Pro-apoptotic activity of lipidic α-amino acids isolated from Protopalythoa variabilis

Article abstract of DOI:10.1016/j.bmc.2010.09.027

Lipidic α-amino acids (LAAs) have been described as non-natural amino acids with long saturated or unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss of mitochondrial potential, and DNA fragmentation.

Full text of DOI:10.1016/j.bmc.2010.09.027

Bioorganic & Medicinal Chemistry 18 (2010) 7997–8004
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pro-apoptotic activity of lipidic
variabilis
a-amino acids isolated from Protopalythoa
Diego Veras Wilke a, Paula Christine Jimenez a, Renata Mendonça Araújo b, Wildson Max Barbosa da Silva b,
Otília Deusdênia Loiola Pessoa b, Edilberto Rocha Silveira b, Claudia Pessoa a, Manoel Odorico de Moraes a,
Mariusz Skwarczynski c, Pavla Simerska c, Istvan Toth c, Letícia Veras Costa-Lotufo a,
⇑
a Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, 60430-270 Fortaleza-Ce, Brazil
b Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60021-940 Fortaleza-Ce, Brazil
c The University of Queensland, School of Chemistry and Molecular Biosciences, St. Lucia, Qld 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipidic
a-amino acids (LAAs) have been described as non-natural amino acids with long saturated or
Received 31 March 2010
Revised 6 September 2010
Accepted 13 September 2010
Available online 18 September 2010
unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine
sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa
variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor
cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage
of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic
effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss
of mitochondrial potential, and DNA fragmentation.
Keywords:
Protopalythoa variabilis
Lipidic a-amino acids
Cytotoxicity
Ó 2010 Elsevier Ltd. All rights reserved.
Apoptosis
1. Introduction
synthetic LAAs along with the anti-proliferative and pro-apoptotic
effects of 1a/1b isolated from P. variabilis on tumor cells.
Lipidic a-amino acids (LAAs) are classically described as non-nat-
ural amino acids with long saturated or unsaturated aliphatic
chains.1,2 In our continuing search to discover anticancer agents
from marine sources,3–6 we have isolated a cytotoxic LAAs mixture
(1a and 1b) (Fig. 1) from the zoanthid Protopalythoa variabilis Duer-
den 1898 collected at a northeast Brazilian beach.7 1a/1b has a long-
er aliphatic chain than any other known synthetic LAA. Although
highly lipophilic, LAAs also have a polar behavior, showing chemical
and conformational characteristics of amino acids and peptides.8,9
The utility of these compounds in the industry is wide: as lubri-
cants,10 cosmetics,11 and polishes.12,13 LAAs may also have thera-
peutic application, as they have been shown to inhibit both
pancreatic14 and platelet phospholipase A2,15 being potential anti-
inflammatory agents. LAAs and their oligomers have been used in
several drug delivery systems.16 In addition, a LAAs based Lipid-
Core-Peptide system was designed and used for the development
of self-adjuvanting peptide vaccines against various diseases.17,18
Despite their wide use, the cytotoxicity of LAAs has not been
addressed. Herein we have investigated the cytotoxic activity of 14
2. Materials and methods
2.1. Chemicals and reagents
Propidium iodide (PI) and rhodamine 123 (rh123) were pur-
chased from Sigma–Aldrich. Doxorubicin (Doxolen) was purchased
from Zoadiac. All reagents were of analytical grade.
2.2. Lipidic a-amino acids (general procedures)
The isolation of 1a/1b (Fig. 1) from the zoanthid P. variabilis was
performed as previously described.7 Its structures were well char-
acterized by 1H and 13C NMR spectral data and HR-ESI-TOFMS. The
1H NMR spectra showed a signal at d 3.57 (br t) characteristic of a
nitrogenated methine, an intense signal at d 1.27 (br s) for several
methylene groups, and a triplet at d 0.88 characteristic of the ter-
minal methyl of fatty acids. The 13C NMR (CPD and DEPT 135) spec-
tra exhibited a signal at d 54.7 (CH), which in the HMBC spectrum
showed correlation with the signal at d 3.57, compatible with an
amino acid moiety. In addition, several methylene signals in the
range of d 32.5–23.3, and a carbon signal at d 14.6 for the terminal
methyl group were observed. The HR-ESI-TOFMS spectrum showed
two peaks at m/z 490.4566 [M+Na]+ indicating the molecular
⇑
Corresponding author. Address: Departamento de Fisiologia e Farmacologia,
Universidade Federal do Ceará, Rua Cel. Nunes de Melo, 1127, 60430-270 Fortaleza,
Ceará, Brazil. Tel.: +55 85 3366 8255; fax: +55 85 3366 8333.
E-mail address: lvcosta@secrel.com.br (L.V. Costa-Lotufo).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.027

7998
D. V. Wilke et al. / Bioorg. Med. Chem. 18 (2010) 7997–8004
aforementioned spectroscopic data and functionalities suggested
that compounds 1a/1b were indeed two lipidic -amino acids.
O
a
All the other LAAs tested (2–15), for their in vitro anti-prolifer-
ative properties, were synthesized by Gibbons et al. 1990,8 as
shown in Scheme 1. The synthetic procedure includes the reaction
catalyzed by EtONa of diethyl acetamidomalonate with 1-bromo
alkanes, under reflux overnight, followed by the reaction with
OH
OH
NH2
1a
O
HCl to give
a-amino acids with an alkyl side chain (2, 4, 7, 10,
12, and 14). These lipoamino acids could be N-Boc protected by
reacting with Boc2O to afford the derivatives 3, 5, 8, 11, 13, and
15, or further O-methylated by reacting with SOCl2 in MeOH to
yield 6 and 9.
NH2
1b
6
O
O
2.3. Cell culture
NH2 HCl
O
Anti-proliferative activity of all LAAs was evaluated for four
human tumor cell lines obtained from the National Cancer Institute
(Bethesda, MD, USA): HL-60 (promyelocytic leukemia), MDA-MB-
435 (melanoma), SF-295 (CNS glioblastoma), and HCT-8 (colon car-
cinoma). Cells were grown in RPMI-1640 medium supplemented
O
NH2 HCl
O
9
with 2 mM glutamine, 10% fetal calf serum, 100 lg/ml streptomy-
OH
cin, and 100 U/ml penicillin at 37 °C in a 5% CO2 atmosphere. The
cell cultures were regularly split to keep them in a logarithmic
phase of growth.
HN
O
11
Pro-apoptotic effects of 1a/1b were evaluated against the HL-60
cell line (3 Â 105 cells/ml). The 24 h incubation time chosen is suf-
ficient for the cells to complete at least one cell cycle. 1a/1b was
diluted in sterile DMSO and stored at À20 °C. Cell membrane integ-
rity, evaluated by flow cytometry (EasyCyte™ Mini System, Guava
Technologies, CA, USA), was used to assess cell viability. All exper-
iments were performed with cell viability above 95%. Five-thou-
sand events were evaluated per experiment and cellular debris
was omitted from analysis. Experiments were performed three to
five times in triplicate. Doxorubicin (Dox) at a concentration of
O
O
OH
O
HN
HN
13
15
O
O
OH
O
0.3
2.4. Anti-proliferative assay
The effect of 5 g/ml concentrations of 1a/1b and synthetic
lg/ml was used as a positive control.
O
l
Figure 1. Structures of the most active synthetic lipidic
a-amino acids (6, 9, 11, 13,
and 15), including the lipidic -amino acids (1a/1b) from Protopalythoa variabilis.
LAAs (2–15) on the tumor cell growth following 72 h incubation
was evaluated in vitro using the MTT [3-(4,5-dimethyl-2-thiazol-
yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, as described by
Mosmann.19 The inhibitory concentration mean (IC50) of 1a/1b
a
formula C30H61NO2 (1a), while the ion peak at m/z 504.4706
[M+Na]+ suggested the molecular formula C31H63NO2 (1b). The
(test concentrations ranging from 0.019 to 10 lg/ml) against
O
O
O
1. EtONa
(
)
n
+
(
)
OH
NH2 HCl
Br
n
O
2. HCl
HN
2 n = 3
4 n = 5
7 n = 7
10 n = 9
12 n = 11
14 n = 13
O
Boc2O
O
O
O
SOCl2
MeOH
(
)
n
(
)
n
O
OH
O
HN
3 n = 3
5 n = 5
8 n = 7
11 n = 9
13 n = 11
15 n = 13
NH2 HCl
6 n = 5
9 n = 7
Scheme 1. Synthesis of lipidic a-amino acids 2–15.

D. V. Wilke et al. / Bioorg. Med. Chem. 18 (2010) 7997–8004
7999
HL-60 cells was also obtained using the MTT assay with 24 h incu-
bation. The IC50 value was, then, used to drive the concentration
choice for the flow cytometry analysis.
lL and 5000 events were evaluated per experiment. Cellular debris
was omitted from the analysis.20
2.7. Analysis of morphological changes
2.5. Hemolytic assay
HL-60 cells that were untreated, treated with Dox or treated with
1a/1b were examined for morphological changes by light micros-
copy (Olympus, Tokyo, Japan) and flow cytometry. To visualize nu-
clear and cell membrane morphology microscopically, cells were
harvested, transferred to cytospin slides, fixed with methanol for
about 1 min and stained with May-Grünwald-Giemsa. To evaluate
size changes and nuclear condensation, light-scattering characteris-
tics (linear scale) of 5000 events were analyzed by flow cytometry
using FCS express software (De Novo Software, Ontario, CAN).20
Direct membrane disruption potential of 1a/1b (test concentra-
tions ranging from 1.56 to 200 lg/ml) was assayed using a 1%
mouse erythrocyte (Mus musculus swiss) suspension in 0.85% NaCl
containing 10 mM CaCl2. The hemolytic activity was measured col-
orimetrically (at 540 nm) after different incubation times (1, 2, and
4 h), as previously described.3
2.6. Cell viability
Cell viability was estimated on the basis of membrane integrity,
and evaluated by measurement of the exclusion of PI. Cell fluores-
cence was determined by flow cytometry using a 680 nm filter (log
scale) and Cytosoft software. Cells were diluted to about 300 cells/
2.8. Measurement of mitochondrial membrane potential
Mitochondrial membrane potential (wm) was determined by
the retention of rhodamine 123 (rh123) in HL-60 cells. 1 Â 105 cells
HCT-8
SF-295
1a/1b
2
1a/1b
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
11
12
13
14
15
10
11
12
13
14
15
0
25
50
75
100
0
25
50
75
100
HL-60
MDA-MB-435
1a/1b
2
N.T.
1a/1b
2
3
4
5
6
7
8
9
3
4
5
6
7
8
9
10
11
12
13
14
15
10
11
12
13
14
15
0
25
50
75
100
0
25
50
75
100
Growth inhibition (%)
Figure 2. Single dose cytotoxic activity of 1a/1b and synthetic LAAs evaluated against SF-295 (glioblastoma), HCT-8 (colon cancer), MDA-MB-435 (melanoma), and HL-60
(promyelocytic leukemia) cell lines (at 5 g/ml) using the MTT assay after 72 h incubation. Data obtained from three to four experiments and presented as a percentage of
growth inhibition relative to untreated cells. N.T. not tested.
l

8000
D. V. Wilke et al. / Bioorg. Med. Chem. 18 (2010) 7997–8004
were incubated with rh123 (1
l
g/ml) at 37 °C in a 5% CO2 atmo-
natural compounds 1a/1b were more active, completely inhibiting
tumor cell growth at 5 g/ml, while among the synthetic LAAs only
compounds 6, 9, 11, 13, and 15 could be considered active. In fact,
sphere for 15 min in the dark. Cells were then centrifuged, the
supernatant aspirated and the pellet incubated in PBS at room tem-
perature for 30 min in the dark. Fluorescence was measured using
a 625 nm filter (log scale) and analyzed using Cytosoft software.21
l
previously published IC50 values of 1a/1b, for 72 h incubation, were
as low as 0.05, 0.07, and 0.13 lg/ml on HCT-8, SF-295, and HL-60,
respectively.7 Herein it was demonstrated that after 24 h, 1a/1b
still strongly inhibited the growth of HL-60 cells, but with an IC50
2.9. Internucleosomal DNA fragmentation
four times higher (0.53
lg/ml, with CI 95% ranging from 0.48 to
HL-60 cells (3 Â 105) were incubated in the dark for 30 min at
room temperature in a buffered stain/lysis solution containing
0.1% citrate, 0.1% Triton X-100, and PI. DNA fluorescence was then
determined by flow cytometry using a 680 nm filter (log scale) and
analyzed using Cytosoft software.21
0.60 g/ml). Unfortunately, the IC50 for the synthetic LAAs could
not be obtained due to the poor solubility and amphiphilic charac-
ter of these compounds.
l
3.2. Hemolytic activity
The hemolysis assay is a useful test to establish if the cytotoxic
activity is related to direct damage on the cell membrane. No lytic
effect was observed on mice erythrocytes after 1, 2, and 4 h treat-
ment with 1a/1b at the highest concentration tested (200 lg/ml)
(data not shown).
2.10. Statistical analysis
For the MTT assay, the IC50 values and their 95% confidence
intervals (CI 95%) were obtained by non-linear regression. For all
other assays performed by flow cytometry, the differences be-
tween control and experimental groups were determined by anal-
ysis of variance (ANOVA) followed by the Dunnett’s test. All
statistical analyzes were performed using the GraphPad Prism soft-
ware version 5 (Intuitive Software for Science, San Diego, CA) and
the minimal significance level for ANOVA and t-test analysis was
set at p <0.05.
3.3. Morphological changes induced by 1a/1b
May-Grünwald-Giemsa staining showed important morpholog-
ical changes in treated cells. In the untreated cells, the nuclei were
stained homogeneously and there were few vacuoles in the cyto-
plasm (Fig. 3A). Most of the Dox-treated cells showed intense nu-
clear fragmentation accompanied by substantial cell shrinkage.
Treatment with 1a/1b induced chromatin condensation with a
half-moon or ring-like appearance, DNA fragmentation, and mem-
brane blebbing (Fig. 3C and D). All of these morphological changes
present in both the Dox and 1a/1b-treated cells are considered
characteristic of apoptosis (Fig. 3B–D).
3. Results
3.1. Anti-proliferative activity
As shown in the Figure 2, the LAAs, including both natural and
some synthetic ones, were able to inhibit tumor cell growth. The
Figure 3. Morphological changes caused by 1a/1b treatment. Photomicrographs of HL-60 cells untreated (A), treated with doxorubicin at 0.3
at 0.1 and 0.25
g/ml (C and D, respectively), after 24 h, stained with May-Grünwald-Giemsa (400Â). Arrow patterns: solid line denotes half-moon or ring-like nuclear
chromatin condensation; dotted line denotes nuclear fragmentation; dashed line indicates membrane blebbing.
lg/ml (B), or treated with 1a/1b
l

D. V. Wilke et al. / Bioorg. Med. Chem. 18 (2010) 7997–8004
8001
The changes in light scattering properties were quantified by flow
cytometry. Cell shrinkage is related to a decrease in the FSC value
which was detected in the R2 region (Fig. 4A–E). Untreated cells
showed approximately5%cell shrinkage, whereas1a/1b-treatedcells
showed increased cell shrinkage (p <0.05) in a dose dependent fash-
ion, ranging from 10% to 70% (Fig. 4C–F). Possible nuclear condensa-
tion was also investigated. It is related to a FSC decrease and a SSC
increase simultaneously, which was detected in the R3 region.20
Dox treatment also increased cell shrinkage to 60% (Fig. 4B). There
was no considerable increase of nuclear condensation as observed
by the low cell population related to region 3 (Fig. 4A–E).
group of healthy cells from the untreated group. As expected,
the control cells more than doubled their population from
3 Â 105 to about 7 Â 105 cells/ml after 24 h. Treatment with 1a/
1b decreased the cell count in a dose-dependent manner. At
the highest 1a/1b concentration, as well as for the Dox treat-
ment, the number of cells remained near the initial plated cellu-
lar density (Fig. 5A).
The percentage of viable cells was evaluated by their ability to
exclude PI. Membrane permeability to this dye is directly related
to already dead or late-staged apoptotic cells. Viable cells show
low fluorescence intensity due to their ability to exclude the
dye. Untreated cells and 0.1 and 0.25
showed minimal membrane disruption. However, cells treated
with 1a/1b at 0.5 g/ml showed approximately 30% of non-viable
lg/ml 1a/1b-treated cells
3.4. 1a/1b effect on cell viability
l
Determination of cell concentration using flow cytometry was
cells (Fig. 5B). Dox-treated cells did not show any membrane
disruption.
performed based on the light-scattering characteristics of
a
Figure 4. Light-scattering features of 1a/1b-treated cells. (A) Untreated cells; (B) cells treated with doxorubicin at 0.3
0.5 g/ml after 24 h incubation. (F) Cell shrinkage presented as a percentage relative to control. R1, gated region of viable cells; R2, gated region of cell shrinkage; and R3,
gated region of nuclear condensation. p <0.05 compared to negative control by ANOVA followed by Dunnett’s test.
lg/ml; (C, D, and E) treated with 1a/1b at 0.1, 0.25, and
l
*

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D. V. Wilke et al. / Bioorg. Med. Chem. 18 (2010) 7997–8004
A 8.0×105
B100
6.0×105
4.0×105
2.0×105
75
*
*
50
*
*
25
0
0
(μg/mL)
0.5
C-
Dox
0.1
0.25
(μg/mL)
C-
Dox
0.1
0.25
0.5
1a/1b
1a/1b
Figure 5. Anti-proliferative effects of 1a/1b. Cell count (A) and cell viability (B) of HL-60 cells untreated (CÀ) or treated with doxorubicin at 0.3
0.25, and 0.5 g/ml, after 24 h incubation, by the exclusion of propidium iodide (50 g/ml). The graphs show the percentage of viable cells obtained from five experiments
performed in triplicate by flow cytometric analysis. *p <0.05 compared to negative control by ANOVA followed by Dunnett’s test.
lg/ml (Dox) or 1a/1b at 0.1,
l
l
3.5. Loss of mitochondrial membrane potential
4. Discussion
To investigate the involvement of the mitochondrion in the
This study has shown the cytotoxic and the apoptosis-inducing
properties of the natural LAAs isolated from P. variabilis. This class
of molecules (LAAs) share a double behavior, showing chemical
and conformational characteristics of lipid and amino acids.9 De-
spite the wide use of LAAs including their therapeutic applications
since the 90s,9,14 the first cytotoxic Letter of these compounds was
only recently published by our group.7
1a/1b was isolated as a LAAs mixture through a cytotoxic-
guided fractionation from the zoanthid P. variabilis collected in
Ceará State, on the northeast Brazilian coast. They showed potent
activity against three tumor cell lines with IC50 values in the nano-
molar range.7 Herein this activity was confirmed using a shorter
incubation time, although with a lower potency, suggesting a
time-dependence for the observed cytotoxicity.
LAAs display an amphiphilic character, as observed to sphingo-
lipids, that present free amide-group linked to a long alkyl
chain.22,23 Accordingly, the critical aggregative concentrations of
sphingolipids is in the nano- or picomolar range,23–25 and the
amount of free monomers in solution is low mainly at high concen-
trations. Additionally, during cytotoxicity assays these amphiphilic
molecules could also interact with proteins from the fetal calf ser-
um, what drastically reduces the cell uptake.25 These structural
LAAs-induced apoptosis, dissipation of
percentage of depolarized m is shown in Figure 6A. Control cells
elicited higher fluorescence, suggesting that these cells were intact.
1a/1b-treated cells showed m dissipation detected by decreased
fluorescence intensity (Fig. 6A and B). The untreated cells showed
only 9% of wm dissipation, while 1a/1b at 0.1 g/ml increased
dissipation to 22% and to almost 50% at 0.5 g/ml. Dox treatment
induced 60% of wm depolarization.
Dwm was evaluated. The
D
w
D
w
D
l
l
D
3.6. Internucleosomal DNA fragmentation
The internucleosomal DNA fragmentation of HL-60 cells was as-
sessed by flow cytometry. The sub-G0/G1 count was considered to
represent fragmented DNA, whereas debris was omitted from the
analysis. Control cells showed 4% of fragmented DNA. 1a/1b-trea-
ted cells showed no increase in DNA fragmentation at the lowest
concentration tested, however, in the following concentrations
DNA fragmentation did increase as shown in the Figure 7. Dox-
treated cells showed 47% DNA fragmentation. Cell cycle analysis
was also performed, however no changes were observed in the
1a/1b-treated cells (data not shown).
60
B
A
100
M2
M1
45
30
15
0
75
*
*
50
*
*
25
0
100
101
C-
102
103
104
C-
Dox
0.1
0.25
0.5
(μg/mL)
1a/1b
GRN-HLog
1a/1b 0.5
Figure 6. 1a/1b treatment induces mitochondrial membrane potential (
rhodamine 123 (1
test. Doxorubicin (0.3
1b at a concentration of 0.5
D
w
m) loss.
D
w
m depolarization of HL-60 cells, after 24 h incubation, determined by the retention of
*
l
g/ml) dye. (A) Depolarized cells, obtained from five experiments performed in triplicate. p <0.05 compared to the negative control (CÀ) by unpaired t-
l
g/ml) was used as a positive control (Dox). (B) Mitochondrial fluorescence histogram overlay of cells untreated (CÀ, black outline) and treated with 1a/
l
g/ml (shadow). Five thousand events were acquired in each replicate.

D. V. Wilke et al. / Bioorg. Med. Chem. 18 (2010) 7997–8004
8003
100
A 75
B
M2
M1
75
*
50
25
0
*
50
*
25
0
100
(μg/mL)
0.5
102
103
104
C-
Dox
0.1
0.25
101
1a/1b
RED-HLog
C-
1a/1b 0.5
Figure 7. Internucleosomal DNA fragmentation induced by 1a/1b treatment. DNA fragmentation assessed by flow cytometry on HL-60 cells, after 24 h incubation, analyzed
by nuclear fluorescence using propidium iodide (50 g/ml), Triton X-100 (0.2%), and citrate (0.1%). (A) Sub-G0/G1 DNA content of cells obtained from five experiments
g/ml) was used as a positive control (Dox). (B) DNA fluorescence
l
performed in triplicate. *p <0.05 compared to the negative control (CÀ) by unpaired t-test. Doxorubicin (0.3
l
histogram overlay of cells untreated (CÀ, black outline) and treated with 1a/1b at 0.5
l
g/ml (shadow). Five thousand events were acquired in each replicate.
similarities may explain the difficulties in the IC50 calculation,
since the MTT experiments using serial dilutions of the synthetic
LAAs did not result in a concentration–response relationship; thus,
the IC50 value could not be obtained. Since the natural compounds
are much stronger active, the assay was conducted in lower
concentrations. Even though, the preliminary data on the cytotoxic
effects of the 14 synthetic LAAs (2–15) suggest that lipoamino
acids with shorter aliphatic chains, which can be easily synthe-
sized, would also demonstrate anticancer activity. Present findings
do not support any considerations on the structure–activity
requirements for this unusual class of compounds, however they
provide the opportunity for rational design of lipoamino acid-
based anticancer agents.
studies investigating the likely function of LAAs as inhibitors of
sphingolipid metabolism would be a good way to explain the
mechanism of action by which 1a/1b triggers apoptosis.
In summary, the cytotoxicity of 14 synthetic LAAs were evalu-
ated on several tumor cell lines, as well as the anti-proliferative
and pro-apoptotic effects of a cytotoxic LAAs mixture isolated from
the marine zoanthid P. variabilis. This is the first Letter on the
cytotoxic activity of synthetic LAAs. Additionally, present findings
indicate that 1a/1b induces apoptosis, however further studies
are still needed to clarify the exact mechanism of action involved
with 1a/1b-induced apoptosis.
Conflict of interest statement
None.
Additionally, further studies were performed with 1a/1b investi-
gating the mechanisms underlying its anti-proliferative effects on
the promyelocytic leukemia cell line, HL-60. Taking together all
the results, it could be noticed that treated cells at the lowest con-
centration are dying through apoptotic process characterized by
Acknowledgment
D
wm dissipation and peripheral nuclear condensation followed by
The technical assistance of Silvana França is gratefully acknowl-
edged. This research has been supported by the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora
de Estudos e Projetos (FINEP), and International Foundation for
Science (IFS).
DNA fragmentation, membrane blebbing at an intermediate concen-
tration and, finally, at the highest concentration membrane disrup-
tion. Apoptosis is an essential physiological process to maintain
tissue homeostasis and proper function of multicellular organisms.
Cell apoptosis is regarded as the preferred way to eliminate unde-
sired cells and its induction is an important strategy in cancer
therapies.26
References and notes
1. Roberts, D. C.; Vellaccio, F.. In Peptides; Gross, E., Meinhofer, J., Eds.; Academic
Due to the lack of previous studies on the cytotoxic activity of
LAAs, it is impracticable to compare our findings with others of
the same chemical class. However, as previously mentioned LAAs
share important structural characteristics with sphingolipids.
Sphingosine, a precursor and breakdown product of sphingolipids
and related compounds, was found to inhibit protein kinase C
(PKC), an enzyme involved in cell replication, oncogenesis, tumor
promotion, and signal transduction.22 Structure–activity relation-
ship studies showed that a long hydrophobic chain and a free ami-
no group were the structural requirements for the inhibition of
PKC.27 Spisulosine (ES-285) is a natural cytotoxic marine product
that resembles both LAAs and sphingosine. ES-285 acts on PKC
via an atypical cell death pathway,28 and it is currently undergoing
phase 1 clinical studies for cancer treatment under the sponsorship
of PharmaMar.29 Additionally, LAAs inhibit PLA2 because they are
potential anti-inflammatory agents. Since sphingolipid analogs
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