance and progression [10,11]. It has
been previously described that the inhibition of STAT-3 can be directly
related to MEK regulation [12], as well as that STAT-1 activation can
modulate MEK1 [13], then we focused on evaluating such proteins.
First, it was examined, by flow cytometry, the expression of p.STAT-
3 in A2058 cell line with or without the treatment with PEA and pa-
clitaxel. Further, we analyzed the expression of such protein while
using an inhibitor of STAT-3 and its association with both compounds.
Cucurbitacin (JSI-124) a well-known inhibitor of JAK/STAT-3 pathway
L.I. Mambelli et al. Biomedicine & Pharmacotherapy 103 (2018) 18–28

Fig. 2. PEA arrested A2058 cells at G2/M phase of the cell cycle. (A) Representative histograms are indicating selected gates of flow cytometry analysis of A2058 cell
line treated with paclitaxel or PEA. The first histogram represents the control (non-treated cells). The second one represents the paclitaxel treatment, used as positive
control. We can note that A2058 cells were arrested in G0/G1 phase. The last histogram is representative of PEA treatment, indicating the accumulation of cells in
G2/M phase. (B) Cell cycle analysis demonstrated that PEA significantly induced cell cycle arrest of A2058 cells in G2/M phase, while paclitaxel provoked the
accumulation of cells in G0/G1 phase. (C) Panel of proteins analyzed by western blotting indicates the upregulation of p.RB, when A2058 cells were treated with 40
and 80 mM of PEA, but no significative expression alteration was observed when these cells were treated with IC50 value of this compound. Also, we note a
significant increase in the expression of p.CDC2, when cells were treated with the same concentrations above. The increase in the expression of p.P38 was also
observed, when cells were treated with different concentrations of PEA, including IC50 value. β-actin was used as total protein loading control. Proteins quantifi-
cation graph representing the values of each band normalized by the corresponding β-actin expression. (* p < 0.05; ** p < 0.001; *** p < 0.0001, relative to
control).

was used. After the pre-treatment for 12 h with cucurbitacin, it was
noted a considerable amount of non-viable cells in culture, even when
treated with only this inhibitor. Notably, when IC50 values of paclitaxel
(10μM) and PEA (160 mM) were added to the pre-treated culture, more
cells were observed (data not shown). Interestingly and not expected,
flow cytometry analyses demonstrated that JSI-124 significantly in-
creased the expression of p.STAT-3, when compared to control (non-
treated cells; Fig. 4A; *** p < 0.0001). Moreover, the combination of
cucurbitacin with paclitaxel or PEA considerably increased the ex-
pression of p.STAT-3 (Fig. 4A; *** p < 0.0001) even when compared
with both compounds without the inhibitor (Fig. 4A, * p < 0.05 (pa-
clitaxel) and ** p < 0.001 (PEA)). Of note, the basal expression of
p.STAT-3 in A2058 cells is not high (Fig. 4A), indicating that STAT-3 is
not constitutively activated in this cell line.
Finally, we evaluated by western blotting the expression of STAT-1
and MEK1/2 and also their phosphorylated forms on A2058 cells
treated with IC50 value of paclitaxel or with PEA (40, 80, 160 mM). We
showed that IC50 value of PEA (160 mM) reduced the expression of
STAT-1 and p.STAT-1, as well as MEK1/2 and its phosphorylated form,
when compared to control (non-treated cells; Fig. 4B).
serum-free solution. When tumors reached 100 mm3, the animals
started to be treated according to their experimental group: control,
PEA or vemurafenib. These animals were treated daily for 10 days and
then they were euthanized when tumors reached a maximum of 2 cm3.
Such experiment allowed us to validate in vivo therapeutic effec-
tiveness of PEA as a potential new candidate antitumor agent. The
antitumor effect of PEA was compared to vemurafenib (used as positive
control), a well-known chemotherapeutic agent used for non-treated
melanoma. Analyzing the survival rate, we noted that both compounds
PEA and vemurafenib were efficient on increasing lifespan on the two
studied models (Fig. 5A). PEA showed to be more efficient on enhan-
cing survival rate in human model, than in murine model (Fig. 5A) but
only vemurafenib was capable of significantly inhibiting tumor growth
along the treatment in human model injected with A2058 cells (Fig. 5B,
* p < 0.05). However, PEA significantly inhibited tumor growth in
C57BL/6 injected with B16F10 cells when compared to control (ani-
mals treated with placebo) and vemurafenib (positive control; Fig. 5B; *
p < 0.05). Taken together, our data demonstrates that PEA is capable
to inhibit the tumor growth in B16F10 model and prolong survival rate
of mice in both human and murine melanoma models.

2.5. PEA inhibits tumor growth and increases survival rate in melanoma 2.6. Antitumor activity of PEA in B16F10 animal model is immune-system
models dependent

To evaluate the antitumor effect of PEA in vivo, we used two mel-
anoma models: first, a human melanoma model in which female BALB/
c nu/nu mice were injected with 5 × 106 of A2058 cells diluted in 50%
matrigel solution and; second, a model performed in female C57BL/6
mice injected with the same amount of B16F10 cells in RPMI-1640
Since we obtained a promising data on increasing survival rate and
reducing tumor growth, we studied whether PEA could affect the im-
mune system in vivo. Herein, we propose a therapeutic protocol using
bone marrow-derived dendritic cells previously described by Figueiredo
and colleagues [14]. The dendritic cells were first incubated ex vivo

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with PEA in combination with a melanoma cell lysate and then adop-
tively transferred into C57Bl/6 mice with lung metastases of B16F10
cells. We observed that such combination of treatment significantly
protected the animals (Fig. 6A; ** p < 0.001) and lead us to suggest
that PEA stimulated DCs on decreasing the number of metastatic no-
dules in this model (Fig. 6B).
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Biomedicine & Pharmacotherapy 103 (2018) 18–28
(caption on next page)
3. Discussion
Herein, we demonstrated that PEA is capable of inducing caspase-
independent cell death in A2058 human melanoma cell line. According
to our data, PEA was efficient on significantly decrease the expression
of C-RAF and ERK proteins when cells were treated with IC50 value of
this compound. Moreover, we showed in silico that PEA indeed fits into
L.I. Mambelli et al. Biomedicine & Pharmacotherapy 103 (2018) 18–28

Fig. 3. Effects of PEA on cell mobility of A2058 and in silico molecular docking. (A) Cell adhesion assay was used to evaluate the capacity of PEA on interfering at
metastatic ability of A2058 cells. PEA significantly decreased the capacity of A2058 cells of adhering to the matrix and to the plastic, but increased cell-cell
interaction ability when treated with IC50 values. (B) Migration area examined by phase contrast microscopy at 0, 24 and 48 h (magnification, ×100). PEA
significantly delayed the repopulation of “scratch” area, demonstrating similar effects of paclitaxel, used as positive control, in 24 h of treatment. (C) Panel of proteins
analyzed by western blotting demonstrated a significant decrease of p.C-RAF and p.ERK. Other important proteins, as p.BRAF, C-RAF and ARAF were also analyzed,
but no significant alteration concerning PEA treatment was observed. β-actin was used as total protein loading control. Proteins quantification graph representing the
values of each band normalized by the corresponding β-actin expression. (* p < 0.05; ** p < 0.001; *** p < 0.0001, relative to control). Having demonstrated that
PEA could modulate the expression of phosphorylated C-RAF, the main component of RAS/RAF/MEK/ERK signaling/pathway, we performed in silico docking of PEA
and observed that this compound fits well in the C-RAF protein domain. (D) View of crystal structure of C-RAF homodimer. (E) Mapped binding sites (green bubbles)
on the molecular surface of C-RAF homodimer, considering regions larger than 20 Å. Circle represents the binding site with the best score in docking simulations; (F)
Schematic representation for the molecular docking of PEA in the mapped binding site of C-RAF using the CLC Drug Discovery Workbench program. PEA is displayed
as stick model where the carbon atoms are represented in gray, nitrogen in blue, oxygen in red, hydrogen in white and phosphorus in yellow. The amino acid residues
(Arg398(a), Arg401(a), Trp 342(a), Trp342(b)) in the binding site are also shown as stick model (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article).

Fig. 4. Regulation of p.STAT-3 induced by PEA on MEK1/2 signaling pathway. STAT-3 inhibitors have been described as potential targets to promote resistance
phenotypes in melanoma cells and other tumor types, by modulating RAS/RAF/MEK/ERK pathway. (A) A2058 cells treated with JSI-124, paclitaxel or PEA were
analyzed by flow cytometry. Such analysis demonstrated that the basal expression of p.STAT-3 protein in A2058 cells is not high, but after the pre-treatment for 12 h
with cucurbitacin, this protein expression significantly increased. Also, the combination of cucurbitacin with paclitaxel or PEA considerably increased the expression
of p.STAT-3, when compared with both compounds without the inhibitor (B) Panel of proteins analyzed by western blotting indicated the reduction in the expression
of STAT-1 also. Corroborating with previous data, the expression of MEK1/2 was considerably reduced when cells were treated with IC50 value of PEA (* p < 0.05;
** p < 0.001; *** p < 0.0001, relative to control). β-actin was used as total protein loading control.

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L.I. Mambelli et al. Biomedicine & Pharmacotherapy 103 (2018) 18–28

Fig. 5. Antitumor activity in vivo of PEA. We evaluated the antitumor effect of PEA in vivo in two melanoma models: a human melanoma model performed in
immunocompromised mice injected with A2058 cells; and a murine model that was performed in female C57BL/6 mice injected with B16F10 cells. (A) The analysis
of survival rate showed that both PEA and vemurafenib (used as positive control) were efficient on prolonging lifespan in human and murine melanoma models.
Mostly important, PEA showed to be more efficient than vemurafenib on improving survival rate in human model. (B) Vemurafenib was capable of significantly
inhibiting tumor growth in human model, while PEA significantly inhibited tumor growth in murine model. (* p < 0.05, relative to control).

Fig. 6. PEA can act in combination with the immune system to promote metastasis regression in syngeneic murine melanoma model. Bone marrow-derived dendritic
cells were used in combination with PEA in order to test their capacity in reducing lung metastases provoked by B16F10 cells in C57Bl/6 mice. (A) Therapeutic
activity of dendritic cells stimulated by PEA and tumor cells lysate significantly protected the animals and decreased metastatic sites. (B) Significant regression of
metastatic lung nodules is shown demonstrating that PEA may act in combination with the immune system. (** p < 0.001, relative to control).

C-RAF protein binding site with a score of -29,62 kcal/mol. Taken to-
gether, our main results lead us to suggest that the mechanisms in-
volved in this cell death process could be related to RAS/RAF/MEK/
ERK signaling/pathway. Most importantly, we showed that PEA pro-
longs the lifespan of mice-developed human and murine melanomas,
besides of inhibiting tumor growth in murine model.
Mitogen-activated protein kinase (MAPK) pathway is appointed as
RAS/RAF/MEK/ERK cascade and can be considered a well-known and
deeply studied cell signaling/pathway. This pathway is commonly
dysregulated not only in melanoma but also in a wide variety of tumor
types [10]. Importantly, PEA can impair the activation of this pathway
and lead as to assume that this compound is mediating the reduction of
cell proliferation and regulating differentiation and survival of A2058
cells. Of note, lots of efforts have been done to develop antitumor
agents that could target key constituents of RAS/RAF/MEK/ERK
pathway. Surely, PEA represents an important drug candidate to target
this pathway.
Synthetic phosphoethanolamine (Pho-s) has been considered a po-
tential antitumor agent, and it has been shown that its cytotoxic effect
acts selectivity against tumor cells and could be used for the treatment
of a wide range of tumors [1,2,5,6,15,16]. At current study, we tested
the effect of a commercial molecule of PEA in melanoma A2058 cells. In
accordance to what was previously demonstrated for different cell lines
using Phos-s, the commercial compound (PEA) also exhibited cytotoxic

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effect on A2058 cells. PEA not only reduced viability but also blocked
the capacity of A2058 cells of forming new clones, which in turn is
considered an important step for tumor development. The in vitro ef-
fects observed by PEA treatment may suggest that this compound can
reduce tumor cells invasion toward different sites [17]. In accordance
to this, we previously demonstrated that Pho-s is capable of reducing
the proliferative capacity of cells in an acute promyelocytic leukemia
(APL) model [5].
Interestingly, at low concentrations PEA provoked the opposite ef-
fect on A2058 cells proliferation and capacity of forming new colonies.
Compared to control, the concentration of 40 mM of PEA significantly
increased the capacity of these cells to divide. We reasonably start out
from the assumption that there is no cytotoxicity at low concentration
of a compound, then as the concentration increases, the cell kill is
proportional to drug concentration until it reaches a plateau at high
concentrations [18].
Recently, it was proposed a hormesis model which claims that is
necessary to consider the mechanisms acquired by organisms to de-
toxify chemicals and then being resistant to drugs [19]. In respect to
such data, it has been described that breast cancer cell lines exposed to
low concentrations of an experimental compound also showed opposite
effects [20]. On the other hands, PEA at sub-toxic concentration may
enter in a growth-stimulating zone and induces cell proliferation.
Contrary to expectations of what we showed in previous studies
using Pho-s [15,16], here, for the first time, we demonstrated that PEA
did not induce caspase-dependent cell death. Notably, we observed
alterations in the expression of important apoptotic proteins, such as
cytochrome c and BAD, as well as BCL-XL an anti-apoptotic protein.
Although the expression of BAD was similar to the control (non-treated
cells) when IC50 of PEA was used, it is important to highlight that this
protein increased significantly when cells were treated with low values
of PEA. In accordance to such data, the expression of anti-apoptotic
protein BCL-XL significantly decreased compared to control (non-
treated cells), including IC50 value. Both BAD and BCL-XL are im-
portant proteins of BCL-2 family, which are in mitochondria and are
responsible for controlling programmed cell death, such as apoptosis
and autophagy. An increase of BAD, a pro-apoptotic member of BCL-2
family induced by PEA can be responsible for initiating cell death in
A2058 cells. The increase of BAD can blocks BCL-XL activity and pre-
vent the release of cytochrome c, then avoiding cell death. Thus, BAD
increase in response to PEA treatment indicates that PEA provokes the
formation of a mitochondrial membrane pore which in turn initiate the
apoptotic cascade in A2058 cells.
Notably, PEA did not induce the cleavage of proteins that are re-
presentative indicators of caspase-dependent cell death, even following
the treatment with different concentrations, including IC50 value of
PEA. Herein, we did not observe the cleavage of caspase 3 or 8. Indeed,
it was not possible to detect the presence of cleaved PARP. The com-
bination of such data, the deregulation of BCL-XL and the increase of
BAD lead us to suggest that PEA induced cell death in a caspase-in-
dependent manner in A2058 cells.
One interesting finding is that PEA showed to possess an inhibitory
effect on A2058 cells proliferation by inducing cell cycle arrest in G2/M
phase. Some of our data on the expression pattern of proteins related to
cell cycle, such as cyclin proteins and cyclin-dependent kinases besides
of p.RB and p38, corroborated with such result. Cyclins are the main
proteins responsible for controlling the progression of cells division.
These proteins act by triggering cyclin-dependent kinases and forming
complexes which are capable to control the progression of specific
phases of the cell cycle. Herein, we observed the reduction of the ex-
pression of cyclins D1 and D3 and consequently the cyclin-dependent
kinases 4 and 6 which are activated by the cyclin D family. This family
of cyclins are known to be related to the progression of G1 to S phase,
then the reduced signaling to the progression of G1 to S phase can
explain the arrest in G2/M phase of A2058 cells treated by PEA.
Retinoblastoma protein (p.RB) is a tumor suppressor protein, which is
25
Biomedicine & Pharmacotherapy 103 (2018) 18–28
responsible for preventing decontrolled cell proliferation, by the in-
hibition of cell cycle progression. When this protein is phosphorylated,
it becomes inactive and allows cells to divide. Herein, we observed a
significant increase in the expression of such phosphorylated protein,
when A2058 cells were treated with 40 and 80 mM of PEA, but inter-
esting this expression pattern was not confirmed when the IC50 value of
PEA was used. More interestingly, it was possible to note a significant
alteration in the expression of p.CDC2 protein when these cells were
treated with the same concentration of this compound. CDC2, also
known as cyclin-dependent kinase 1 (CDK1) is a cell cycle key protein
responsible for regulating the progression of cell cycle. When phos-
phorylated, this protein forms complexes responsible to phosphorylate
important substrates, which will allow the entry of cell in S phase of cell
cycle, thus permitting cell division. According to the literature, the
increase of p.CDC2 indicates that cells were accumulated in G1/S phase
of cell cycle [21]. Considering our data which showed that PEA arrested
A2058 cells at G2/M, we suggest that p.CDC2 increased significantly as
a reverse mechanism of the cell as an unsure to stimulate cell division
and avoid cells death. Of interest, A2058 cells were not able to reenter
the cell cycle and were arrested in G2/M phase. We also note an in-
crease in the expression of p.P38, which is a kinase from the class of
mitogen-activated protein kinases (MAPK) that are responsive to stress
stimuli, in cell treated with IC50 value of PEA. Such data could be
considered a response to such stress stimuli induced by PEA in A2058
cell line.
Interestingly, we demonstrated that PEA exhibits similar effect of
paclitaxel when considering the inhibition of A2058 cells of adhering to
a matrix or a substrate (plastic). The most important and relevant data
of this finding is that PEA can reduce the capacity of A2058 cells to pass
through the extracellular matrix. The cell adhesion assay is often used
to assess the metastatic ability of a specific tumor cell population, as
well as, to test whether a compound may interfere in such ability [22].
Our finding suggests that PEA can prevent metastasis, which is one of
the reasons for the resultant mortality of patients with cancer [23,24].
In this study, PEA was found to trigger the increase of cell-cell inter-
action properties of A2058 cells. Of note, PEA was more efficient on this
role than paclitaxel. Regarding melanoma and metastasis progression,
such feature exerted by PEA may be valuable if it is considered that the
initially main property to be lost for cell invasion is the ability of a cell
to adhere to its neighbored cells [17].
It was also analyzed whether PEA would be able to interfere at
migration capacity of A2058 cells. Notably, PEA significantly delayed
the repopulation of “scratch” area, corroborating with previous data of
adhesion analyses. Moreover, proteins that are known to be important
for melanoma tumor cells division, maintenance and progression were
also investigated. Most importantly, there was an expressive reduction
in the expression of p.C-RAF protein, and consequently of p.MEK1/2
and p.ERK.
Herein, we observed that PEA significantly decreased p.C-RAF ex-
pression, the main component of MAPK pathway. Thus, the inhibition
of C-RAF phosphorylation by PEA leads to the decrease of ERK1/2
phosphorylation in A2058 cells. Importantly, when p.C-RAF is activated
it is capable of modulating different substrates than those directly in-
volved in MAPK cascade, notably one of these such important targets
that was increased by PEA was the pro-apoptotic protein BAD [25].
Indeed, cell cycle arrest of A2058 cells in response to PEA treatment can
also be related to the inhibition of C-RAF phosphorylation and the re-
duction of MEK activation, which is capable to regulate the cell cycle
progression and consequently cell division. One unanticipated finding
was that PEA has induced cell death in A2058 cells by the reduction of
C-RAF activation, and leads to the increase of BAD expression in a
mechanism independent of caspase cleavage.
Additionally, as previously mentioned, the pro-apoptotic protein
BAD also showed to be downregulated when the same cell line was
treated with IC50 value of paclitaxel and PEA. Notably, when the mo-
lecular docking of PEA was performed in silico, we noted that this
L.I. Mambelli et al.
compound established intermolecular interactions with the amino acids
of the interaction site of C-RAF protein. This analysis showed that PEA
fits well in the binding site located between the dimers of the protein, in
a binding site with 29,62 kcal/mol of score. All these data together, lead
us to suggest that PEA could induce cell death in A2058 cell line by
modulating RAS/RAF/MEK/ERK pathway.
Having demonstrated that PEA could lead cell death by the mod-
ulation of RAS/RAF/MEK/ERK cascade, we attempt to investigate if
important proteins indirectly related to such signaling/pathway could
be altered. It has been previously described that STAT-3 inhibitors
could mediate resistance phenotypes not only in melanoma cells but
also in other tumor types, by modulating RAS/RAF/MEK/ERK pathway
[12,26]. STAT-3 is a transcription factor that although plays an im-
portant role during normal cell development, usually is constitutively
activated in almost 70% of solid and hematological tumors, thus in-
hibitors of this protein appeared as a novel target for cancer drug dis-
covery [27,28]. We showed that A2058 cells possess low expression
levels of p.STAT-3, indicating that STAT-3 is not constitutively acti-
vated in this cell line. But interestingly, when cells are treated with
cucurbitacin, a natural selective inhibitor of p.STAT-3, there is a sig-
nificant increase of this protein expression. Although this inhibitor
alone provoked cell death on A2058 population, the combination with
PEA significantly increased the expression of p.STAT-3. Cichowski and
Jänne mentioned that some inhibitors, such as some of RAF enzymes,
could either suppress or activate the same signaling pathway [29]. The
precise mechanism by which some inhibitors could contrarily act is
unknown, but provides a warning note for targeting anticancer drug
discovery.
In previous studies developed by our group, Pho-s showed in vivo
anti-melanoma properties [3] and anti-metastatic effect in a renal
carcinoma model6. In this model, Pho-s inhibited lung metastasis in
nude mice and blocked endothelial cells proliferation, migration and
tube formation, besides provoked cell cycle arrest at the G2/M phase.
Herein, we evaluated the antitumor effect of PEA in vivo in two
melanoma models: a human melanoma model in which female BALB/c
nu/nu mice were injected with A2058 cells; and a second model that
was performed in female C57BL/6 mice injected with B16F10 cells. We
showed that PEA triggered an important therapeutic effect in both
melanoma models in vivo. Considering the survival rate, it was observed
that both compounds PEA and vemurafenib (used here as positive
control) were efficient on increasing lifespan in human and murine
melanoma models. Most important, PEA showed to be more efficient
than vemurafenib on improving survival rate in human model. On the
other hand, only vemurafenib was capable of significantly inhibiting
tumor growth in such model. It is important to highlight that PEA
significantly inhibited tumor growth in murine model, while vemur-
afenib was not efficient on inhibiting tumor growth. Vemurafenib is a
well-known BRAF enzyme inhibitor used for non-treated melanoma and
part of our results, such as the fact that this chemotherapeutic was ef-
ficient on inhibiting tumor growth in human model, could be explained
by this feature. As previously mentioned, the human model consisted in
immunocompromised mice injected with A2058 cells. This cell popu-
lation is known to be a BRAF-mutant cell line and then is expected that
vemurafenib would be the most efficient on regretting tumor growth.
Taking into consideration that PEA was capable of prolonging sur-
vival rate in both animal models proposed here, but only in the murine
model showed to be efficient on delaying tumor growth, while in im-
munocompromised animals had no effect, we decided to explore whe-
ther the immune system could be associated to this effect. Herein, we
used bone marrow-derived dendritic cells, previously incubated ex vivo
with PEA and melanoma cell lysate, to test their adoptive transfer into
C57Bl/6 mice with lung metastases provoked by B16F10 cells.
Interestingly, we observed that this combination of treatment sig-
nificantly protected the animals and lead us to suggest that PEA was
capable to stimulate the dendritic cells to decrease the number of me-
tastatic nodules in this syngeneic metastatic melanoma model,
26
Biomedicine & Pharmacotherapy 103 (2018) 18–28
demonstrating that this compound may act in combination with the
immune system.
Herein, we presented for the first time, new data that PEA is capable
of inducing caspase-independent cell death in A2058 cells by reducing
C-RAF expression and modulating RAF/MEK/ERK signaling/pathway.
Nevertheless, further analyses are required to clarify the antitumor
mechanisms of action in more details. Most importantly, PEA showed
therapeutic effects in animal melanoma models. Thus, PEA represents a
potential prototype as a new chemotherapeutic agent for the treatment
of untreated melanoma.
4. Methods
4.1. Chemical and reagents
O-Phosphorylethanolamine (cat.27640) was purchased from Sigma-
Aldrich (St. Louis, MO, USA) and stock solution was diluted in phos-
phate buffered saline (PBS) at a concentration of 0.6 M. The pH of the
solution was adjusted to 7.2 with sodium hydroxide (NaOH) and then
stored at 4 °C. For in vivo and in vitro tests PEA was again diluted in PBS-
vehicle or directly in the basal medium, respectively. Paclitaxel (cat.
T7191) was purchased from Sigma-Aldrich and diluted in dimethyl
sulfoxide (DMSO) according to manufacture’s instructions.
Cucurbitacin I (JSI-124) was purchased from Sigma-Aldrich, as well as
Thiazolyl Blue Tetrazolium Bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; MTT), Propidium Iodide (PI) and RNase.
4.2. Cell culture
The A2058 (human melanoma; CRL-11147) and B16F10 (murine
melanoma; CRL-6475) cell lines were obtained from American Type
Culture Collection (ATCC). All cell lines were routinely maintained in a
basal medium composed by RPMI-1640 (Sigma-Aldrich) supplemented
with 10% (v/v) fetal bovine serum (FBS) (Gibco, Karlsruhe, Germany),
100U/ml penicillin and 100 μg/ml streptomycin at 37 °C under a hu-
midified atmosphere of 5% CO2.
4.3. Cell viability assay
The MTT cell viability assay was conducted according to manu-
facturer’s instructions (Sigma-Aldrich). A2058 cells were seeded in
triplicate into 96-well plates at a density of 1 × 104 cells/100 μL/well
and allowed to attach overnight. Cells were then treated with different
concentrations of PEA (from 4.69 to 300 mM) or paclitaxel and in-
cubated for 24 h. Cell viability was monitored by the MTT solution at a
final concentration of 5 mg/mL. Then, the medium was discarded,
DMSO was added and absorbance was measured in a plate reader
(Thermoplate Q4 TP Reader, Tokay Hit, Japan) at 540 nm. The data
obtained were further analyzed using Prism nonlinear regression soft-
ware (GraphPad Software, Inc.) for curve fitting and the determination
of absolute IC50 values.
4.4. Cell cycle analysis
The A2058 cell line was seeded into 6-well plates at a density of
1 × 104 cells/well, synchronized by deprivation of serum for 24 h and
induced to reenter the cell cycle by the subsequent addition of serum.
Then, cells were treated for 24 h with PEA or paclitaxel at concentra-
tions of 160 mM and 10μM, respectively. Next, cells were collected and
fixed with cold 70% ethanol and stored at -20 °C for 24 h. Cells were
washed and resuspended in PBS for being incubated at 37 °C for 15 min
with 10 mg/ml RNase and 1 mg/ml PI. After, they were washed and
resuspended in PBS for analysis. A total of 10.000 cells per analysis
were examined by flow cytometry (FACSCalibur) and analyzed using
Modfit LT software (Verity Software House, Topsham, ME). Data re-
present the mean ± SD of three independent experiments.
L.I. Mambelli et al.
4.5. Colony forming units assay
To analyze the capacity of A2058 cell line to form new colonies
when treated with 160 mM PEA or 10μM paclitaxel, 102 cells were
plated in triplicate in 6-well culture plates. The cultures, both experi-
mental and control were maintained until day 15 and the medium was
changed every two or three days. After 15 days, cells were fixed with
4% paraformaldehyde for 2 h and stained by crystal violet.
4.6. Cell lysate and Western Blot analysis
Lysates were prepared from A2058 cell line treated with different
concentrations of PEA (40, 80 or 160 mM) or 10μM paclitaxel with lysis
buffer containing protease and phosphatase inhibitors. Briefly, cells
debris were removed by centrifugation at 12.000 rpm for 5 min, and
after protein separation by gel electrophoresis, the proteins were
transferred to PVDF membranes. Membranes were then blocked with
5% bovine serum albumin (BSA, Sigma-Aldrich) in TBS-Tween 20 so-
lution for 1 h, followed by overnight agitating incubation with appro-
priate primary antibodies obtained from cell signaling at 4 °C. Finally,
membranes were incubated for 1 h with HRP-conjugated secondary
antibodies. Detection was by WesternSure ECL Substrate (Li-Cor,
Lincoln, NE, USA). Images were acquired by ChemiDoc MP System (Bio-
Rad, California, USA) and the blots were normalized for β-actin in each
lane.
4.7. Cell adhesion assay
The binding capacity of A2058 cell line when treated with 160 mM
PEA and 10μM paclitaxel was tested. In order to analyze the interaction
among A2058 cell line and the plate surface, the Geltrex Matrix
(Thermo Fisher, Massachusetts, USA) and itself, 104 cells were seeded
in 96-well culture plate. First, Geltrex Matrix was prepared by in-
cubation for 30 min at 37 °C in 5% CO2 humidified chamber. At the
same time, a new culture plate was prepared with 105 cells, which were
allowed to adhere for 2 h to test cell-cell interaction. Next, the same
quantity of cells (104 cells/well) was plated on top of Geltrex, or on top
of previously adhered cells, or directly onto the bottom of the plate. At
this moment, each treatment was added to the corresponding well. Cells
were treated for 24 h, then the supernatants were individually cen-
trifuged and the number of viable cells was counted by using trypan
blue solution.
4.8. Migration (wound-healing) assay
To evaluate the capacity of A2058 cell line to migrate in vitro under
specific treatment, 160 mM PEA and 10μM paclitaxel was used. For this
experiment, 5 × 105 cells were plated in 24-well culture plates until
they reached total confluence. At this point, the “wound” was per-
formed and the corresponding treatment added. Pictures were obtained
at the moment the wound was performed and after 24 and 48 h.
4.9. Expression of p.STAT-3 by flow cytometry
The A2058 cell line was seeded into 6-well culture plates and pre-
treated for 12 h with 5μM of Cucurbitacin I (JSI-124), a selective in-
hibitor of JAK/STAT-3 [27,30,31]. After this period, it was added in the
culture medium 160 mM PEA or 10μM paclitaxel for 24. Next, cells
were fixed and permeabilized with Triton X-100 0.02%, then stained
with p.STAT-3 antibody and the corresponding secondary antibody
both obtained from cell signaling. The percentage of cells expressing the
protein was analyzed by flow cytometry (FACSCalibur). A total of
10.000 cells/sample were analyzed using FlowJo software (TreeStar,
Version X).
27
Biomedicine & Pharmacotherapy 103 (2018) 18–28
4.10. In vivo preclinical studies
All animal experiments were conducted in agreement with ethical
principles for animal research and approved by the ethics committee for
animal research of the University of Sao Paulo, Sao Paulo, SP, Brazil
(process number 31/2014). Females BALB/c nu/nu, 6- to 8-week old
were injected subcutaneously with a suspension of A2058 cells
(5 × 106/cells per animal) in a 50% growth factor-reduced Matrigel
(BD Biosciences, Franklin Lakes, NJ) solution. These experiments were
also conducted using female C57BL/6 mice, 6- to 8-week old, which
were injected subcutaneously with a suspension of B16F10 cells
(1 × 106/cells per animal) in RPMI-1640 serum-free solution.
Melanoma tumors were allowed to reach 100mm3 and randomized
(n = 5 per group) into control (vehicle solution only), PEA 100 mg/kg
or vemurafenib 5 mg/kg groups. All groups were treated daily for 10
days, tumors were measured with a manual caliper every 2 days and
volumes were calculated using the formula: tumor weight
(mg) = [length (mm) × width2 (mm2)]/2. The tumor doubling time,
i.e. the time for a tumor to double in volume and tumor growth delay,
that was the difference between the time required for tumors in treated
and untreated animals to reach at least 1cm3, was evaluated. Animals
were euthanized when tumors reached a maximum of 2cm3. Then, the
tumors were surgically excised and masses were recorded.
4.11. Bone marrow-derived dendritic cells (BMDCs) and adoptive transfer
therapy
In order to evaluate whether PEA could act in combination with the
immune system to prolong survival rate in animal models, we per-
formed adoptive transfer analysis. BMDCs were obtained from femurs
of C57Bl/6 mice after euthanasia. These femurs were flushed with
RPMI-1640 medium and red blood cells were lysed for 5 min. The cells
from each femur were suspended in 20 mL of basal medium supple-
mented with 50 ng/mL of GM-CSF and 25 ng/mL of IL-4 (BD
Bioscience) and incubated in a Petri dish at 37 °C with 5% CO2. After
five days in culture, part of the medium was replaced by fresh medium
with GM-CSF. Non-adherent blood cells were discarded and adherent
cells were used in the experiment. Dendritic cells were pulsed with the
lysate of a subcutaneous B16F10 tumor in the proportion of 10:1 (10 of
dendritic cells and 1 of tumor cells lysate (L)) and with 100 mM of PEA.
Then, mature dendritic cells (maDCs) were used for adoptive transfer in
C57BL/6 syngeneic mice, with previously developed-melanoma by the
injection of 5 × 105 B16F10 cells. After seven days of the adoptive
transfer, animals were treated with 5 × 105 maDCs and separated in
two groups (n = 5 per group): Control (vehicle + L) and experimental
(100 mM PEA + L). On the day 14, mice were euthanized and meta-
static lung nodules were quantified.
4.12. Molecular docking of PEA
A molecular docking approach was performed to predict a binding
mode for PEA on C-RAF kinase. The C-RAF co-crystallized structure
bounded to the (1E)-5-(1-piperidin-4-yl-3-pyridin-4-yl-1H-pyrazol-4-
yl)-2,3-dihydro-1H-inden-1-one oxime inhibitor (SM5), entry code
3OMV at 4.0 Å resolution [32] was retrieved from Brookhaven Protein
Data Bank (PDB) [33] and used as geometric reference for constructing
the biomacromolecule model.
PEA was docked at the C-RAF kinase mapped binding sites. Docking
studies were developed with the CLC Drug Discovery Workbench pro-
gram (version 2.4, Qiagen Aarhus A/S, 2014). The protocol used was
1000 interactions, considering rigid interactions, and the minimization
was calculated with the Nelder-Mead Simplex Minimization function
implemented in the software [34]. The classification or score function
considers the total potential energy of the complex generated (Gibbs
interaction free energy, ΔG) with the PLANTSPLP [35] algorithm and
also considers the square root mean square deviation (RMSD). Negative
L.I. Mambelli et al.
values of score indicate the energetic favoring of formation of the en-
zyme-ligand complex. The more negative value of ΔG, the more fa-
vorable is the formation of the enzyme-ligand complex.
4.13. Statistical analysis
GraphPad Prism 5.0 (San Diego, CA) was utilized for all tests. All
values were expressed as mean ± SD. Each value is the mean of at least
three independent experiments in each group. Two-way analysis of
variance (ANOVA) with Tukey Post-Hoc test or Student’s t-test was
carried out to determine significant differences from untreated controls.
The asterisk (*) indicates the values that are significantly different from
control (* p < 0.05, ** p < 0.001 and *** p < 0.0001). The absence
of * indicates non-significant differences.
Author contributions
A.K.F designed the research. L.I.M, S.F.T, S.D.J, B.K, R.A.A and
A.K.F performed the experiments. L.I.M, S.D.J, J.A.B, R.A.A and A.K.F
analyzed the data. L.I.M and A.K.F wrote the main text and prepared all
figures. All authors reviewed and approved this manuscript.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgments
The authors thank the financial support of FAPESP on this work
(grants 2015/18528-7, 2013/07273-2; post-doctoral fellowships 2016/
07519-0; 2014/14267-1 and 2017/01265-9; and doctoral fellowship
2016/09392-7).
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