Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells

b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 2 7 – 3 3 5

Irreversible cytoskeletal disarrangement is independent ofcaspase activation during in vitro azaspiracid toxicity inhuman neuroblastoma cells

Natalia Vilarino a, K.C. Nicolaou b,c, Michael O. Frederick b, Mercedes R. Vieytes d,Luis M. Botana a,*aDepartamento de Farmacologıa, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, SpainbDepartment of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,

10550 North Torrey Pines Road, La Jolla, CA 92037, USAcDepartment of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USAdDepartamento de Filsiologıa, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain

a r t i c l e i n f o a b s t r a c t

avai lab le at www.sc iencedi rec t .com

journal homepage: www.e lsev ier .com/ locate /b iochempharm

Article history:

Received 26 January 2007

Accepted 4 April 2007

Keywords:

Azaspiracid

Phycotoxin

F-Actin cytoskeleton

Caspase

Structure–activity

Human neuroblastoma

Azaspiracid-1(AZA-1) isamarinetoxindiscoveredin1995.Besidesdamagetoseveral tissuesin

vivo, AZA-1 has been shown to cause cytotoxicity in a number of cell lines and alterations in

actin cytoskeleton and cell morphology. We studied the reversibility of AZA-1-induced

morphological changes in human neuroblastoma cells and their dependence on caspases

and signaling pathways involved in cytoskeleton regulation. Morphological/cytoskeletal

changes were clearly observed by confocal microscopy 24 h after the addition of toxin, without

recoveryupontoxinremoval. Interestingly,2 minof incubationwithAZA-1wasenoughforthe

cytoskeleton to be altered 24–48 h later. The activation of caspases by AZA-1 was studied next

using a fluorescent caspase inhibitor. A cell population with activated caspases was observed

after 48 h of exposure to the toxin, but not at 24 h. Two fragments and a stereoisomer of AZA-1

were tested to analyze structure–activity relationship. Only ABCD-epi-AZA-1 was active with a

similar effect to AZA-1. Additionally, regarding the involvement of apoptosis/cytoskeleton

signaling in AZA-1-induced morphological effects, inhibition of caspases with Z-VAD-FMK did

not affect AZA-1-induced cytoskeletal changes, suggesting, together with the activation

kinetics, that caspases are not responsible for AZA-1-elicited morphological changes. Mod-

ulation of PKA, PKC, PI3K, Erk, p38MAPK, glutathione and microtubules with inhibitors/

activators did not inhibit AZA-1-induced actin cytoskeleton rearrangement. The JNK inhibitor

SP600125 seemed to slightly diminish AZA-1 effects, however due to the effects of the drug by

itself the involvement of JNK in AZA-1 toxicity needs further investigation. The results suggest

that AZA-1 binds irreversibly to its cellular target, needing moieties located in the ABCDE and

FGHI rings of the molecule. Cytotoxicity of AZA-1 has been previously described without

reference to the type of cell death, we report that AZA-1 induces the activation of caspases,

commonly used as an early marker of apoptosis, and that these proteases are not responsible

for AZA-1-induced cytoskeleton disarragement in human neuroblastoma cells.

# 2007 Elsevier Inc. All rights reserved.

* Corresponding author. Tel.: +34 982 252 242; fax: +34 982 252 242.E-mail address: [email protected] (L.M. Botana).

Abbreviations: AZA-1, Azaspiracid-1; EMEM, Eagle’s Minimum Essential Medium; HBSS, Hank’s balanced salt solution; FLICA, fluor-escent inhibitor of caspases; DMSO, dimethylsulfoxide; BSA, bovine serum albumin; PBS, phosphate buffered saline; DSP, diarrheticshellfish poisoning; i.p., intraperitoneal; p.o., per os or oral administration; PMA, phorbol 12-myristate 13-acetate0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.bcp.2007.04.004

mailto:[email protected]

http://dx.doi.org/10.1016/j.bcp.2007.04.004


1. Introduction

Azaspiracid-1 was the first molecule identified as the

causative toxin of an outbreak poisoning related to shellfish

consumption in Killary Harbour, Ireland in 1995 [1]. Although

the symptoms of this toxic episode in humans were similar to

diarrhetic shellfish poisoning (DSP), the neural symptoms

observed during mouse bioassays, absent after DSP i.p.

administration, suggested a different etiology. AZA-1 was

isolated by Satake in 1997 who performed the first structural

studies [2]. Later, the initially proposed structure was proved

incorrect, and after degradative and synthetic studies, the

Nicolaou’s group would prove the correct structure of AZA-1 to

be that seen in Fig. 3 [3–9]. Since 1997, 11 azaspiracids have

been isolated from contaminated shellfish, their structures

differing in the presence of methyl and hydroxyl groups

through the backbone of AZA-1 [2,10–13]. AZA-1, AZA-2, AZA-

3, AZA-4 and AZA-5 have been proved to be toxic to mice by i.p.

administrations, and AZA-1 also p.o. [2,11,12,14,15]. No

toxicology data are available regarding the other compounds

due to the small quantities of toxin obtained after purification.

In vivo and in vitro experiments have helped to understand

the effects of azaspiracids both at the whole organism and

single cell levels, however the mechanism of action of this

group of toxins is still unknown. Toxicology studies have

demonstrated that AZA-1 targets several tissues causing

atrophic lamina propria and shortening and erosion of villi

in the intestine, necrosis of lymphoid tissues, fatty liver and

lung tumors [14,15]. Remarkably, the injuries caused by acute

administration of AZA-1 take extremely long periods of time to

recover, mainly in the intestine, where lesions are still present

3 months after a single AZA-1 administration [15]. In vitro

studies have shown that AZA-1 induces cytotoxicity and

inhibition of cell proliferation in several cell models [16,17]

without any inhibition of PP2A [16], the target of DSP toxins.

Additionally, some aspects of cell physiology have been

described to be affected by AZAs, such as intracellular

calcium, cAMP and pH in lymphocytes [18–21] and bioelec-

trical activity in spinal cord neuronal networks [22]. Regarding

the cytoskeleton, AZA-1 alters the actin cytoskeleton of non-

adherent and adherent cell models at concentrations that do

not depolymerize F-actin [16–18]. In human neuroblastoma

cells the alteration of cytoskeleton arrangement is simulta-

neous to a rounding up of cell shape and detachment,

reminiscent of apoptotic morphological changes [17].

Although AZA-1 is cytotoxic to many cellular models, the

activation of apoptotic mechanisms by AZAs, which might be

responsible for cytoskeletal disarrangement, has not been

reported in any cell type. Besides apoptosis, modulation of

many intracellular signaling pathways could be implicated in

the alterations of actin cytoskeleton induced by AZA-1, since

the regulation of the cytoskeletal function is complex.

In this study we explored four aspects of AZA-1 toxicity in

human neuroblastoma cells in order to obtain information

about the characteristics of its target. One of the character-

istics of the drug-target interaction is its reversibility. There-

fore, we first studied the reversibility of AZA-1 effects on

human neuroblastoma cytoskeleton. Next, we analyzed AZA-

1-induction of caspase activation, an early marker of apop-

tosis, to understand if protease activation is a primary event

responsible for morphological changes in AZA-1 toxicity.

Additionally the structure–activity correlation for caspase

activation was studied using an AZA-1 steroisomer and two

fragments of the molecule. Finally, the involvement in AZA-1

toxicity of several signaling pathways that regulate actin

cytoskeleton and apoptotic death was explored using phar-

macological inhibitors/activators in human neuroblastoma

cell cultures.

2. Methods and materials

2.1. Reagents

Eagle’s Minimum Essential Medium (EMEM), Ham’s F12,

glutamine, non-essential amino acids, gentamycin, ampho-

tericine B and penicillin/streptomycin were purchased from

Biochrom AG (Berlin, Germany). Oregon Green1 514 Phalloi-

din, Image iT live pan caspase kit and Texas red DNAse I were

obtained from Molecular Probes (Eugene, Oregon, USA). Fetal

bovine serum (FBS), paraformaldehyde, Triton X-100, DMSO,

SQ 22,536, H-89, forskolin, dibutyril cAMP, wortmannin,

LY294002, SB202190, PD98059, SP600125, colchicine, nocoda-

zol, glutathione ethyl ester (GSH-MEE), DL- buthionine-S, R-

sulfoximine, bisindolylmaleimide I, cheleritrine, PMA (phorbol

12-myristate 13-acetate) and glycerol were obtained from

Sigma (St. Louis, MO, USA). Z-VAD-FMK was from BD

Biosciences (Erembodegem, Belgium). BSA was obtained from

ICN Biomedicals (Aurora, Ohio, USA). PBS was 137 mM NaCl,

8.2 mM Na2HPO4, 3.2 mM KCl and 1.5 mM KH2PO4, pH 7.4.

2.2. AZA-1 and enantiomer/fragment synthesis

AZA-1, AZA-1 isomer, and AZA-1 fragments were synthesized

by Nicolaou et al. [3–9].

2.3. Cell line culture

Human neuroblastoma BE(2)-M17 cell line (European Collec-

tion of Cell Cultures) were cultured in EMEM:Ham’s F12 (1:1)

supplemented with 2 mM glutamine, 1% non-essential amino

acids, 10% FBS, 50 mg/L gentamycin and 50 mg/L amphoter-

icine B. For imaging assays, neuroblastoma cells were plated

on glass coverslips at a density of 2.5–5 � 104 cells/well and

used after 5–7 days. Cell cultures were kept at 37 8C in 5% CO2.

2.4. Cytoskeleton imaging

Actin cytoskeleton was stained with Oregon Green1 514

Phalloidin that binds specifically to F-actin. Cells previously

incubated with toxin or carrier were fixed with 4% parafor-

maldehyde and permeabilized with 0.1% Triton X-100 in PBS.

After blocking for 30 min with 1% BSA/PBS the cells were

incubated with 165 nM Oregon Green Phalloidin in 1% BSA/PBS

for 20 min and washed three times with PBS. The coverslips

were then mounted in 50% glycerol/PBS and sealed with nail

polish. Images were captured with a Nikon Eclipse TE2000-E

confocal microscope as described before [17]. Confocal images

are shown as volume render with maximum projection

method.

b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 2 7 – 3 3 5 329

2.5. Quantification of caspase activation

Caspase activation was measured with the Image-iTTM LIVE

poly caspases detection kit which is based in binding of

fluorescent Z-VAD to activated caspases by a covalent bond.

Neuroblastoma cultures were incubated with toxin or carrier

for 24 or 48 h. Following the company instructions, the cells

were then washed twice with culture medium and incubated

for 1 h with the FLICA (fluorescent inhibitor of caspases)

reagent. Cells were washed, fixed with 4% paraformaldehyde

and permeabilized with 0.1% Triton X-100. After blocking with

1% BSA/PBS cells were incubated with 20 mM Texas Red1

DNAse I, which was used as counterstain. The preparation

was then washed and mounted. FLICA green fluorescence was

measured in a CompuCyte laser scanning cytometer (Cam-

bridge, MA) using an argon laser as the excitation light source.

Detection parameters were set to identify red fluorescent cells

as events. Data were analyzed as histogram of green

fluorescence maximum pixel. Texas Red1 DNAse I does not

emit green fluorescence in this experimental setting. Experi-

ments were performed in duplicate.

3. Results

3.1. Irreversibility of AZA-1 toxic effects on neuroblastomaactin cytoskeleton

The reversibility of AZA-1 effects on morphology and actin

cytoskeleton of human neuroblastoma cells was studied by

Fig. 1 – Irreversibility of AZA-1-induced changes in actin cytosk

incubated for 2, 10 and 30 min, respectively with 50 nM AZA-1 an

the toxin. (D) Human neuroblastoma cells incubated with DMSO

AZA-1 for 30 min. (F) Human neuroblastoma cells incubated with

with paraformaldehyde before actin cytoskeleton staining with

experiments.

incubation in the presence of 50 nM AZA-1 for variable times

and posterior removal of the toxin from the culture medium.

Two washes with toxin-free culture medium were performed

before toxin-free culture to ensure complete removal of AZA-

1. Previous kinetic studies [17] demonstrated that morpholo-

gic/cytoskeletal changes induced by AZA-1 were clearly

observable at 24–48 h, but not so apparent at shorter times.

Interestingly the kinetics of AZA-1 effect on morphology was

not dependent on the toxin concentration, since the effects

appeared at the same time at concentrations of 50 nM and

1 mM (data not shown). Initial observations showed that

removal of toxin after 24 and 48 h of exposure to AZA-1 did

not induce a recovery of the morphologic and cytoskeletal

changes already present at the time, on the contrary several

hours (24–48) of toxin free culture later the cells looked more

affected. Our next experiments were directed to determine the

minimum time of toxin-exposure required for AZA-1 toxicity

to occur. Therefore our experimental design consisted in

incubating the cell cultures with 50 nM AZA-1 for reduced

periods of time and, after the removal of toxin, we let them

complete a total of 48 h of culture. The results showed that

exposure times of 12, 6, 3, 1 h (data not shown), 30, 10 and,

surprisingly, 2 min (Fig. 1), were enough for the cells to show

the typical morphological changes 48 h later. In every

experiment a control of cells exposed for 48 h to AZA-1 was

included for comparison and there was no observable

difference in cell appearance between short and long

exposures. A control at the time of toxin removal was included

as well in every experiment with no morphological/cytoske-

letal changes at times shorter than 12 h.

eleton morphology. (A–C) Human neuroblastoma cells were

d then incubated for a total period of 48 h in the absence of

. (E) Human neuroblastoma cells incubated with 50 nM

50 nM AZA-1 for 48 h. Incubation was stopped by fixation

Oregon Green Phalloidin. Representative of three

Fig. 2 – Caspase activation during AZA-1 in vitro toxicity. Human neuroblastoma cells were incubated in the presence of

toxin for 24 or 48 h, then washed and incubated for 1 h with the FLICA reagent as described in the methods section. (A)

Representative histograms of DMSO-treated cells (black line) and 50 nM AZA-1-treated cells (grey line) after 48 h of toxin

exposure. (B) Percentage of the cell population with activated caspases (region 2 of (A)) after 48 h of incubation in the

presence of 50 nM AZA-1 or carrier (n = 12). (C) Percentage of the cell population with activated caspases after 24 and 48 h of

incubation with 50 nM AZA-1 or carrier (n = 4) (The symbol (*) shows statistically different vs. DMSO control p < 0.05).


3.2. Caspase activation during AZA-1 treatment

A fluorescent probe (FLICA) was used to detect activation of

caspases in live cells, including caspases 1 and 3–9. After 48 h

of incubation with 50 nM AZA-1, a cell population with

activated caspases appeared in the histogram as a population

with increased green fluorescence (FLICA labeling, Fig. 2A, grey

line). Fig. 2B summarizes the results of 12 experiments,

showing that the percentage of the cell population with

activated caspases was significantly higher in AZA-1-treated

cells than in carrier-treated cells. The mean green fluores-

Fig. 3 – Caspase activation by AZA-1 stereoisomer and fragmen

natural ABCDE fragment and natural FGHI fragment, respective

caspases after 48 h of incubation with carrier or 50 nM AZA-1,

natural FGHI (n = 6) (The symbol (*) shows statistically different

cence in AZA-1 treated cells was 3.45 � 0.57 times higher than

in control cells. No caspase activation was detectable by this

method at 6, 12 (data not shown) and 24 h (Fig. 2C), whereas it

was significantly increased at 48 h in the same experiments.

The typical morphological changes induced by AZA-1 were

clearly observed in the preparations used for caspase activa-

tion measurements both at 24 and 48 h.

Additionally, a stereoisomer of ABCD rings and two

fragments of AZA-1 molecule (Fig. 3B–D) were tested for

caspase activation induction and compared to AZA-1 (Fig. 3A).

A 50 nM concentration of ABCD-epi-AZA-1 induced an

ts. (A–D) Chemical structures of AZA-1, ABCD-epi-AZA-1,

ly. (E) Percentage of the cell population with activated

50 nM ABCD-epi-AZA-1, 50 nM natural ABCDE and 50 nM

vs. DMSO control p < 0.05).


increase of caspase activation similar to 50 nM AZA-1 (Fig. 3E).

The two fragments of AZA-1, natural ABCDE and natural FGHI,

did not induce caspase activation at concentrations of 50 nM

(Fig. 3E) or 200 nM (data not shown). Additionally, 50 nM AZA-1

was combined with 150 nM natural ABCDE or natural FGHI

fragments and added to the cell culture for 48 h. None of the

fragments inhibited AZA-1-induced activation of caspases

(data not shown).

3.3. Effects of signaling modulation on AZA-1 toxicity

Inhibitors and activators of several signaling pathways were

tested for similar effects to AZA-1 or inhibition of AZA-1-

induced morphological/cytoskeletal changes. The following

drugs were tested (maximum concentration tested in brack-

ets): adenylate cyclase inhibitor SQ 22,536 (10 mM), PKA

inhibitior H-89 (1 mM), adenylate cyclase activator forskolin

(30 mM), cAMP analog dibutyril cAMP (250 mM), PI3K inhibitors

wortmannin (100 nM) and LY294002 (10 mM), p38MAPK inhi-

bitor SB202190 (1 mM), MEK-ERK inhibitor PD98059 (20 mM), JNK

inhibitor SP600125 (40 mM), microtubule disruptors colchicine

(3 mM) and nocodazol (10 mM), intracellular antioxidant glu-

tathione ethyl ester (GSH-MEE, 300 mM), DL- buthionine-S, R-

sulfoximine (300 mM) which depletes cellular glutathione, PKC

inhibitors bisindolylmaleimide I (1 mM) and cheleritrine

Fig. 4 – Effect of Z-VAD-FMK on AZA-1-induced actin cytoskelet

incubated in the presence of Z-VAD-FMK and 10 nM AZA-1 duri

between 25 and 75 nM all through the experiment by repeated a

(C) AZA-1. (D) AZA-1 and Z-VAD-FMK. Representative of three e

(1 mM), PKC activator PMA (100 ng/ml) and caspase inhibitor

Z-VAD-FMK (100 mM). The experiments were performed both

with a preincubation in the presence of the drug before AZA-1

addition or with simultaneous addition of drug and AZA-1. In

the case of Z-VAD-FMK the drug was added every 8–12 h along

the 48 h duration of the experiment, and for wortmannin and

LY294002 the addition was repeated every 5–7 h due to their

short half-lives [23–25]. The concentration of AZA-1 in these

experiments was 10 nM or 50 nM, and the cells were checked

at 48 h for morphological/cytoskeletal changes. Although the

concentration of AZA-1 used in the initial experiments was

50 nM, a lower concentration of 10 nM was also tested in

combination with the inhibitors to ensure that the inhibition

of AZA-1 effects by these drugs was not hampered by a too

high concentration of the toxin. None of the drugs tested had

an effect on morphology or actin cytoskeleton that resembled

the changes induced by AZA-1. Actually, only forskolin,

SP600125, the PI3K inhibitors wortmannin and LY294002,

and microtubule disruptors had observable effects on mor-

phology. Forskolin induced an increase in the length of

neurites (data not shown), SP600125 seemed to increase stress

fibers associated to cell flattening (Fig. 5B) and microtubule

disruptors induced rounding up, brush-like prolongations and

loss of membrane integrity (data not shown). Wortmannin

and LY294002 induced a less pronounced rounding up and

on disarrangement. Human neuroblastoma cells were

ng 48 h. The concentration of Z-VAD-FMK was maintained

dditions of Z-VAD-FMK. (A) DMSO control. (B) Z-VAD-FMK.

xperiments. Bar size 10 mM.

Fig. 5 – Effect of SP600125 on AZA-1-induced actin cytoskeleton disarrangement. Human neuroblastoma cells were

preincubated for 1 h in the presence of 40 mM SP600125 and then incubated for 48 h with 10 nM AZA-1. The concentration of

SP600125 was maintained all through the experiment. (A) DMSO control. (B) SP600125. (C) AZA-1. (D) AZA-1 and SP600125.

Representative of three experiments. Bar size 10 mM.


detachment without loss of neurites (data not shown). When

AZA-1 was present in the incubation medium none of these

drugs reverted AZA-1-induced changes in cytoskeleton mor-

phology. Only data regarding the caspase inhibitor Z-VAD-

FMK and the JNK inhibitor SP600125 are shown in Figs. 4 and 5,

respectively, representing an example of the results obtained

for all the inhibitors tested, except for wortmannin and

LY294002 that exerted an apparent additive effect when toxin

and inhibitor were present together. Z-VAD-FMK did not

inhibit the morphological changes induced by 10 nM (Fig. 4) or

50 nM AZA-1 (data not shown) at 24 or 48 h. The concentration

of Z-VAD-FMK was maintained all through the experiment

between 25 and 75 mM by periodic additions of the inhibitor

due to its short half-life (8 h) and the concentration of DMSO

(carrier) was always lower than 0.2% as recommended by the

commercial source. SP600125 effect on AZA-1-induced mor-

phological/cytoskeletal changes was tested also with 10 and

50 nM concentrations of AZA-1, although only the results

obtained with 10 nM AZA-1 are shown in Fig. 5. AZA-1 treated

cells still rounded up in the presence of 10 mM SP600125

although the effect seemed slightly less pronounced (data not

shown), increasing the dose of SP600125 to 40 mM did not

eliminate AZA-1-induced effects (Fig. 5).

4. Discussion

In a previous study we described the slow kinetics of AZA-1-

induced morphological and cytoskeletal changes in human

neuroblastoma cells [17]. This work demonstrates that AZA-1-

induced morphological changes are irreversible. Moreover,

although a long time is necessary for morphological/cytoske-

letal changes to appear, a continuous exposure of the cells to

the toxin is not necessary. Incubation times as short as 2 min

are enough for morphological changes to appear 24–48 h after

the toxin has been removed. Additionally, contrary to what

happens with okadaic acid (manuscript in preparation), a 100-

fold increase in the concentration of AZA-1 did not speed up

kinetics. Overall, the results point to an irreversible binding of

AZA-1 to its cellular target. They also suggest that AZA-1 target

in the cell has a slow turnover or its turnover is inhibited by the

toxin. Otherwise, the toxin-bound target would be quickly

removed and replaced by a new non-toxin-bound molecule.

This theory would be in agreement with the slow recovery of

AZA-1-induced lesions in mice, with injuries to the intestine

taking several months to recover [15]. The only partial

recovery of membrane potential inhibition by AZA-1 in mouse

spinal cord neuronal networks supports the idea of an


irreversible effect of AZA-1 [22]. The partial recovery of cell

function in these primary, non-dividing cells might be due to

implication of a second target or a higher rate of target

turnover in this cell type, which physiology differs from tumor

proliferating cells in many aspects.

During the process of apoptosis the cell undergoes several

morphological changes that resemble those induced by AZA-1.

One of the mechanisms responsible for apoptotic morphology

is the activation of protease enzymes that chew up proteins

involved in cytoskeleton dynamics, such as caspases, which

activation is considered an early marker of apoptosis [26,27]. In

fact, the inhibition of capases has been shown to inhibit

apoptosis triggered by several means, including pectenotoxin-

induced apoptosis, and in many cell models, among them

neuroblastoma cells [28–33]. AZA-1 induced the activation of

caspases in human neuroblastoma cells. However the kinetics

of caspase activation are slower than the kinetics of

morphology changes, suggesting that caspases are not

responsible for actin cytoskeleton disarrangement. It could

be argued that the sensitivity of the caspase detection

technique was not enough to account for low levels of

activation at earlier time points, but caspase inhibition with

Z-VAD-FMK did not inhibit AZA-1-induced cytoskeletal

changes. Therefore, although AZA-1 toxic effects include

activation of caspases, and thus apoptosis, the activity of these

proteases do not seem responsible for the morphological/

cytoskeletal effects observed after exposure to this toxin. It

would be possible that cytoskeletal changes were responsible

for apoptosis induction, however, as far as we know, apoptosis

could also be a cytoskeleton-independent event. Therefore our

future studies will include caspase activation as second read

out in the search for AZA-1 mechanism of action.

Structure/activity studies using the AZA-1 enantiomer

ABCD-epi-AZA-1 and two fragments of the AZA-1 molecule

containing the ABCDE rings and the FGHI rings, revealed that

none of both fragments induced caspase activation, and that

the stereochemistry of the ABCD rings of the AZA-1 molecule

is irrelevant for AZA-1 toxicity. These results are in agreement

with the previously reported structure–activity relationship

found for cytoskeleton effects [17]. In that study, 15 different

molecules that were modifications of the AZA-1 molecule

were tested for induction of cytoskeletal/morphological

changes. None of the fragments of AZA-1, including the two

fragments tested in this work for caspase activation, induced

any changes of actin cytoskeleton/cell morphology or cyto-

toxicity. The only molecule that induced cytoskeletal changes

was also the stereoisomer ABCD-epi-AZA-1, with similar

effects to AZA-1. These similarities in structure–activity

relationship for cytoskeleton alteration and caspase activation

suggest that AZA-1-induced cell death could be a consequence

of cytoskeleton alterations.

Several inhibitors and activators of signaling pathways

implicated in cytoskeleton and apoptosis regulation were

tested for cytoskeletal/morphological effects in neuroblas-

toma cells. The signaling pathways targeted in this work

included PKA/cAMP, PKC, ERK, p38MAPK, JNK, PI3K, glu-

tathione (intracellular anti-oxidant) and microtubules [34–47].

Previous studies in mouse cerebellar neurons indicated that

AZA-1-induced modifications of intracellular calcium are not

involved in cell death triggering by AZA-1 based on disparities

regarding effective concentration and structure–activity rela-

tionship [48]. Since the concentration dependency and

structure–activity relationship are the same for cytoskeletal

changes in neuroblastoma cells and cell death in cerebellar

neurons, these criteria exclude the involvement of calcium in

cytoskeletal alterations as well, and, therefore, it was not

explored further in this work. Intracellular pH was not

considered as a possible mechanism of action since AZA-1

has been reported not to induce changes of intracellular pH in

neurons or lymphocytes [21,48]. Binding of AZA-1 to G-actin is

also unlikely, as discussed previously [17], based on the lack of

effect of AZA-1 on the levels of G-actin and F-actin.

Unfortunately, the amount of AZA-1 available is too scarce

to perform direct binding studies to G-actin or F-actin in order

to completely rule out this possibility. None of the drugs used

to modulate the above mentioned signaling pathways had

similar effects to AZA-1 or eliminated AZA-1-induced changes

in neuroblastoma cell morphology, suggesting that none of

these signalling routes is a major participant in AZA-1-

induced cytoskeletal effects. Six of the drugs, the PKA activator

forskolin, the microtubule disruptors nocodazole and chol-

chicine, the PI3K inhibitors wortmannin and LY294002, and

the JNK inhibitor SP600125, changed the morphology of

neuroblastama cells. The PKA activator forskolin and micro-

tubule disruptors changed neuroblastoma morphology

accordingly to previously published reports on the role of

PKA in neurite outgrowth [43,44] and apoptosis induction in

neuronal cells [45,49], respectively. The morphological altera-

tions induced by the PI3K inhibitors are also in agreement with

the well-known role of this enzyme in actin cytoskeleton

regulation [38]. Regarding the JNK inhibitor SP600125, it

increased cell flattening and stress fiber spreading by itself,

an effect opposite to AZA-1. Therefore, even though a 10 mM

concentration of the inhibitor seemed to slightly diminish

AZA-1-induced changes in morphology, we cannot conclude

so far that JNK is implicated in AZA-1 mechanism of action,

since it is possible that both the inhibitor and the toxin were

acting in parallel routes converging in cell adhesion regula-

tion. The apparently additive effects of PI3K inhibitors and

AZA-1 on cytoskeleton/morphology are probably a conse-

quence of two mechanisms of cytoskeleton regulation, since

the morphology in the presence of both toxin and inhibitor

looked like a mixture of the morphologies induced by both

drugs separately. A more detailed analysis of JNK and PI3K

signaling is necessary to clarify their possible role in AZA-1

toxicity and will be addressed in our future studies. The

possibilities in the search for the mechanism of action of AZA-

1 are countless. There are many other aspects of cell

physiology that remain to be explored, such as ATP depriva-

tion or protein expression.

In summary, AZA-1 toxicity to human neuroblastoma cell

cytoskeleton is irreversible, and although the alterations of

cytoskeleton morphology appear slowly, a very short exposure

to AZA-1 is enough for toxic activity. These morphological/

cytoskeletal changes precede the activation of caspases, an

early marker of apoptosis. The whole molecule of AZA-1 is

required for caspase activation regardless of the stereochem-

istry of the ABCD rings, as it has been reported previously for

cytoskeleton alterations. The results strongly indicate that

cytoskeletal toxicity is not dependent on caspase activation.


Whether cytoskeletal effects are responsible for AZA-1-

induced cell death is still to be determined. The mechanism

of action of AZA-1 remains unknown although it does not

seem to involve some major signaling pathways such as PKA,

PKC, ERK or p38MAPK.

Acknowledgements

Professor Nicolaou acknowledges financial support for this

work from The Skaggs Institute for Chemical Biology, the

National Institutes of Health (USA), a predoctoral fellowship

from the National Science Foundation (to M.O.F.), and grants

from Amgen and Merck. The spanish work was funded with

grants from the following agencies: Ministerio de Ciencia y

Tecnologıa, Grant Number: SAF2003-08765-C03-02, REN2001-

2959-C04-03, REN2003-06598-C02-01, AGL2004-08268-02-O2/

ALI, INIA CAL01-068. Xunta de Galicia, Spain; PGID-

T99INN26101, PGIDIT03AL26101PR and PGIDIT04TAL261005PR.

Fondode InvestigacionesSanitarias,GrantNumber:FISSREMA-

G03-007. EU VIth Frame Program; Grant Number: IP FOOD-CT-

2004-06988 (BIOCOP), STREP FOOD-CT-2004-514055 (DETEC-

TOX) and CRP 030270-2 (SPIES-DETOX).

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Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells

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