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Marisa Rangel a,b,c,*, Marisa P. Prado b, Katsuhiro Konno c, Hideo Naoki d, José C. Freitas a,c,
Glaucia M. Machado-Santelli b
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a
Department of Physiology, Biosciences Institute, University of Sao Paulo, Sao Paulo 05508-900, Brazil
Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of Sao Paulo, Sao Paulo 05508-900, Brazil
c
Center for Applied Toxinology, Butantan Institute, Sao Paulo 05503-900, Brazil
d
Okinawa Health Biotechnology Research Development Center, 12-75 Suzaki, Gushikawa, Okinawa 904-2234, Japan
b
article info
abstract
Article history:
Crude extracts of the marine sponge Geodia corticostylifera from Brazilian Coast have previously
Received 17 February 2006
shown antibacterial, antifungal, cytotoxic, hemolytic and neurotoxic activities. The present
Received in revised form
work describes the isolation of the cyclic peptides geodiamolides A, B, H and I from G.
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Cytoskeleton alterations induced by Geodia corticostylifera
depsipeptides in breast cancer cells
5 April 2006
corticostylifera (1–4) and their anti-proliferative effects against sea urchin eggs and human
Accepted 6 April 2006
breast cancer cell lineages. Its structure–activity relationship is discussed as well. In an initial
CT
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journal homepage: www.elsevier.com/locate/peptides
series of experiments these peptides inhibited the first cleavage of sea urchin eggs (Lytechinus
variegatus). Duplication of nuclei without complete egg cell division indicated the mechanism
of action might be related to microfilament disruption. Further studies showed that the
Cancer cell
geodiamolides have anti-proliferative activity against human breast cancer cell lines (T47D
Cytoskeleton
and MCF7). Using fluorescence techniques and confocal microscopy, we found evidence that
Sea urchin egg
the geodiamolides A, B, I and H act by disorganizing actin filaments of T47D and MCF7 cancer
Marine sponge
cells, in a way similar to other depsipeptides (such as jaspamide 5 and dolastatins), keeping the
Depsipeptide
normal microtubule organization. Normal cells lines (primary culture human fibroblasts and
Geodiamolide
BRL3A rat liver epithelial cells) were not affected by the treatment as tumor cells were, thus
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Keywords:
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Studies on marine life forms in the last few years have led to
the discovery of a variety of organic compounds with known
or novel pharmacological and toxic activities on mammalian
species. Available evidence suggests that the sea offers a rich
source of new organic molecules which, either structurally
modified or not, may be used as medicines, or as biochemical,
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Introduction
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indicating the biomedical potential of these compounds.
# 2006 Elsevier Inc. All rights reserved.
physiological or pharmacological tools in biomedical research
[17,29,40].
Chemical defense through synthesis or accumulation of
large amounts of toxic or deterrent natural products is
usually found in Porifera [5]. Many of the compounds
isolated from marine sponges exhibit neurotoxic, bactericidal, ichthyotoxic, cytotoxic, haemolytic and other toxic
properties [37].
* Corresponding author at: Laboratorio Especial de Toxinologia Aplicada, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900 Sao Paulo,
SP, Brazil. Tel.: +55 11 37261024; fax: +55 11 37261024.
E-mail address: [email protected] (M. Rangel).
0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2006.04.021
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evidence for the biomedical potential of these compounds, the
cytoskeleton proteins of two different normal cell lineages
(human fibroblasts and rat liver cells) were also analyzed
under confocal microscope after incubation with the sponge
depsipeptides.
2.1.
Extraction and isolation of compounds
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Specimens of G. corticostylifera (2.7 kg) were collected by
Dr. Marcio dos Reis Custodio on June 2001 off the coast of
Sao Paulo State, Brazil, then homogenized in methanol
(1:3, w/v) and filtered. The filtered material was evaporated
and partitioned with water/methylene chloride (1:1, v/v).
The non-polar fraction volume was reduced in a vacuum
evaporator and partitioned in methanol–water (9:1, v/v)/
n-hexane (1:2, v/v). The methanol–water fraction was
fractionated by a Sep Pak Vac C18 cartridge with step-wise
elution of 20, 50 and 90% CH3CN in water. Successive
purification of the 50–90% acetonitrile fractions by
reversed-phase HPLC using CAPCELL PAK C18 (10 mm 250 mm) with isocratic elution of 45% CH3CN/H2O at a
flow rate of 2.5 ml/min over 40 min, monitored by UV
absorption at 215 nm yielded the anti-proliferative compounds geodiamolide A (8.8 mg), B (4.6 mg), H (12.2 mg) and I
(5.9 mg).
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Materials and methods
2.2.
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CT
Bioassay guided fractionation is the most frequently used
technique to isolate sponge peptides, which are usually
cyclic and lipophilic [18]. The depsipeptides isolated from
marine sponges or associated organisms are usually
described as cytotoxic substances, such as jaspamide, or
jasplakinolide [14,45,46], geodiamolides [10,12,13,15,43],
hemiasterlins and criamides [12], halicilindramides [23].
Nevertheless, there are sponge depsipeptides which presented anti-inflammatory activity, as the halipeptins A and B
[36], and anti-HIV activity, as papuamides A and microspinosamide [16,44].
From the genus Geodia, compounds other than depsipeptides have also presented interesting biological activities; for
example, the brominated cyclopeptides from the marine
sponge Geodia barretti, barretin and 8,9-dihydrobarettin,
which showed potent antifouling effects inhibiting the
settlement of cyprid larvae of the barnacle Balanus improvisus
[41]. A Geodia species collected from southern Australian
waters of the Great Australian Bight has yielded a potent
new in vitro nematocidal agent identified as geodin A Mg salt
(1), a new macrocyclic polyketide lactam tetramic acid
magnesium salt [9]. Additionally, two proteins from
Geodia mesotriaena, named geodiastatins 1 and 2, presented
antineoplastic activity against the murine P388 lymphocytic leukemia [33], and geodiatoxins 1 and 2, related
proteins from the same species, were very toxic to mice
(i.p.)[34].
The present is focused on the anti-proliferative compounds
from the marine sponge Geodia corticostylifera (Porifera,
Demospongiae). This species can be found in Southeastern
Brazilian coast and Venezuela [21]. Its orange color, the
apparent lack of predators and other sponge species nearby,
suggests the presence of chemical defense mechanisms.
Previous studies indicated that the crude extracts of G.
corticostylifera displayed bactericidal and fungicidal activities
[30], and cytotoxicity to sea urchin eggs, neurotoxicity to crab
sensory nerve, and haemolytic activity in mice erythrocytes
[38]. Neurotoxic and haemolytic activities were recently
related to a pore-forming substance in the extract, which
incorporates channels on artificial lipid bilayers. These
channels have small conductance levels, are rectifiers and
cation selective [37].
The cyclic depsipeptides geodiamolides A (1), B (2), H (3)
and I (4) were previously isolated and characterized from the
Caribbean marine sponge Geodia sp. [10,43]. Geodiamolides A
and B presented antifungal activity [10], while geodiamolide
H was active against cancer cell lineages (lung, HOP 92;
central nervous system, SF-268; ovarian, OV car-4; kidney,
A498 and UO-31; breast, MDA-MB-231/ATCC and HS 578T),
although geodiamolide I was considered inactive in the same
screening [43]. Compounds 1–4 were also isolated from the
marine sponge G. corticostylifera, collected on the Brazilian
coast, during our studies on purifying the pore-forming
substance, and the anti-proliferative effects of these peptides were investigated against sea urchin eggs (Lytechinus
variegatus), and T47D and MCF7 human breast cancer cells
lineages.
Using fluorescence techniques and confocal microscopy
the effects of the geodiamolides A, B, I and H on cancer cell
cytoskeleton and nucleus were observed. In an attempt to give
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Spectroscopic analysis
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The compounds purified by HPLC were detected by positive
electrospray ionization (ESI) mass spectrometry. Typical
conditions were a capillary voltage of 2.4 kV, a cone voltage
of 32 V, and a desolvation gas temperature of 150 8C. The
experiments were done with a Q-ToF mass spectrometer
(Micromass, UK) in Qq-orthogonal time-of-flight configuration. The spectra were recorded by the use of MassLynx 4.0
software (Micromass).
NMR spectra were recorded on a JEOL EX-400 (at 400 MHz)
or a Bruker DMX-750 spectrometer (at 750 MHz) in CD3OD or
CDCl3.
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2.3.
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Sea urchin eggs development experiments
Antimitotic activity was monitored initially as the ability of
the geodiamolides A, B, H and I to inhibit the first cleavage of
L. variegatus sea urchin eggs. The animals were collected in
Sao Sebastiao, off the north coast of Sao Paulo State, Brazil.
Germ cell release was induced by KCl injections (0.5 M, up to
3 ml) into the perivisceral cavity of the sea urchins. The eggs
were washed three times in filtered seawater to remove the
jelly coat. The sperm was maintained under refrigeration
and not diluted until it was used. The geodiamolides A, B, H
and I were diluted in filtered sea water/MeOH (19:1; v/v) in
different concentrations. The final volume was 200 ml of
seawater/MeOH mixture, plus 200 ml of egg suspension
(prepared with 50 ml of washed eggs + 50 ml of sperm and
observation of formation of fertilization layer). Control
tubes contained 200 ml of sea water/MeOH mixture. When
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the first division in control tubes occurred, the material was
fixed in formaldehyde 10%, observed under microscope and
photographed [25].
The results were analyzed according to their logarithms of
mean and respective standard errors (n = 3). Dose response
curves were plotted, and the EC50 values were calculated by
means of non-linear regression.
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The T47D and MCF7 human breast cancer cells were grown
in Sigma culture medium (DMEM) with 10% fetal bovine
serum (Cultilab) in cellular culture multiplates (24 wells)
with an initial density of 5 104 cells/well and incubated
during 24 h. After this period, the medium was changed to a
new one with the geodiamolides A, B, H and I. After 48 h the
medium was removed, each well was washed with PBS, and
trypsin was added to release the cells from the bottom. Then
the trypsin was neutralized with medium, and 20 ml of
Trypan Blue were added. The living cells were counted in a
Neubauer chamber.
The results were analyzed according to their mean log and
respective standard errors (n = 3). Dose–response curves were
plotted and EC50 were calculated by means of non-linear
regression.
For the fluorescence techniques, cells were plated on
coverslips within culture Petri dishes. After 48 h incubating
with either geodiamolides or control, the medium was
removed, and the cells were fixed with formaldehyde (3.7%).
After washing with PBS, the cells were treated with RNAase,
stained with phalloidin-FITC (actin), and propidium
iodide (nuclei). Monoclonal anti-tubulin and secondary antimouse-CY were used in immunofluorescence reactions. The
preparations were mounted on slides with anti-fading (VectaShield, Vector). The fluorescent images were obtained by
confocal laser scanning microscope (Zeiss LSM 510) with lasers
of argon (458, 488 and 514 nm), helium–neon 1 (543 nm) and
helium–neon 2 (633 nm) connected to an inverted fluorescence
microscope, Zeiss Axiovert 100 M [26].
The effect of geodiamolides A and H (50 ng/ml) on microfilaments of T47D cells was also observed at different incubation
times (2, 4, 8, 12 and 48 h), using phalloidin-FITC stained cells
and confocal laser scanning microscope, as described above.
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Primary culture human fibroblasts and BRL3A rat liver
epithelial cells were tested against geodiamolides A and H,
stained with phalloidin-FITC and observed under a confocal
laser scanning microscope, as described above.
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3.2.
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comparison with literature revealed that the structures of
GC503/901-1, -2, -3, and -4 were identical to geodiamolides B
(2), A (1), I (4), and H (3), respectively, cyclic depsipeptides
previously described [10,43].
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Biological activities
In the experiments with L. variegatus sea urchin eggs, the
geodiamolides A, B, H and I inhibited the first cleavage in a
dose-dependent form (Fig. 1), and the geodiamolides A and B
acted at much lower concentrations than H and I (Table 1).
Light microscope images of sea urchin eggs revealed a
particularity of the depsipeptides anti-mitotic effect: nuclei
duplications without complete cytokinesis at lower concentrations, and cells deformations at high concentration treatment (Fig. 2).
In the breast cancer cells experiments, the values of EC50
for the geodiamolides A, B, H and I were also obtained in
nM range (Table 1). Geodiamolides A and H were more
effective against T47D cells, while geodiamolides B and A
had a stronger effect on MCF7 cells. T47D and MCF7 cells
growth inhibition curves by the compounds are shown in
Fig. 3.
The observation of T47D cells stained for actin filaments
and nuclei in confocal microscope showed that geodiamolides A, B, H and I act upon F-actin, disorganizing the
filaments and gathering them in the cytoplasm, in a dosedependent manner (Fig. 4). At the concentration of 100 ng/ml
(135–170 nM), nuclei were displaced from central position in
the cytoplasm and their shape changed when compared to
control cells (Fig. 4). Notwithstanding, microtubule organization remained unchanged, as observed in immunofluor-
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3.
Results
3.1.
Extraction and isolation of compounds
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Fig. 1 – Dose–response curves of sea urchin eggs first
cleavage inhibition induced by the geodiamolides (n = 3).
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Normal cells experiments
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Breast cancer cells experiments
Reversed-phase HPLC purification of Sep Pak Vac prepared
extract fractions GC503 and GC901 yielded four anti-mitotic
peaks (screened in sea urchin eggs), named 1–4. Further mass
spectrometry and NMR analysis of these peaks as well as
Table 1 – EC50 values of geodiamolides (in nM) tested in L.
variegatus sea urchin eggs, and in T47D and MCF7 human
breast cancer cell lineages
Compound
Geodiamolide
Geodiamolide
Geodiamolide
Geodiamolide
A
B
H
I
Sea urchin eggs
T47D
MCF7
101.2
98.8
595.6
620.0
18.82
113.90
38.36
115.30
17.83
9.82
89.96
65.70
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Fig. 2 – Light microscope observation of sea urchin eggs first cleavage in the absence of (control (A)) and under treatment
with the geodiamolides A ((B) and (C)) (0.1 and 15 mg/ml), B ((D) and (E)) (0.1 and 15 mg/ml), H ((F) and (G)) (0.5 and 50 mg/ml)
and I ((H) and (I)) (0.5 and 15 mg/ml).
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escence preparations under confocal microscope, showing
that the geodiamolides do not disassemble microtubules
(Fig. 5).
Disorganization of microfilaments of T47D cells induced by
the geodiamolides A and H was perceived within 2 h of the
treatment (the first time interval chosen in our experiment),
and progressed along the incubation time (Fig. 6).
In our experiments using normal cells lines, only
geodiamolide A induced a slight disorganization of the
human fibroblasts microfilaments at 100 ng/ml concentration, while geodiamolide H did not cause cytoskeleton
alterations (Fig. 7(A)–(C)). The same concentration of both
peptides had no effect on rat liver epithelial cells (BRL-3A) Factin (Fig. 7(D)–(F)).
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Fig. 3 – Dose–response curves showing the growth inhibition of T47D and MCF7 human breast cancer cells induced by the
depsipeptides of the sponge G. corticostylifera (n = 3).
Cyclic peptides and depsipeptides are metabolite classes that
have not been reported in sponges until recent years. These
metabolite classes were described in species of four different
orders: Axinellida, Choristida (to which the genus Geodia
belongs), Halichondrida and Lithistida. A number of peptides
and depsipeptides are characterized by the presence of new
amino acids [39]. Depsipeptides, besides presenting unusual
amino acids in their molecules, are also characterized by ester
bonds and carboxylic acid.
According to Tinto et al. [43], who first described the
structures of the geodiamolides H and I, only geodiamolide H
presented cytotoxic activity, inhibiting the growth of tumor
cells in vitro, while geodiamolide I was completely devoid of
activity. Our results with sea urchin eggs (Figs. 1 and 2) showed
that both peptides are active under similar concentrations.
Additionally, geodiamolides A and B from G. corticostylifera
inhibited first division at smaller concentrations than geodiamolides H and I (Fig. 1).
Observation under light microscope of sea urchin eggs
showed that at the higher concentrations, the geodiamolides
induced cell deformation, and at lower concentrations multinucleated cells were present (Fig. 2). Inhibition of sea urchin
egg cleavage may occur in several processes in different stages
of the cell cycle; for example, DNA, RNA or protein synthesis.
Nevertheless, initial cleavage inhibition in these cells is due to
either DNA synthesis blockade or inhibition of cytoskeleton
protein organization [19], since in the first period of development the embryo does not synthesize RNA once all the mRNA
it needs comes from the oocyte [6]. When there is microtubule
disorganization, spots corresponding to nuclei duplication are
perceptible in the cytoplasm, without having fulfilled mitosis
[19]. Moreover, actin filaments sustain the cytoplasm, and
form contractile rings during cell division [1]. Thus the effects
induced by the geodiamolides in the sea urchin eggs seem to
be due to a disorganization of cytoskeleton, specially the
microfilaments.
Similar results were found when the macrolides, isolated
from an unidentified nudibranch whose chemical structures
are similar to swinholide A produced by the Red Sea sponge
Theonella swinhoei, inhibited the development of starfish
embryos, producing multinucleated cells and unusually
shaped nuclei [19]. Also, the diterpenoids of the sponge
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Fig. 4 – Laser scanning confocal microscope images of T47D cells stained with phalloidin-FITC (actin, green) and propidium
iodide (nuclei, red); controls, and after 48 h incubation with the geodiamolides A, B, H and I.
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Strongylophora strongylata from Japan inhibited the maturation
of starfish oocytes Asterina pectinifera, and this effect may be
due to either cyclinB/cdc2 or microtubule assembly inhibition
[24].
Though a series of cyclic depsipeptides geodiamolides have
been found and described as in vitro cytotoxic substances
[10,12,13,15,43], their mechanism of action was not investigated in as much detail as jaspamide (5) [7,8,11,28,31,35],
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Fig. 5 – Laser scanning confocal microscope images of T47D cells stained with phalloidin-FITC (actin) and Cy-5 (tubulin);
controls, and treated with 100 ng/ml of the geodiamolides A, B, H and I.
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Fig. 6 – Laser scanning confocal microscope images of T47D cells stained with phalloidin-FITC (actin), treated with 50 ng/ml
of geodiamolides A and H and observed at different time intervals: ((A) and (G)) control; geodiamolide A: (B) 2 h, (C) 4 h, (D)
8 h, (E) 12 h, (F) 24 h; geodiamolide H: (H) 2 h, (I) 4 h, (J) 8 h, (K) 12 h, (L) 24 h.
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Fig. 7 – Laser scanning confocal microscope images of normal cells stained with phalloidin-FITC (actin). Human fibroblasts
from primary culture: (A) control; (B) geodiamolide A and (C) geodiamolide H (100 ng/ml) and rat liver epithelial cells BRL-3A:
(D) control; (E) Geodiamolide A and (F) Geodiamolide H (100 ng/ml).
whose chemical structure is very similar to the geodiamolides
(1–4).
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The cytoskeleton is one of the main targets of substances in
the tests applied to search for new compounds with potential
anti-tumor activity [2,27]. It is known that jaspamide, isolated
from the sponge Jaspis johnstoni, exerts its cytotoxic activity
through actin filament stabilization, competing with phalloidin
binding to F-actin [7,8,32]. Other cytotoxic marine depsipeptides, isolated from the sea hare Dolabella auricularia, dolastatin
11 [4] and doliculide [3], also stabilize actin filaments, in a way
similar to jaspamide. More recently, bistramide A, a cyclic
polyether from the ascidia Lissoclinum bistratum was found to
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induce actin depolymerization by direct binding to F-actin [42].
Laser scanning confocal microscope analysis of T47D cells
stained for actin and nucleus showed that the geodiamolides
A, B, H and I act by affecting the cell microfilaments,
disorganizing them and forming aggregates in the cytoplasm,
in a dose-dependent manner (Fig. 4). In addition, under high
doses the nucleus shapes were altered, as well as their
location in the cytoplasm. In the present work a- and b-tubulin
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The authors wish to thank Dr. Marcio Reis Custodio, from the
Department of Physiology of Biosciences Institute, University
of Sao Paulo, for collecting and identifying the sponge. This
work was supported by FAPESP. MR and MPP received FAPESP
fellowships and GMMS a CNPq fellowship.
Conclusions
The geodiamolides A, B, H and I presented anti-proliferative
activity against breast cancer cells. This effect is related to the
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Stereostructures of geodiamolides A and B, novel
cyclodepsipeptides from the marine sponge Geodia sp.. J
Org Chem 1987;52:3091–3.
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line cells. Cell Mol Life Sci 2002;59:1377–87.
[12] Coleman JE, de Silva E, Kong F, Andersen RJ, Allen TM.
Cytotoxic peptides from the marine sponge Cymbastela sp..
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6.
F
actin depolymerization, as observed in the confocal microscopy analysis of stained cells. Normal cell lines, however, did
not show cytoskeleton alterations after treatment with the
peptides, which is beneficial to the biomedical potential of
such compounds. Interestingly, differences in peptide potencies are associated with an amino acid substitution or with the
presence of bromide or iodide.
CT
immunofluorescence was utilized to show that the geodiamolides do not affect microtubule assembly (Fig. 5).
Usually, the microtubule and microfilament networks
interact during a variety of cellular processes, including
vesicle and organelles transport, cleavage orientation, cell
migration control, mitotic spindle rotation, and nuclear
migration. Thus, substances that specifically affect microtubules or microfilaments may impair the processes accomplished by the cooperation of both of them [20]. Since the
geodiamolides A, B, H and I disorganize actin microfilaments,
they must be impairing the mechanisms involved in mitosis,
and may be inducing cell death by apoptosis, like jaspamide
does [11,28,31,35].
The disassembly of microfilaments of T47D cells induced
by the geodiamolides A and H (Fig. 6) was perceived within
2 h of the treatment, and progressed along the incubation
time. Jaspamide effect was evident after 2 h incubation, and
continued to grow until 24 h of treatment [8]. Other peptides
can affect the microfilaments in shorter incubation time,
such as doliculide [3] and dolastatin 11 [4], which start to
disrupt actin filaments of kangaroo rat kidney epithelial
cells (PtK1 and 2) within 30 min of the beginning of
treatment.
The results of our experiments using normal cells lines
(Fig. 7) are encouraging, considering that at the same
concentration of the geodiamolides 70% of the breast cancer
cells died in culture, and at 25 ng/ml a microfilaments
disorganization was very clear (Fig. 4), thus indicating the
biomedical potential of these marine depsipeptides.
It is noteworthy that the structure–activity relationships
of these compounds seem to vary according to the cell type.
For instance, in sea urchin eggs, geodiamolides A and B are
much more potent than geodiamolides H and I, a difference
which apparently resides in the structural difference of
alanine versus b-tyrosine. In contrast, this structural
difference does not matter with T47D cells, but the halogen
substituent X in the phenol ring of N-methyltyrosine moiety
is crucial in this case because geodiamolides A and H (X = I)
are much more potent than geodiamolides B and I (X = Br).
More interestingly, in case of MCF7 cells, the trend is similar
to that of sea urchin eggs, but distinct from that of T47D
cells, despite the fact that these are mammalian cancer cell
lines similar to T47D cells. Thus, we found that small
structural alteration in geodiamolides largely affects the
rank order of potency in each cell line, indicating that there
would be different cellular sensitivity depending on its
phenotype.
Disruption of cytoskeleton elements such as microtubule
and microfilament has been shown to interfere with the
invasiveness and adhesion of tumor cells during the initial
phases of metastasis formation [22]. New drugs acting in
specific manner of this process may contribute to establishing
new therapeutic approaches focusing on different phases of
tumor progression.
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peptides xxx (2006) xxx–xxx
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