The oleanolic acid derivative, 3-: O -succinyl-28- O -benzyl oleanolate, induces apoptosis in B16-F10 melanoma cells via the mitochondrial apoptotic pathway

Article abstract of DOI:10.1039/c6ra18879f

Oleanolic acid (1) is a pentacyclic triterpene present in olive pomace, which is known to induce apoptosis and to have anti-tumor properties; however, high concentrations of this product are necessary to produce cytotoxic effects. The 3-O-succinyl-28-O-benzyl oleanolate derivative (4) presents greater cytotoxicity and apoptosis effects than its natural precursor, oleanolic acid, or its benzyl derivative (2). This study examines the response of B16-F10 melanoma cells to treatment with compound 4, in comparison to 1 and 3. Our studies show that treatment with 4 results in a significant inhibition of cell proliferation in a dose-dependent manner and causes apoptotic cell death. At concentrations inhibiting cell growth by 50% and 80%, compound 4 induces strong G₀/G₁ cell-cycle arrest, around 72-95% apoptosis, and mitochondrial disturbances confirmed by FACS analysis, which probably involve the activation of the intrinsic apoptotic route. Morphological changes including cell shrinkage, chromatin condensation, and loss of nuclear architecture were also observed. In this report, we demonstrated for the first time that in melanoma cancer cells, compound 4 exerts a significant anti-proliferation effect by inducing the apoptotic process with mitochondrial depolarization. These findings support the role of compound 4 as a new, potential therapeutic tool against aberrant cell proliferation in melanoma.

Full text of DOI:10.1039/c6ra18879f

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M. Medina-O'Donnell, R. M. Ferrer-Martín, E. E. Rufino-Palomares, S. Martin-Fonseca, F. Rivas, A.
Martinez, A. Garcia-Granados, A. Pérez-Jiménez, L. García-Salguero, J. Peragon, K. Mokhtari, P. P.
Medina, A. Parra and J. A. Lupiáñez, RSC Adv., 2016, DOI: 10.1039/C6RA18879F.
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Page 1 of 37
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The Oleanolic Acid Derivative, 3-O-Succinyl-28-O-benzyl Oleanolate,
Induces Apoptosis in B16-F10 Melanoma Cells via the Mitochondrial
Apoptotic Pathway
*a
b
Fernando J. ReyesꢀZurita, Marta MedinaꢀO’Donnel, Rosa M. Ferrerꢀ
c
a
b
Martin, Eva E. RufinoꢀPalomares, Samuel MartinꢀFonseca, Francisco
b
b
b
Rivas, Antonio Martínez, Andrés GarcíaꢀGranados, Amalia Pérezꢀ
a
a
d
a,e
Jiménez, Leticia GarcíaꢀSalguero, Juan Peragón, Khalida Mokhtari,
a,f
*b
*a
Pedro P. Medina, Andrés Parra, and José A. Lupiáñez
a
Department of Biochemistry and Molecular Biology I, Faculty of Science, University
of Granada, 18071 Granada, Spain. E-mail: ferjes@ugr.es, jlcara@ugr.es; Tel/Fax:
+34 958 243252, +34 958 243250
b
c
d
e
f
Department of Organic Chemistry, Faculty of Science, University of Granada, 18071
Granada, Spain. E-mail: aparra@ugr.es; Tel/Fax: +34 958 240480
Department of Cellular Biology, Faculty of Science, University of Granada, 18071
Granada, Spain
Department of Experimental Biology, Biochemistry and Molecular Biology Section,
University of Jaen, 23071 Jaen, Spain
Department of Biology, Faculty of Sciences, Mohammed I University, BP 717 60000
Oujda, Morocco
Centre of Genomic and Oncologic Investigation (GENyO), Pfizer Pharmaceutical-
University of Granada-Government of Andalusia, Technological Park of Health
Sciences, 18016 Granada, Spain
1

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Abstract
Oleanolic acid (1) pentacyclic triterpene present in olive pomace, is known to induce
apoptosis and to be antiꢀtumor, but high concentrations of this product are necessary to
produce its cytotoxic effects. The 3ꢀOꢀsuccinylꢀ28ꢀOꢀbenzyl oleanolate derivative (4),
present a greater cytotoxicity and apoptosis effects than its natural precursor, oleanolic
acid, or its benzyl derivative (2). This study examines the response of B16ꢀF10
melanoma cells to compound 4’ treatment, in comparison with 1 and 3. Our results
show that treatment with 4 results in a significant inhibition of cell proliferation in a
doseꢀdependent manner and causes apoptotic cell death. At concentrations inhibiting
cell growth by 50% and 80%, compound 4 induce strong G
0 1
/G cellꢀcycle arrest, over
72ꢀ95% of apoptosis, and mitochondrial disturbs confirmed by FACS analysis, probably
involving the activation of the intrinsic apoptotic route. Morphological changes
including cell shrinkage, chromatin condensation, and loss of nuclear architecture were
also observed. In this report, was demonstrated for the first time that, in melanoma
cancer cells, compound 4 exerts a significant antiꢀproliferation effect by inducing
apoptotic process with mitochondrial depolarization. These finding support a role of
compound 4 as possible new therapeutic tool against aberrant cell proliferation in
melanoma.
Key words: 3-O-succinyl-28-O-benzyl oleanolate, Triterpene derivatives, Cytotoxicity
activity, Cell cycle, Apoptosis, Anti-cancer, Green chemistry
.
2

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1. Introduction
Triterpenoid compounds are present in a wide range of plants that are used in
1
ꢀ8
traditional medicine and known to have antiꢀtumor properties. They are also involved
5
, 9ꢀ14
15
16ꢀ22
in metabolic regulation
, metabolic syndrome and growth processes.
All these
compounds belong to a broad family of compounds obtained by cyclic reactions from
23
2
,3ꢀoxidesqualene. Triterpenoids, in plants, are secondary metabolites that are not
essential for growth or development, but might contribute to their survival (e.g.
24
phytoalexin), and are involved in the protection of plants against pathogens or pests.
Currently, about oneꢀfourth of all medications contain an active ingredient derived from
plants. The most important triterpenoid structures are oleanane, ursane, lupane and
25
dammarane.
Oleanolic acid (1), an oleananeꢀtype triterpenoid, is a natural compound that
belongs to the pentacyclic triterpene family and is widely distributed in the plant
kingdom. Oleanolic compounds for the current study were isolated, in high amounts,
from the oliveꢀpressing residues. Oleanolic acid (1) possesses many biological
26
properties, including antiꢀinflammatory, antiꢀHIV and cytotoxic activities. This
2
6
compound (1) has also been described as an inhibitor of skin tumor progression. This
natural compound (1) provides very useful scaffold to obtain derivatives with improved
2
7ꢀ29
biological properties.
chemical modifications of 1 to improve its biological activities.
Most pentacyclic triterpenes are not sufficiently water soluble, which hampers
Our research group has performed a series of systematic
30, 31
32ꢀ34
the biological tests, and results in their low bioavailability.
One option to improve
the solubility of these triterpene compounds is to semiꢀsynthesize derivatives by
attaching diverse functional groups to the hydroxyl groups of the A ring and/or to the
carboxyl group at Cꢀ28. Our group has demonstrated that acyl oleanolic acid derivatives
3

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with a polar dicarboxylic fragment as acyl group on Cꢀ3 present major cytotoxic and
3
0, 31, 35
apoptotic effects than their precursor (1).
Chemical modifications of natural
triterpenoids compounds have resulted in products that have improved biological
activities than their natural precursors. Our group has shown that several triterpene
derivatives, with monoꢀ or diꢀpeptidyl groups at Cꢀ28, and acyl groups at Cꢀ2 or Cꢀ2/Cꢀ
3
of the triterpene skeleton, exhibit cytotoxic properties on B16−F10, HT29, or Hep G2
3
0
cancer cell lines.
Apoptosis or programmed cell death is defined as an active physiological
process of cell selfꢀdestruction. Agents that suppress the proliferation of malignant cells,
by inducing apoptosis, may be useful in both chemoprevention and chemotherapy of
cancer. Two major pathways have been described during apoptotic activation: the
mitochondrial or intrinsic pathway, and the deathꢀreceptor or extrinsic pathway.
The intrinsic pathway involves the mitochondrial disruption and a decrease in
35, 36
the mitochondrial membrane potential.
This apoptotic route involves the
mitochondriaꢀdependent activation of initiator caspases, which in turn activate
downstream executive caspases, such as caspaseꢀ3. Our research group has
demonstrated that, the oleananeꢀtype triterpenoid, maslinic acid selectively induces antiꢀ
1
tumor and proꢀapoptotic effects in HT29 colon cancer cells.
The activation of the
intrinsic apoptotic pathway is mediated by mitochondrial Bax activation and Bclꢀ2
inhibition, resulting in the mitochondrial membrane disruption, increase of ROS,
2
,
3
activation of caspaseꢀ9, caspaseꢀ3 and caspaseꢀ7.
Maslinic acid induced
1
, 2
differentiation in this cell line before the activation of the apoptotic process.
The extrinsic deathꢀreceptor pathway is triggered by death ligand through the
formation of the deathꢀinducing signaling complex that results in caspaseꢀ8 and
32, 37, 38
caspaseꢀ10 activation, which initiates the downstream apoptotic signaling.
4

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Maslinic acid also produced the activation of apoptosis directly through extrinsic
apoptotic pathway in Cacoꢀ2 colon cancer cells, involving caspaseꢀ8 activation, Bid
5
cleavage and caspaseꢀ9 and caspaseꢀ3 activation, with a lower cellꢀcycle arrest.
8
Whereas caspaseꢀ9 and ROS generation were not observed.
The evaluation of mitochondrial potential, at the cytotoxic and apoptotic
concentrations can help to determine the possible molecular mechanism involved in the
3
1
apoptotic response. We describe here the antiꢀcancer effects of three oleanolic acid
derivatives. In order to increase the apoptotic activity of these products, compound 1
was benzylated to obtain 28ꢀbenzyl derivative (2) and the 3ꢀsuccinylꢀ28ꢀbenzyl
derivative (4) (Figure 1).
(Figure 1. should be listed here)
Showed, following to 4 treatment, a potent apoptotic and antiꢀproliferation
effects in melanoma B16ꢀF10 cancer cells, including cellꢀcycle arrest in the G phase
0
with the disappearance of S phase. The compound 4 also produced over 90% of
apoptosis induction, through mitochondrial disturbances, with clear morphological
changes in the nucleus and the cytoplasm. On the other hand, the compound 2 produced
40% of apoptosis induction, similar to the natural triterpene, without affecting the
mitochondrial membrane potential.
Agents that suppress the proliferation of malignant cells, by inducing apoptosis,
could be useful in cancer therapy. Consequently, the compound 4 may provide a useful
new therapeutic strategy for skin melanoma.
5

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2. Experimental section
2.1. Apparatus and Materials
Purity of compounds was determined by a Waters Acquity UPLC system (ultraꢀ
performance liquid chromatography) coupled with a Waters Synapt G2 HRMS
spectrometer (highꢀresolution mass spectra) with ESI (electrospray ionization). The
purities of all compounds were confirmed to be ≥ 95%. Measurements of NMR spectra
1
13
(
300.13 MHz H and 75.47 MHz C) were made in CDCl (which also provided the
3
1
13
lock signal) using a VARIAN Inova unity (300 MHz H NMR). The C chemical shifts
were assigned with the aid of distortionless enhancement by polarization transfer
o
(
DEPT) using a flip angle of 135 . IR spectra were recorded on a MATTSON
SATELLITE FTIR spectrometer. Optical rotations were measured on a PerkinꢀElmer
o
2
41 polarimeter at 25 C. Melting points (mp) were determined using a Kofler
(Reichter) apparatus and were uncorrected.
All reaction solvents and chromatography solvents were distilled prior to use.
Commercially available reagents were used without further purification. Merck silicaꢀ
gel 60 aluminum sheets (ref. 1.16835) were used for TLC, and spots were rendered
o
visible by spraying with H SO –AcOH, followed by heating to 120 C, and also
2
4
visualized under UV at 254 nm. Merck silicaꢀgel 60 (0.040–0.063 mm, ref. 1.09385)
was used for flash chromatography. CH Cl (Fisher, ref. D/1852/17), CHCl (Fisher,
ref. C/4960/17), or nꢀhexane (Merck, ref. 1.04374), with increasing amounts of acetone
Fisher, ref. A/0600/17), MeOH (Fisher, ref. M/4000/17), or EtOAc (Fisher, ref.
2
2
3
(
E/0900/17), were used as eluents (all the solvents had an analytical reagentꢀgrade
purity).
Plant Material, a specimen of the plant of Olea europaea (order Lamiales,
family Oleaceae) was collected in Almegíjar, Granada, Spain in May 2001. This plant
6

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was identified by Laura Baena from the herbarium of the University of Granada. A
voucher specimen (53489ꢀ1ꢀ1) was deposited at the University of Granada Herbarium,
Granada, Spain.
2
.2. Synthesis and Characterization of 3-O-Succinyl-28-O-Benzyl Oleanolate and its
intermediates (1-4)
Oleanolic Acid (1): This compound (1) was isolated from solid oliveꢀoilꢀ
production wastes, which were extracted in a Soxhlet with hexane. Hexane extracts
were concentrated and 1 was purified from these residues by column chromatography
over silica gel, eluting with a CHCl
3
/MeOH or CH
White solid, mp 306ꢀ308 °C; [α] = +80 (c 1, CHCl
438, 2930, 2869 y 1690 cm . The compound (1) presents a mass spectrum that
corresponds to a molecular formula of C H O and, therefore, has a molecular weight
2
Cl
2
/acetone mixtures of increasing
3
9, 40
polarity.
D
3
); IR (KBr) νmax:
ꢀ1
3
30
48
3
1
of 456 amu. In its H NMR spectrum, the more deshielded signals appear at 5.25 ppm
1H, dd, J =J = 3.6 Hz) for the olefinic proton of Cꢀ12, 3.16 ppm (1H, dd, J =4.8 Hz,
=11.2 Hz) for the equatorial proton of Cꢀ3 geminal to the hydroxyl at this position and
.84 ppm (1H, dd, J =3.7 Hz, J =13.6 Hz) for the allylic hydrogen of Cꢀ18: Finally, the
(
1
2
1
J
2
2
1
2
signals of the seven angular methyl groups are observed at δ 1.15 (s, 3H Cꢀ27), 0.97
H
(s, 3H Cꢀ23), 0.93 (s, 3H Cꢀ25 and 3H Cꢀ30), 0.90 (s, 3H Cꢀ29), 0.80 (s, 3H Cꢀ26) and
1
3
0
.77 (s, 3H, Cꢀ24). This structure was confirmed by studying the C NMR, COSY,
HMBC and HMQC monoꢀ and biꢀdimensional spectra exhaustively and is completely
concordant with those in the literature.
2
8-O-Benzyl Oleanolate (2): BnCl (418 ꢁL) was added in 2:1 ratio to a solution
of 1 (912 mg, 2 mmol) in DMF (8 mL) with K CO (0.61 g). The reaction was stirred
for 4 h at 55 °C. The mixture was diluted with water and extracted with CH Cl , and the
. The solvent was removed under
2
3
2
2
2 4
organic layer was dried with anhydrous Na SO
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reduced pressure, and the residue was purified by column chromatography using
41
CH Cl /acetone (10:1) to give 2 as a white solid (710 mg, 83%) (Scheme 1). White
2
2
ꢀ1
solid, mp 221ꢀ223 °C; [α]
D
+64 (c 1, CHCl
3
:MeOH, 2:1); IR νmax (KBr)/cm : 3371,
1
2
940, 2891, 1668. This compound has a molecular mass of 561 amu and in its H NMR
spectrum presents, in addition to the above mentioned signals, at 7.48 ppm (m, 5H) the
signal of the aromatic protons, at 5,00 ppm (2H, AB system, J=12.0 Hz) of the
methylene benzyl group. Moreover, the more significant signals of compound 2 in its
1
3
C NMR spectrum are shown at 177.6 ppm (Cꢀ28, this signal is shielded from 184.5
ppm for OA as consequence of the benzyl ester formation), 136.7 ppm (a quaternary
carbon of the benzyl group), around 128.0 ppm (five methane carbons of the benzyl
group) and 66.1 ppm (methylene group of the benzyl group)
3-O-Succinyl Oleanolic Acid (3): Succinic anhydride (236 mg, 2 mmol) was
slowly added to a solution of 1 (456 mg, 1 mmol) in pyridine (5 mL). The reaction was
maintained at room temperature while stirring for 24 h. Cold water was added to the
reaction mixture, followed by an extraction with CH
dried with anhydrous Na SO . The solvent was removed under reduced pressure and the
residue was purified by column chromatography using CH Cl /acetone (10:1) to give 3
2 2
Cl , and the organic layer was
2
4
2
2
4
2
as a white solid (90%) (Scheme 1). White solid, mp 190–192 °C; [α]
D
+ 49 (c 1 in
ꢀ1
CHCl
3
:MeOH, 2:1); IR νmax (KBr)/cm : 3394, 3018, 2854 and 1712. This compound
has a molecular mass of 565 amu. The main spectroscopic difference between the
succinylated compound (3) and the corresponding substrate (1) was the deshielding of
the signal of the Hꢀ3 geminal proton because of the presence of succinyl group on Cꢀ3,
in this sense, this signal was situated at δ
H
3.19 for (1) and δ
H
4.51 for (3). Moreover, in
1
3
the C NMR spectrum of 3 the four signals of the four carbons of the succinyl group
appear at δ
H
178.6 and 171.8 due to free carboxylic group and carboxylate group, and
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δ
H
29.6 and 29.4 corresponding to the two methylene groups. The other signals in this
spectrum of this compound remain practically unaltered.
-O-Succinyl-28-O-Benzyl Oleanolate (4): BnCl (210 ꢁL) was added in 2:1
ratio to a solution of 3-O-succinyl oleanolic acid (3, 556 mg, 1 mmol) in DMF (4 mL)
with K CO (0.30 g). The reaction was stirred for 4 h at 55 °C. The mixture was diluted
with water and extracted with CH Cl , and the organic layer dried with anhydrous
Na SO . The solvent was removed under reduced pressure, and the residue was purified
3
2
3
2
2
2
4
by column chromatography using CH
Colourless oil, [α] + 38 (c 1 in CHCl :MeOH, 2:1); IR νmax(film)/cm : 3377, 3020,
859 and 1720. This compound (4) has a molecular mass of 670 amu and presented the
2 2
Cl /acetone (10:1) to give 4 (Scheme 1).
–1
D
3
2
above mentioned NMR spectroscopic characteristics when both functional groups of the
molecule were modified as a succinyl derivative at Cꢀ3 (deshielding of Hꢀ3 from 3.19
ppm to 4.48 ppm due to the esterification the hydroxyl group at this position) and as a
benzyl ester at Cꢀ28 (apparition of the methylene group signal at 5.05 ppm and a
multiplet signal at 7.31 ppm). On the other hand, all the carbons of two new
1
3
functionalizations at Cꢀ3 and Cꢀ28 appear adequately in its C NMR spectrum. This
bifunctional compound (4) was also obtained by succinylation of 28ꢀbenzyl oleanolate
(
2). Succinic anhydride (73 mg, 0.732 mmol) was slowly added to a solution of 2 (100
mg, 0.183 mmol) in pyridine (2 mL). The mixture was treated as described above for
3
1
the succinylation of 1 and thus compound 4 was again formed.
2.3. Anti-cancer test on colon cancer cells
The different compounds used in cell treatment (1, 2, 3, and 4) (Figure 1) were
dissolved before use at 10 mg/mL in 50% DMSO and 50% PBS. A stock solution was
frozen and stored at ꢀ20 °C. Prior to the experiments, this solution was diluted in cellꢀ
culture medium. Apoptosis, cellꢀcycle, mitochondrialꢀmembrane potential and Hoeschtꢀ
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stained were measured at the IC50 and IC80 concentrations (concentrations causing 50%
and 80% reduction in growth). All the experiments were measured and compared to the
controls after 72 h of treatment.
2.3.1. Cell-proliferation Activity Assay
Murine melanoma cell line B16ꢀF10 (ECACC CRLꢀ6475) was cultured in
DMEM supplemented with 2 mM glutamine, 10% heatꢀinactivated FCS, 10,000
units/mL of penicillin and 10 mg/mL of streptomycin, and incubated at 37 °C in an
2
atmosphere of 5% CO , and 95% humidity. Subꢀconfluent monolayer cells were used in
all experiments.
The cell line was provided by the cell bank of the University of Granada, Spain.
B16ꢀF10 cells were derived from a melanoma from the skin of a C57BL/6 strain mouse,
are adherent cells with fibroblastꢀlike appearance, capable of producing melanin, and
4
3
form metastatic tumor nodules in lung upon injecting mice. The clone F10 of B16
44
cells are susceptible to lysis mediated by syngeneic lymphocytes.
The effect of treating of products 1, 2, 3, and 4 on proliferation of B16ꢀF10
melanoma cells was measured using the MTT assay (Sigma, MO, USA), which is based
on the ability of live cells to cleave the tetrazolium ring, thus producing formazan,
which absorbs at 570 nm. Cell viability was determined by measuring the absorbance of
3
MTT dye staining of living cells. For this assay, 11x10 B16ꢀF10 cells were grown in a
96ꢀwell plate and incubated with the different derivatives (0ꢀ80 µg/mL). Following 72
h, 100 mL of MTT solution (0.5 mg/mL) was added to each well. After 2 h of
incubation, the cells were washed twice with PBS and formazan was resuspended in
1
00 µL DMSO.
Relative cell viability, with respect to untreated control cells, was measured by
absorbance at 550 nm on an ELISA plate reader (Tecan Sunrise MR20ꢀ301, TECAN,
1
0

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Austria). Derivatives that showed high cytotoxicity were used to perform apoptosis,
cellꢀcycle, mitochondrialꢀmembrane potential analysis and studies on morphological
changes with Hoeschtꢀstaining.
2
.3.2. Cell-cycle Analysis
The cellꢀcycle was analyzed with flow cytometry by using a fluorescenceꢀ
activated cell sorter (FACS) at 488 nm in an Epics XL flow cytometer (Coulter
34
4
Corporation, Hialeah, FL, USA), as described previously . For this assay 11x10 B16ꢀ
F10 cells cells/well were plated in 12 well plates with 2 ml of medium. After 24 h, the
different compounds (1, 2, and 4) were added and the cells were incubated for 72 h. The
doses used for each compound were their respective IC50 and IC80 values as determined
by the cell proliferation activity assay.
Cells were washed twice with PBS and harvested by tripsinization, resuspended
in TBS 1X (10mM Tris, 150 mM NaCl) and subsequently Vindelov Buffer (100 mM
Tris, 100 mM NaCl, 10 mg/mL Rnase, 1 mg/mL Propidium Iodide [PI], pH 8) was
added. Just before measurements, the total DNA content was stained with 1 mg/mL PI.
Cellꢀcycle was analyzed using Multicycle software. The data were analyzed to
determine the percentage of cells at each phase of the cellꢀcycle (G /G , S and G /M).
0
1
2
3
Approximately 10x10 cells were analyzed in each experiment. All experiments were
performed three times with two replicates per experiment.
2.3.3. Annexin VeFICT/PI Flow-cytometric Analysis
The extent of apoptosis was analyzed with flowꢀcytometry by using a FACScan
flowꢀcytometer (fluorescenceꢀactivated cell sorter) (Coulter Corporation, Hialeah, FL,
4
USA). Briefly, 11x10 B16ꢀF10 cells were plated in 12 well plates with 2 mL of
medium following treatment with the different compounds (1, 2, and 4) for 72 h, at the
1
1

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IC50 and IC80 concentrations calculated previously. The cells were collected and
resuspended in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM
2
CaCl ). Annexin VeFITC conjugate (1 ꢁg/mL) was added and incubated for 30 min at
RT in the dark. Just before FACS analysis, cells were stained with 20 µL of 1 mg/mL PI
3
solution. In each experiment, approximately 20x10 cells were analyzed and the
experiment was performed twice in triplicates.
2.3.4. Flow-cytometry Analysis of the Mitochondrial-membrane Potential
Oxidative damage was studied by flowꢀcytometry analysis of the mitochondrial
membrane potential using dihydrorhodamine 123 (DHR) which is oxidized to the highly
fluorescent product, rhodamine (Rh123). The formation of Rh123 can be monitored by
fluorescence spectroscopy using excitation and emission wavelengths of 500 and 536
nm, respectively. The intracellular measurement of the mitochondrial membrane
potential was made by cytometry determination of Rh123. The intensity of fluorescence
from Rh123 and PI was determined using a FACScan flowꢀcytometer (fluorescenceꢀ
activated cell sorter) (Coulter Corporation, Hialeah, FL, USA).
4
Similar to the apoptosis assays, 11x10 B16ꢀF10 cells were plated in 12ꢀwell
plates and treated with different compounds (1, 2, and 4), at their IC50 and IC80
concentrations. Following the treatment, the medium was removed and a fresh medium
with DHR at a final concentration of 5 µg/mL was added. After 30 min of incubation,
the medium was removed and the cells were washed and resuspended in PBS with 5
µg/mL of PI.
2.3.5. Hoechst-staining
The morphological changes were analyzed by fluorescent microscopy using
4
Hoechst staining. For this, 11x10 B16ꢀF10 cells were plated onto a coverslip in 12ꢀwell
1
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plates. After 24 h, different compounds (1, 2, and 4) were added and the cells were
incubated for further 72 h at their respective IC50 and IC80 values. The cells were
washed twice with PBS and treated with cold 100% MeOH for 3 min. Subsequently, the
cells were washed in PBS and incubated in 500 µL of Hoechst solution (50 ng/mL) in
PBS for 15 min in the dark. The samples were prepared with mounting medium,
Mowiol, and visualized by fluorescent microscopy (DMRB, Leica Microsystems,
Wetzlar, Germany) with a DAPI filter.
2.4. Statistical analysis
Statistical analyses were performed with the GraphPad Prism 5.0 software. All
quantitative data are summarized as the mean ± standard deviation (SD) or mean
standard error (SEM). For each assay Student’s t test was used for statistical comparison
with the untreated control cells. A limit of p ≤ 0.05 was considered to be statistically
significant. Key: p < 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***). All data shown here
are representative of at least three independent experiments performed in duplicate.
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3
3
3
. Results and Discussion
.1 Chemistry synthesis
.1.1 Synthetic route to 3-O-Succinyl-28-O-Benzyl Oleanolate
The synthetic route to the target 3ꢀOꢀsuccinylꢀ28ꢀOꢀbenzyl oleanolate was
depicted in Scheme 1. The starting compound 1, oleanolic acid (3βꢀhydroxyoleanꢀ12ꢀ
enꢀ28ꢀoic acid) is natural compound present in different plants. For the current study,
this compound was isolated from the oliveꢀpressing residues by extraction processes
with different solvents. The benzylation by treatment with benzyl chloride in DMF of 1
to achieve 28ꢀOꢀbenzyl oleanolate (2), was carried to protect the carboxylic group at Cꢀ
2
8 of the oleanane skeleton and to obtain a derivative that has an aromatic group
attached at this position. Following, the succinylation process was performed by treating
1) with succinyl anhydride (2:1 ratio) in pyridine for 24 h at RT.
(
(Scheme 1. should be listed here)
Thus, 3ꢀOꢀsuccinyl oleanolic acid (3) was obtained in high yield. Finally, 28ꢀOꢀ
benzyl oleanolate (2) was succinated following similar conditions to those indicated
above for 1 and, thus the bifunctional derivative 3ꢀOꢀsuccinylꢀ28ꢀOꢀbenzyloleanolate
(
4) was obtained in high yield. This bifunctional derivative (4) of 1 was also obtained
by succinylation of 28ꢀbenzyloleanolate (2) in previously indicated similar conditions.
After the target compound was successfully prepared, structural characteristics of the
compound were examined on UV, MS and NMR spectroscopies.
3.2 Anti-cancer activities
3
.2.1 Inhibition of B16-F10 skin-melanoma cell proliferation
In this work the potential cytotoxic effect of these semiꢀsynthesized derivatives
(
2, 3, 4) was examined, on the proliferation of B16ꢀF10 murine melanoma cell line,
1
4

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compared with its natural precursor (1), using the MTT assay. B16ꢀF10 cells were
treated with increasing doses of each compound. Their viability was determined by
formazan dye uptake and expressed as percentage of untreated control cell proliferation.
Compounds 1, 2 and 4 induced a doseꢀdependent decrease in viable formazan
accumulating cells after 72 h of treatment, ranging from 0 to 80 µg/mL.
The concentration (expressed in ꢁg/mL) of products required for 50% and for
8
0% growth inhibition (IC and IC ) were determined for all products (Figure 2). The
50 80
treatment with 1 showed quite high values of IC50 and IC80 (IC50 = 46.2 ± 1.4 and IC80
=
50.2 ± 2.0). For 2 treatment, IC50 value was reduced almost to half (IC50 = 29.0 ± 1.7
and IC80 = 47.4 ± 2.4). The treatment with 3 had no effect on cancer cell proliferation,
not reaching IC50 value.
(Figure 2. Should be listed here)
Finally, the treatment with 4 had the lowest IC50 value (IC50 = 15.3 ± 0.6 and
IC80 = 44.3 ± 3.1), being three times lower than that of its natural precursor (1), and was
half that of 2. These effects could to be related to the increase polarity of the whole
molecule, without a significant increase in molecular weight or volume. The compounds
1
, 2 and 4 that showed cytotoxic effects in the range of concentrations assayed were
selected for the rest of assays: cellꢀcycle analysis, characterization of apoptosis, changes
in mitochondrial membrane potential and Hoechstꢀstaining.
The effects of the selected products, 1, 2 and 4 on cellꢀcycle distribution were
investigated to determine a possible cytostatic effect attached to the cytotoxic response.
The distribution of cells in different cellꢀcycle phases was analyzed after 72 h of
treatment, by the incorporation of propidium iodide (PI). B16ꢀF10 melanoma cells were
treated with the products at IC50 and IC80 concentrations, determined previously.
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3.2.2. Cell-cycle arrest and distribution
Flow cytometry was used to measure DNA ploidy as well as alterations in cellꢀ
cycle profiles. In this way, Cell subpopulations with differing DNA content, allowing
determining the percentage of cells in each cellꢀcycle phase. Changes in DNA
concentration are characteristic of apoptosis or cellꢀcycle arrest. DNA histogram
analysis revealed that the two derivatives, 2 and 4, produced a significant cellꢀcycle
arrest (Figure 3), increasing the number of cells in the G /G phase. 2 increased this
0
1
population up to 68% at IC50, and 75% at IC80 concentration. 4 produced a major effect
with an increase up to 79% at IC50, and 84% at IC80 concentration. These increases were
accompanied by a decrease in the percentage of proliferating cells in S phase (up to
2
25% for 2 and 16% for 4 at IC80 concentrations). Changes in the G /M phase were less
significant. The cellꢀcycle arrest observed could also be related to the induction of
differentiation by 4 in this cell type.
(Figure 3. Should be listed here)
A wide range of studies in recent years have shown that triterpenoids hinder
carcinogenesis by intervening in pathways such as carcinogen activation, DNA repair,
1, 45, 46
cellꢀcycle arrest, cell differentiation and the induction of apoptosis in cancer cells.
The identification of new cytotoxic agents that enhance or restore the capability of
malignant tumor cells to undergo apoptosis may be crucial for more effective anticancer
1
,39,40
therapies.
Apoptosis is one of the body’s most potent defenses against cancer; the
pathogenesis of many forms of this degenerative disease is closely connected with
aberrantly regulated apoptotic cell death. Different mechanisms that regulate apoptosis
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6

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associated with mediators that trigger or inhibit cell death have led to the development
of therapeutic strategies against cancer.
In most of the pentacyclic triterpenoids, the cytotoxic effects are often
accompanied by cytostatic effect. These cytostatic effects are frequently associated to
apoptosis induction or to differentiation cell process activation. The cellꢀcycle
machinery is an alternative target for the identification of novel biomarkers for cancer
detection and prognosis, providing target validation for cellꢀcycleꢀdirected therapies. As
the products (1, 2 and 4) inhibited cell growth, we investigated their effect on cellꢀcycle
distribution in B16ꢀF10 skinꢀmelanoma cells after 72 h of treatment. In this respect, two
derivatives, 2 and 4, produced cellꢀcycle arrest at G
The compound 4 induces greater cellꢀcycle arrest in G
5% at IC concentration and 20% at IC concentration, coinciding with apoptotic cell
0 1
/G phase.
0
1
/G phase, approximately
1
50
80
death, thus contributing to the inhibition of cell growth. This could indicate a likely
induction of cell differentiation in response to the antiꢀcancer properties of product 4.
Further studies, such as the determination of the expression of the differentiation marker
enzymes, would be necessary to assert this point.
3.2.3. Apoptosis induction in B16-F10 murine melanoma cells
Annexin VꢀFITC staining and PI accumulation were used to determine the
percentage of apoptotic cells. Early events in the apoptotic process are loss of plasma
membrane asymmetry accompanied by translocation of phosphatidylꢀserine (PS) from
the inner to the outer membrane leaflet, thereby exposing PS to the external
47
environment. The phospholipidꢀbinding protein annexin V has a high affinity for PS
and binds to cells fluorescently labelled with FITC (fluorescein isothiocyanate). The
percentages of apoptosis, as determined with annexin VꢀFITC/PI flow cytometry
analysis are shown in Figure 4.
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Assessment of apoptosis in B16ꢀF10 cells was performed 72 h after treatment
with the compounds 1, 2 and 4 at IC and IC concentrations. FACS analysis using
5
0
80
Annexin VꢀFITC staining and PI accumulation was used to differentiate normal cells
ꢀ
ꢀ
+
ꢀ
(
AnnexinꢀV and PI ), early apoptotic cells (AnnexinꢀV and PI ), late apoptotic cells
+
+
ꢀ
+
(
AnnexinꢀV and PI ) and necrotic cells (AnnexinꢀV and PI ).
The three compounds showed apoptotic effects on the treated cells, with high
percentage of total apoptosis (early apoptotic together with late apoptotic cells), reached
at IC50 concentration of 42% (28.1% early apoptosis and 16.4% late apoptosis) in
response to compound 1, 28% (12.2% early apoptosis and 29.1% late apoptosis) for
compound 2, and 69% (48.8% early apoptosis and 20.5% late apoptosis) for compound
4
.
(Figure 4. Should be listed here)
At IC80 concentration these percentages were 47% (12.4% early and 29.1% late
apoptosis) for 1, 42% (18.6% early apoptosis and 23% late apoptosis) for 2, and 92%
16.6% early apoptosis and 73.9% late apoptosis) in the treatment with 4. In addition,
(
the percentages of the necrotic population were not noteworthy. The apoptotic effect in
respond to 4, with the lowest IC concentration was the more significant.
50
In most cases, the highest percentages of apoptosis were achieved at IC80
concentrations. In all cases, the number of apoptotic cells increased concomitantly with
dose concentration. The low values of IC50 concentrations and good percentages of
apoptosis attained by these derivatives, especially by 4, show that they could be used as
promising anticancer drugs, further studies will be necessary to strengthen this point.
The apoptotic process frequently does not function in tumor cells. The intrinsic
and extrinsic apoptotic activation routes have been described in the apoptotic effect of
1
8

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numerous triterpenoids such as maslinic, acid ursolic acid, betulinic acid, OA and its
1
ꢀ3, 5
derivatives.
With alterations in the Bclꢀ2 protein family balance, loss of
4
8,49
mitochondrial membrane potential, and the release of mitochondrial cytochromeꢀc.
3
.2.4. Mitochondrial membrane potential disturbances
Mitochondria constitute a key target in synthetic and natural triterpenoids.
Pentacyclic triterpene compounds have proved to be able to activate the intrinsic
50
2
, 3, 51
apoptotic pathway, mediated by mitochondrial disruption.
In a first approximation
to molecular mechanism, we analyzed the mitochondrialꢀmembrane potential (MMP).
Changes in this membrane potential can be associated with the intrinsic mechanism of
apoptosis activation, while apoptosis induction without MMP changes may suggest
activation of the extrinsic apoptotic pathway.
We investigated the MMP to determine the possible mechanism involved in the
apoptotic responses. The changes in MMP were analyzed by monitoring the cell
fluorescence following double staining with Rh123 and PI. Rh123 is a membraneꢀ
permeable fluorescent cationic dye that is selectively taken up by mitochondria directly
52
proportional to MMP.
Changes in MMP were analyzed by flowꢀcytometry staining with Rh123/IP after
treatment with the compounds 1, 2, and 4 for 72 h, at IC50 and IC80 concentrations. The
apoptosis induction by 2 did not produce any changes in MMP, suggesting that the
apoptosis was triggered by the extrinsic apoptotic pathway activation. Compounds 1
and 4 clearly produced MMP disturbances with loss of MMP and negative Rh123
staining.
The treatment with these products showed a decrease in the Rh123 positive cells
stained, 63.8% at IC50 and 80.1% at IC80 concentrations for 1, and 78.1% at IC50 and
69.8% at IC80 concentration in response to 4, with a concomitant increase in Rh123
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negative cells stained (Figure 5). Therefore, the mitochondrial disruption together with
apoptosis could indicate the activation of the intrinsic apoptotic route in B16ꢀF10 cell
line in response to 1 and 4. Nevertheless, no changes in mitochondrial potential were
observed following treatment with 2.
These results clearly show mitochondrial disturbances following treatment with
1
and 4 in B16ꢀF10 murine melanoma cells, which could be related to the activation of
the mitochondrial or intrinsic apoptotic pathway by these products. Further molecular
studies will be necessary to confirm this assertion.
(Figure 5. Should be listed here)
3.2.5. Morphological apoptotic changes
The Hoechst procedure stains nuclei that contain nicked DNA, a characteristic
exhibited by cells in apoptotic cell death. Fluorescence microscopy can determine
changes in the cell morphology. The fluorescence microscopic observations showed that
a significant number of cells treated with the selected compounds acquired apoptotic
features (Figure 6).
The morphological analysis of Hoechstꢀstained cells, in B16ꢀF10 cells, indicated
that they had undergone remarkable morphological changes. After 72 h exposure to
compounds 1, 2, and 4 (at IC50 and IC80 concentrations), the cells showed typical
apoptotic changes, including cell shrinkage, chromatin condensation, and loss of normal
nuclear architecture. Fluorescence microscopic observation after Hoechst staining
showed that a significant number of cells treated with compound 4 acquired apoptotic
features, as evident from nuclear fragmentation, and the prominent disruption of the
cellꢀmembrane integrity.
(Figure 6. Should be listed here)
2
0

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4. Conclusions
This study is the first to investigate the apoptotic effects of the oleanolic acid
derivatives, 2, 3 and 4, in comparison to its natural precursor. Our data provide evidence
that these products exert antiꢀproliferative and proꢀapoptotic effects on B16ꢀF10 skinꢀ
melanoma cells, a model for malignant melanoma cells. Moreover, they induce
morphological changes that are characteristic of apoptosis, such as chromatin
condensation and fragmentation as well as cell shrinkage, and loss of cellꢀmembrane
asymmetry, as demonstrated by fluorescence microscopy. These effects being greater in
respond to 4, compared to its precursor, with a smaller IC50 and IC80 values and greater
percentage of apoptosis induction. In conclusion, the 3ꢀOꢀsuccinylꢀ28ꢀOꢀbenzyl
oleanolate (4), a novel oleanolic acid derivative is able to induce apoptosis in murine
skinꢀmelanoma cells, probably via the intrinsic mitochondrial pathway. The precise
molecular signaling pathway by which this compound induces apoptosis needs to be
investigated. Nevertheless, our data could contribute to the development this compound
and related compounds for use as cancer chemotherapeutics or chemopreventive agents.
The apoptotic potency of 4 suggests that it may be an effective compound in the
prevention or treatment of the skinꢀmelanoma.
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Acknowledgements
This study was supported by grants Group BIO 157 from the Technology and
Innovation Council of the Andalucian regional government and AGL2006ꢀ12210ꢀC03ꢀ
02/ALI, SAF2005ꢀ01627, ISCIIIꢀRTICC (RD06/0020/0046) from the Spanish
government and European Union FEDER funds.
Authors’ Contribution
Fernando J. ReyesꢀZurita, Andrés Parra and José A. Lupiáñez conceived and designed
the study protocol. Fernando J. ReyesꢀZurita, Marta MedinaꢀO’Donnell, Rosa M.
FerrerꢀMartín, Eva E. RufinoꢀPalomares, Amalia PérezꢀJiménez, Leticia Garcíaꢀ
Salguero, Khalida Mokhtari, Juan Peragón, Pedro P. Medina and José A. Lupiáñez
performed all cell culture (in vitro) and other biological experiments. Samuel Martínꢀ
Fonseca, Francisco Rivas, Antonio Martínez, Andrés GarcíaꢀGranados and Andrés
Parra performed all the synthesis and purification experiments of SBOA and OA.
Fernando J. ReyesꢀZurita, Marta MedinaꢀO’Donnell, Rosa M. FerrerꢀMartín, Eva E.
RufinoꢀPalomares, Amalia PérezꢀJiménez, Khalida Mokhtari, Leticia GarcíaꢀSalguero
and Pedro P. Medina contributed to reagents, materials, and analysis tools. Fernando J.
ReyesꢀZurita, Eva E. RufinoꢀPalomares, Leticia GarcíaꢀSalguero, Juan Peragón, Pedro
P. Medina, Andrés Parra and José A. Lupiáñez conducted the data analysis. Fernando J.
ReyesꢀZurita, Andrés Parra and José A. Lupiáñez contributed to the paper preparation.
2
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Scheme and figure legends
Scheme 1. Synthetic route of 3ꢀOꢀSuccinylꢀ28ꢀOꢀBenzyl Oleanolate. In first time
oleanolic acid (1) is treatment with benzyl chloride to achieve 28ꢀOꢀbenzyl oleanolate
(
2), or it can be succinylated with succinyl anhydride to achieve 3ꢀOꢀsuccinyl oleanolic
acid (3). Then, 2 is succinylated, and 3 is benzylated, following similar conditions
described for 1, obtained the 3ꢀOꢀsuccinylꢀ28ꢀOꢀbenzyl oleanolate (4).
Figure 1. Structure of the triterpene skeleton of oleanolic acid (1), and its derivatives:
28ꢀOꢀbenzyl oleanolate (2), 3ꢀOꢀsuccinyl oleanolic acid (3), and 3ꢀOꢀsuccinylꢀ28ꢀOꢀ
benzyl oleanolic acid (4).
Figure 2. Effects of oleanolic acid (1), 28ꢀOꢀbenzyl oleanolate (2), 3ꢀOꢀsuccinyl
oleanolic acid (3), and 3ꢀOꢀsuccinylꢀ28ꢀOꢀbenzyl oleanolate (4), on the viability of
B16ꢀF10 murine skinꢀmelanoma cells. After treatment with the compounds for 72 h in
an range of 0 to 80 ꢁg/mL, each point represents the mean value ± S.D. at least two
independent experiment performed in triplicate. IC50 and IC80 are the concentrations
required for 50% and 80% of growth inhibition, respectively.
Figure 3. Top: Histograms of cellꢀcycle of B16ꢀF10 skinꢀmelanoma cells, after 72 h
treatment with compounds 1, 2 and 4, at IC50 and IC80 concentrations. Bottom: Increase
of the percentage of cells in each cellꢀcycle phase with respect to untreated control cells.
Cellꢀcycle was conducted after propidium iodide (PI) staining as it is described in
Experimental section. Values represent means ± S.E.M of at least three independent
experiments performed in duplicate. Key: p < 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001
(***), respect to untreated control cells.
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Figure 4. Effects of compounds 1, 2, and 4 on apoptosis in B16ꢀF10 skinꢀmelanoma
cells, after 72 h treatment, at IC50 and IC80 concentrations. Top: Diagrams of annexin
V/propidium iodide (IP) flow cytometry. B16ꢀF10 cells were labeled with annexin Vꢀ
FICT/PI as described in Experimental section. The right quadrants of each diagram
represent apoptotic cells (Q2, late apoptosis; Q4, early apoptosis). Early apoptotic cells
+
ꢀ
+
+
were annexin V PI , whereas late apoptotic cells were annexin V , PI . Bottom: Flow
cytometry analysis of Annexin VꢀFITC staining and PI accumulation, values represent
means ± S.E.M of three experiments in duplicate. Key: p < 0.05 (*), p ≤ 0.01 (**), and p
≤
0.001 (***), respect to untreated control cells.
Figure 5. Effects of compounds 1, 2, and 4 on mitochondrial membrane potential in
B16ꢀF10 skinꢀmelanoma cells, after 72 h treatment, at IC50 and IC80 concentrations.
Top: Diagram of rhodamine 123/propidium iodide flowꢀcytometry. B16ꢀF10 cells were
labeled with Rh123/IP as described in Experimental section. The right quadrants of each
diagram (Q2 and Q4) represent positive cells stained with Rh123. Bottom: Percentage
of Rh123 positive and negative B16ꢀF10 cell population, in comparison with untreated
control cells (CT). Values are expressed as means ± S.E.M of three experiments in
duplicate. Key: p < 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***), respect to untreated
control cells.
Figure 6. Morphology changes in B16ꢀF10 skinꢀmelanoma cells after exposure to
compounds 1, 2 and 4 for 72 h, at IC50 concentrations. The samples were prepared with
mounting medium, Mowiol, and the cells were examined using fluorescence
microscopy following Hoechstꢀstaining as described in Experimental section.
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