Modulation of pro-apoptotic effects and mitochondrial potential on B16F10 cells by DODAC/PHO-S liposomes
Luna et al. BMC Res Notes
Modulation of pro-apoptotic effects and mitochondrial potential on B16F10 cells by DODAC/PHO-S liposomes
Arthur Cássio de Lima Luna 0 2
José Roberto de Assis Santos Filho 0
Henrique Hesse 0
Salvador Claro Neto 1
Gilberto Orivaldo Chierice 1
Durvanei Augusto Maria 0 2
0 Biochemistry and Biophysical Laboratory, Butantan Institute , 1500, Vital Brasil Avenue, Sao Paulo 05503-900 , Brazil
1 Department of Chemistry and Molecular Physics, University of Sao Paulo , Sao Carlos , Brazil
2 Department of Medical Sciences, Medical School, University of Sao Paulo , Sao Paulo , Brazil
Objective: We aimed to evaluate the potential of DODAC/PHO-S liposomes on the modulation of the expression of pro-apoptotic proteins, loss of lysosomal integrity and the mitochondrial electrical potential, compared with phosphoethanolamine. Results: The results of this study demonstrate that DODAC/PHO-S liposomes have exhibited broad cytotoxic potential in B16F10 murine melanoma cells, with significantly greater proportions than treatment with PHO-S. The treatment with the DODAC/PHO-S 2.0 mM liposomal formulation was more efficient in decreasing mitochondrial electrical potential at the same concentrations and treatment time than PHO-S The liposomal formulation DODAC/PHO-S (2.0 mM) was more efficient to promote morphological changes in the cells, without presenting intact lysosomes, at the same time of treatment and concentration as PHO-S Our results demonstrated that the liposomal formulation increased DR4 receptor expression and activated caspases 8 and 3, resulting in the release of cytochrome c in B16F10 tumour cells, when compared to treatment with PHO-S The data obtained prove that the use of DODAC as carrier can maximize the cytotoxic effects of PHO-S This was demonstrated by the translocation of cytochrome c to the cytoplasm and activation of caspase-3 and 8, decreasing the mitochondrial electrical potential and generating morphological changes, in B16F10 cells.
Liposomes; Nanotechnology; Melanoma; Phosphoethanolamine
Taking into account the limiting factors of the therapies
available for the treatment of melanoma, new treatments
that are more effective and less harmful are necessary.
Thus, synthetic phosphoethanolamine (PHO-S), a
phosphoric ester known as amino-ethyl phosphoric ester, has
previously been synthesized by our group. Our results
demonstrated that PHO-S was cytotoxic on melanoma
tumor cells associated with apoptotic effect, without any
effect on normal cells, as such human normal
endothelial, fibroblasts and lymphocytes cells [
]. However, its
cytotoxic effect and selectivity against tumor cells could
increase with encapsulation in cationic liposomes, such
as dioctadecyldimethylammonium chloride (DODAC),
due to electrostatic interactions between these liposomes
and tumor cell membranes [
Recently our group encapsulated PHO-S in DODAC
liposomes (DODAC/PHO-S), because this cationic
liposomes can interact with the PHO-S due to
electrostatic attraction, in aqueous solution after being
subjected to phase transition temperature and subsequent
sonication. Physical–chemical analysis showed that these
liposomes were stable [
The treatment of B16F10 and Hepa1c1c7 cells with
DODAC/PHO-S liposomes demonstrated the lowest
values of IC50% for tumour cells, compared with PHO-S
alone, with an IC50% of 0.8 mM for B16F10 cells and
0.2 mM for Hepa1c1c7 cells, and without significant
effects on endothelial cells [
]. Thus, we aimed to
evaluate the potential of DODAC/PHO-S liposomes on
modulates the expression of pro-apoptotic proteins and the
mitochondrial electrical potential.
Materials and methods
Liposomal formulation DODAC/PHOS‑
Synthetic phosphoethanolamine was synthesised in
accordance with the previous studies [
Posteriorly, DODAC (Sigma, St. Louis, MO, EUA) powder
was weighed and suspended in 10 mL of water, in order
to obtain final concentrations of 0.3; 0.6; 1.0; 1.3; 1.6;
2.0 mM. Further, PHO-S was included in the solution,
at the same concentration of DODAC
(DODAC:PHOS = 1/1 molar ratio). The dispersions were maintained at
57 °C, for 20 min., until complete homogenization.
Samples were vortexed and sonicated using a Braun Sonic
1510 ultrasonic apparatus (Branson, EUA) equipped with
a titanium tip (70 watts, 3–4 min, at 60 °C). After
sonication, the samples were centrifuged at 1500 rpm for 5 min
to remove residual titanium released from the probe. The
liposomes were sterilised by filtration through a 0.22 µm
Millipore filter [
B16F10 murine melanoma cells (ATCC® CRL-6475) was
cultured in RPMI medium (LGC Biotecnologia, Cotia,
SP, Brazil). Medium was supplemented with 2 mM l-glu
tamine (Cultilab, Campinas, SP, Brazil), 10 mM HEPES
(Cultilab, Campinas, SP, Brazil), 24 mM sodium
bicarbonate, 0.01% of antibiotics and 10% fetal bovine serum
(Cultilab, Campinas, SP, Brazil). Cells were cultivated
in 5% CO2 atmosphere at 37 °C as monolayers cultures.
Cells were checked for viability using the Trypan Blue
Analysis of changes in mitochondrial electrical potential (ΔmΨ) and lysosomal integrity by confocal laser scanning microscopy
The analysis of the mitochondrial potential was realized
by confocal laser scanning microscopy. B16F10 cells were
plated on sterile glass slides in 24-well plates, at a
concentration of 105. After 24 h, the cells were treated with
PHO-S and DODAC/PHO-S at 0.3 and 2.0 mM, for 6 h.
Then, the cells were washed three times with culture
medium RMPI (Cultilab, Campinas, SP, Brazil) without
supplementation, at 37 °C and incubated with 15 µg/mL
of rhodamine-123 (Assay Designs Inc., Ann Arbor, MI,
USA), for 10 min. in the dark, at 37 °C. The excess
rhodamine-123 was washed with culture medium at 4 °C.
For lysosomal integrity assay, the cells were incubated
with 10 µg/mL of acridine orange (Molecular Probes Inc.,
Eugene, OR, USA), for 10 min. in the dark, at 37 °C. The
excess of fluorochrome was washed with culture medium
at 37 °C. The analysis was realized by confocal laser
scanning microscopy (Carl Zeiss LSM 700; Leica, Mannheim,
Analysis of expressions of markers by flow cytometry
The B16F10 cells (1 × 105 cells/well, the cell
density reached 80–90% confluence), treated with PHO-S
(2.0 mM), DODAC/PHO-S 1:1 (0.3–2.0 mM), and empty
DODAC (0.3–2.0 mM), for 12 h, were washed with PBS
and re-suspended in FACS buffer with 2.5%
paraformaldehyde for 1 h. The cell membrane was
permeabilized in 0.1% Triton ×100 for 30 min. on ice. Cells were
washed and re-suspended in FACS buffer. After
washing, cells were re-suspended in a primary antibody
anticytochrome c (ab13575, Abcam, Cambridge, MA, United
States), anti-caspase-8 (sc70501, Santa Cruz
Biotechnology Inc, Santa Cruz, EUA) and anti-caspase-3 (sc7272,
Santa Cruz Biotechnology Inc, Santa Cruz, EUA) and
anti-DR4 (ab9809, Abcam, Cambridge, MA, United
States) at a concentration of 1 μg/mL at 4 °C, for 1 min.
The corresponding isotope antibody was used as negative
control and as a secondary antibody: Goat anti Mouse
IgG (H/L): Ficoeritrina–Invitrogen (Invitrogen,
Carlsbad, EUA) was used. The analysis were performed on
BD Biosciences FACs Calibur flow cytometer (Becton–
Dickinson, San Jose, CA, United States) using Cell Quest
All values were expressed as mean ± standard deviation
(SD). Each value is the mean of at least three independent
experiments in each group. One-way analysis of variance
(ANOVA) and Tukey–Kramer multiple comparisons
test was performed. Graphics were obtained by Prism
version 5.0 (CEO and Founder, La Jolla, CA, USA)
software. P values < 0.05, < 0.01, and < 0.001 are statistically
DODAC/PHO‑S liposomes increase PHO‑S cytotoxicity
in B16F10 cells
The cells of the control group presented fibroblast-like
appearance, with cytoplasmic extensions and evident
nucleus (Fig. 1a). After the treatment period, cells treated
with PHO-S underwent morphological changes, such as
cytoplasmic retraction (Fig. 1b). In contrast, treatment
with liposomes culminated in a greater change in B16F10
cells, such as loss of its morphology, cytoplasmic
retraction and formation of apoptotic bodies between adherent
cells (Fig. 1c).
Evaluation of mitochondrial electrical potential (ΔΨm)
and lysosome integrity
It was possible to observe that the cells of the control
group had a positive marking for rhodamine 123,
demonstrating the large number of active mitochondria
in these cells and homogeneously distributed through
the cytoplasm (Fig. 1d). After treatment with PHO-S,
in higher concentration, it was possible to observe that
there was a reduction in the fluorescence emission and
a modification in the morphology of the
mitochondria in the cytoplasm of the cells. It was also possible
to observe that mitochondria fused after treatment
(Fig. 1e). Treatment with DODAC/PHO-S liposomes,
in all concentrations, reduced fluorescence emission
compared to isolated PHO-S, exhibiting low
fluorescence emission and loss of cell morphology (Fig. 1f ).
Liposomes led to disorganisation and depolarisation of
In the untreated B16F10 tumour cells, it was
possible to indicate the nucleus of the cells and the
organisation of the condensed (green) chromatin structure and
few lysosomes in the cytoplasm (red) (Fig. 1g). The cells
that were treated with PHO-S and DODAC/PHO-S
(2.0 mM) liposomes did not present intact lysosomes
(Fig. 1h, i). However, only treatment with the DODAC/
PHO-S liposomes led to chromatin disruption and
destruction of cells, at the evaluated time of treatment,
as well as a significant decrease in cell density (Fig. 1i).
Expression of pro‑apoptotic proteins after treatment
of B16F10 cells
The concentrations used in the treatment were those
that showed the greatest effectiveness in inducing
significant cytotoxicity in B16F10 cells. The expression
of TRAIL-DR4 receptor increased significantly only
in treatments with DODAC and DODAC/PHO-S 1:1
(2.0 mM) in B16F10 melanoma cells. The respective
percentages were 7.5 ± 1.3 and 8.4 ± 0.4% (Fig. 2a).
Treatment with DODAC/PHO-S (2.0 mM) was
effective in modulating the expression of active caspases 3
and 8, with mean values of 11.7 ± 0.3 and 29.8 ± 5.5%,
respectively (Fig. 2b, c). The DODAC 2.0 mM also
modulated the expression of caspase 3, with a
significant increase in this treatment, with the percentage of
3.4 ± 0.6%, but to a smaller extent than the liposomal
formulation (Fig. 2b).
The free cytochrome c in the cytoplasm was high in the
treatments with DODAC/PHO-S 2.0 mM and DODAC
2.0 mM, the mean values presented are 4.4 ± 0.6 and
2.5 ± 0.7%, respectively (Fig. 2d).
Recently, the PHO-S was encapsulated in DODAC
liposomes by our research group, aiming to maximize the
antitumor effect and providing a greater availability of
PHO-S in the tumour microenvironment [
The results of this study demonstrate DODAC/PHO-S
liposomes have exhibited broad cytotoxic potential
in B16F10 murine melanoma cells, with significantly
greater proportions than treatment with PHO-S. This
result demonstrates the efficacy of liposomal formulation
DODAC/PHO-S to promote B16F10 cell death. Recently,
these liposomes were evaluated on normal and did not
promote significant cell death [
]. Studies have shown
that the use of nanocarriers are promising drug delivery
systems, presenting several advantages such as low skin
irritation and increased protection of the encapsulated
drug. The use of nanocarriers may also increase the
penetration of anti-tumour drugs into the skin [
Cytotoxic compounds for tumour cells can lead to the
decreased mitochondrial electrical potential (ΔmΨ) [
]. Studies have shown that PHO-S has the ability to lead
to the loss of ΔmΨ and morphological changes in
Ehrlich’s tumour . Therefore, treatment with the DODAC/
PHO-S 2.0 mM liposomal formulation was more
efficient in decreasing mitochondrial electrical potential at
the same concentrations and treatment time than
PHOS. Corroborating our findings, other results showed that
PHO-S could modify the distribution of mitochondria in
the cytoplasm, leading to its fragmentation and
]. Several studies have shown that mitochondrial
dysfunctions represent a central process in the induction
of apoptosis [
It was verified in our study, that after treatment with
the PHO-S 2.0 mM it was not possible to verify intact
lysosomes, probably due to its rupture, since it is
evident in the more intense green coloration in the treated
cells. However, treatment with the liposomal formulation
DODAC/PHO-S (2.0 mM) was more efficient to promote
morphological changes in the cells, without presenting
intact lysosomes, at the same time of treatment and
concentration as PHO-S.
Taking into consideration of the morphological,
structural and energetic changes that the cells suffered after
treatment with PHO-S and liposomal formulation, the
data indicate that the liposomal formulation can lead
the cells to activate mechanisms of programmed cell
death, faster and efficient. The changes described after
the treatments are like those occurring during the
apoptotic process [
]. Therefore, the proteins involved
in the apoptotic caspase 3 and 8, DR4 receptor and free
cytochrome c treatments were quantified after the
treatments with PHO-S and DODAC/PHO-S liposomes.
Our results demonstrated that the liposomal
formulation increased DR4 receptor expression and activated
caspases 8 and 3, resulting in the release of cytochrome
c in B16F10 tumour cells, when compared to treatment
The increase of the active 8 and 3 caspases, TRAIL
receptor-DR4 expression and the release of cytochrome
c in B16F10 tumour cells, demonstrated the efficacy of
the liposomal formulation in maximising the apoptotic
potential mediated by PHO-S. Most of the apoptotic
events are required to participate in the caspases, which
are divided into initiators (8, 9 and 12) and executors (3, 6
and 7), where the latter are responsible for morphological
changes in the nucleus and cytoskeleton [
]. The PHO-S
induced apoptosis in several tumour cell lines, increasing
the expression of pro-caspase 8 that can be classified as a
caspase-8-like. Previous studies have shown that PHO-S
induced cell death by apoptosis, with increased
expression of pro-caspase 3 and the presence of tBid, indicating
that the intrinsic pathway was recruited in response to
activation of caspase 8 [
The data obtained prove that the use of DODAC as
carrier can maximize the cytotoxic effects of PHO-S. This
was demonstrated by the translocation of cytochrome
c to the cytoplasm and activation of caspase-3 and 8. In
B16F10 cells, the laser confocal microscopy analysis
demonstrated the efficiency of DODAC/PHO-S liposomes
in decreasing the mitochondrial electrical potential and
generating morphological changes.
The results of mitochondrial electrical potential and
lysosome integrity were measured by confocal microscopy.
Thus, the quantification of results and bright field can be
needed. Furthermore, western blot analysis of each
apoptotic protein can be needed to confirm the fact.
PHO-S: DODAC Dioctadecyldimethylammonium Chloride; ATCC: American
Type Culture Collection; ΔmΨ: mitochondrial electrical potential.
ACLL and JRASF carried out the research and wrote the manuscript. SCN and
GOC assisted in the research work and revised the manuscript. HH revised
the manuscript. DAM designed the protocol and revised the manuscript. All
authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the present study are available from
the corresponding author on reasonable request.
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