aplidintm induces apoptosis in human cancer cells via glutathione depletion and sustained activation...

Aplidin Induces Apoptosis Through EGFR, Src, JNK and p38MAPK

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Aplidin Induces Apoptosis in Human Cancer Cells via Glutathione Depletion

and Sustained Activation of Epidermal Growth Factor Receptor, Src, Jun N-

Terminal Kinase and p38 Kinase

Ana Cuadrado1,2,*, Luis F. García-Fernández1,2,*, Laura González2, Yajaira Suárez2, Alejandro

Losada1, Victoria Alcaide2, Teresa Martínez2, José María Fernández-Sousa1,

José María Sánchez-Puelles1, and Alberto Muñoz2,3

1Drug Discovery Department, Pharma Mar S.A., E-28760 Tres Cantos, Madrid, Spain;

2Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones

Científicas-Universidad Autónoma de Madrid, E-28029 Madrid, Spain

3Corresponding author: Instituto de Investigaciones Biomédicas "Alberto Sols", Arturo

Duperier, 4, E-28029 Madrid, Spain. Phone: #34-91-585 4640. Fax: #34-91-585 4587

e-mail: [email protected]

* Both authors contributed equally to this work

Running title: Aplidin induces apoptosis through EGFR, Src, JNK and p38MAPK

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 31, 2002 as Manuscript M201010200 by guest on June 14, 2016

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We report that Aplidin , a novel antitumor agent of marine origin presently

undergoing Phase II clinical trials, induces growth arrest and apoptosis in

human MDA-MB-231 breast cancer cells at nanomolar concentrations. Aplidin

induces a specific cellular stress response program including the sustained

activation of the epidermal growth factor receptor (EGFR), the non-receptor

protein tyrosine kinase Src and the serine/threonine kinases Jun N-terminal

kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK). Aplidin -

induced apoptosis is only partially blocked by the pan-caspase inhibitor Z-

VAD-fmk, and is also sensitive to: AG1478, an EGFR inhibitor, PP2, an Src

inhibitor, and SB 203580, an inhibitor of JNK and p38MAPK in MDA-MB-231

cells. Supporting a role for EGFR in Aplidin action, EGFR-deficient mouse

embryo fibroblasts undergo apoptosis upon treatment more slowly than wild-

type EGFR fibroblasts, and also show delayed JNK and reduced p38MAPK

activation. N-acetylcysteine and ebselen, but not other antioxidants such as

diphenylene iodonium, Tiron, catalase, ascorbic acid or vitamin E reduced

EGFR activation by Aplidin . N-acetylcysteine and PP2 also partially inhibited

JNK and p38MAPK activation. The intracellular level of glutathione (GSH)

affects Aplidin action: pretreatment of cells with GSH or N-acetylcysteine

inhibited, whereas GSH depletion caused hyperinduction of EGFR, Src, JNK

and p38MAPK. Remarkably, Aplidin also induced apoptosis and activates

EGFR, JNK and p38MAPK in two cell lines (A-498, ACHN) derived from

human renal cancer, a neoplasia that is highly refractory to chemotherapy.

These data provide a molecular basis for the anticancer activity of Aplidin .

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INTRODUCTION

Aplidin (a cyclic depsipeptide: C57H89N7O15; Mr 1112) is a novel antitumor agent

isolated in 1990 from the Mediterranean tunicate Aplidium albicans (1). In vitro, Aplidin has

potent cytotoxic activity against breast, colon, non-small cell lung and prostate carcinoma and

melanoma cells (2, 3). It has also shown strong antitumor activity in various xenograft models

(4). Aplidin has recently entered Phase II clinical trials in a variety of solid tumors upon

showing promising toxicity and pharmacological properties in Phase I studies (5).

Little is known about the mechanism of action of Aplidin. Early studies indicated that

it exerts an antiproliferative effect through the inhibition of protein synthesis and reduction of

ornithine decarboxylase activity (6, 7). In addition, Aplidin inhibits vascular endothelial factor

secretion and autocrine stimulation in a human leukemic cell line (8).

Many anticancer drugs elicit apoptosis in cancer cells (9-11). Several studies have

reported the activation of one or more members of the mitogen-activated protein kinase (MAPK)

family of intracellular signaling kinases by cytotoxic agents. Among them, the Jun N-terminal

kinase (JNK) and the p38MAPK, and less frequently the extracellular signal-regulated kinases

(ERK1/2) play important roles in these apoptotic processes (12-18). However, JNK and

p38MAPK are not always involved in apoptosis. They also promote proliferation,

differentiation or survival depending on the cell type and external stimulus (19-22).

Interestingly, JNK is also activated by chemopreventive agents such as the isothiocyanates

present in cruciferous vegetables (23) and has been shown to lie upstream of the caspase

proteases that constitute the effector system in the apoptotic pathway (14). However, in other

cases, such as apoptosis induced by doxorubicin or γ-irradiation, caspase activation is

independent of JNK (24).

To study the mechanism of action of Aplidin in human cancer cells we chose the

MDA-MB-231 breast cell line, which contains mutated p53 and ras genes and is highly invasive


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and proliferative. Here we report that Aplidin induces rapid and sustained activation of the

epidermal growth factor receptor (EGFR), the non-receptor tyrosine kinase Src, and JNK and

p38MAPK. SB 203580, an inhibitor of both JNK and p38MAPK in these cells, substantially

inhibits the cytotoxic effect of Aplidin, suggesting an important role of these kinases in

Aplidin action. These effects depend on a decrease in intracellular glutathione (GSH). In

addition, we found that EGFR activation by Aplidin is partially mediated by Src but somehow

abnormal since it does not lead to the same binding of Shc and Grb2 adaptor proteins as that

induced by EGF. In view of the promising effects observed in patients in phase I studies, we

extended the study to two lines, ACHN and A-498 cells (both p53 wild-type), of renal cancer, a

neoplasia highly refractory to chemotherapy. In these cells Aplidin also activates EGFR, JNK

and p38MAPK, but does not affect the high endogenous level of active Src.

EXPERIMENTAL PROCEDURES

Cell lines, Antibodies, and Reagents.MDA-MB-231, ACHN and A-498 cells were

obtained from the American Type Culture Collection (Rockville, USA) and grown in

Dulbecco's modified Eagles' medium supplemented with 10% FCS and 1 mM glutamine (all

from GIBCO-BRL). Wild-type and egfr-/- mouse embryonic fibroblasts were obtained from Dr.

E. Wagner (Institut für Molekulare Pathologie, Vienna, Austria). Aplidin is manufactured by

Pharma Mar S.A. (Madrid, Spain). Stock solutions were freshly prepared in dimethylsulfoxide

and diluted in the cell culture to final concentrations as indicated. Ascorbic acid, diphenelene

iodonium (DPI), Tiron, vitamin E ((+)-α-tocopherol acid succinate), catalase, hydrogen

peroxide (H2O2), glutathione (GSH), N-acetylcysteine (NAC), ebselen (EBS), L-

buthionine,S,R-sulfoximine (BSO), 4´,6-diamino-2-phenylindole (DAPI) were from Sigma

Chemical Co. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2),


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tyrphostin AG1478, SB 203580, and Z-VAD-fmk caspase inhibitor were from Calbiochem.

Human epidermal growth factor was from PreproTech Ltd.

Antibodies used were: anti-JNK1, anti-p38MAPK, anti-ERK, anti-human EGFR, anti-Grb2,

and anti-Src (for western blotting) were from Santa Cruz; anti-Src (for immunoprecipitation)

was generously donated by Dr. J. Brugge; anti-phospho-JNK1, anti-phospho-p38MAPK, anti-

phospho-ERK, anti-phospho(Tyr418)-Src, anti-phospho(Ser348)-Akt, anti-caspase 3 (active

form), and anti-β-actin from New England Biolabs; anti-PDGFR was from Upstate

Biotecnology International; anti-phosphotyrosine and anti-Shc were from Transduction

Laboratories; and anti-phosphotyrosine specific-EGFR (Tyr1068, Tyr1086, Tyr1148 and Tyr1173)

from BioSource International.

Nuclear staining assays. After treatment, cells were washed once in PBS and fixed

in a solution of chilled methanol:acetic acid (1:1) for 2 min. The fixed cells were washed in

PBS, placed on slides and stained with 2 µg/ml DAPI for 15 min. Excess dye was washed off

with PBS. Nuclear morphology was observed under a fluorescence microscope (Zeiss

Axiophot).

Flow Cytometry Analysis. Cells after different treatments with Aplidin and/or

inhibitors were stained with propidium iodide (PI) and analyzed by flow cytometry (FACScan,

Becton Dickinson). For staining, one million cells were harvested, washed in PBS and then

fixed in 70% ethanol. Fixed cells were treated with DNAse-free RNAse for 30 min at 37 ºC,

washed in PBS, centrifuged, and incubated in PBS-containing PI (25 µg/ml; Sigma). Forward

light scatter characteristics were used to exclude the cell debris from the analysis. Apoptotic

cells were determined by their hypochromic, subdiploid staining profiles. To estimate early

apoptotic cells Annexin V Alexa Fluor 488 conjugate (Molecular Probes) was used together

with PI dead cell counterstain following manufacturer´s recommendations.

DNA Synthesis Measurements. Thirty-five thousand cells were seeded in 24-well

dishes (Nunc). One day later, cells were treated or not with the Aplidin in normal growth

medium and pulsed with 5 µCi/ml [3H]-thymidine (Amersham-Pharmacia Biotech) for 30 min


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at the indicated times post-treatment. At the end of the labeling period, the medium was

removed and the cells were rinsed twice in PBS and fixed with chilled 10% trichloracetic acid

for 10 min. Trichloracetic acid was then removed and the monolayers were washed in ethanol

and air-dried at room temperature for 20 min. Thereafter, precipitated macromolecules were

dissolved in 500 µl of 0.5 N NaOH-0.1% SDS and 450 µl of each sample was diluted in 5 ml

of scintillation solution OptiPhase HighSafe (Wallac Scintillation Products). Radioactivity was

measured on a 1209 RackBeta counter (LKB Wallac).

Crystal violet staining method. To estimate cell mass, medium was removed and

24-well dishes were washed in PBS, fixed with 1% glutaraldehyde for 15 min, washed again in

PBS twice and stained with 100 µl 0.1% aqueous crystal violet for 20 min. Dishes were rinsed

four times in tap water and allowed to dry. One hundred µl 10% acetic acid was added and the

content of each well was mixed before reading A595.

Immunoprecipitation and Western Blot Analysis. To study the effect of Aplidin on

the activity of different kinases (Src, p38MAPK, JNK, Erk, Akt) cells were preincubated for

24 h in serum-free medium. For immunoprecipitation cells were lysed in modified RIPA buffer

(50 mM Hepes pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 1%

Triton X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 25 mM β-glycerolphosphate,

100 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF). Lysates were precleared

by incubation with protein G-Sepharose at 4ºC for 30 min and then incubated overnight with

the corresponding antibody (1 µg/ml). After 1 h incubation with protein G-sepharose, immune

complexes were washed three times in the same RIPA buffer lacking deoxycholic acid and SDS

and three times with HNTG (50 mM Hepes pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton

X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 25 mM β-glycerolphosphate, 100

mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF) and electrophoresed in 8%

(EGFR, PDGFR) or 12% (Src) acrylamide gels. For western blot analysis cell protein extracts


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were prepared following standard procedures (25). Protein extracts were electrophoresed in 8%

or 15% polyacrylamide gels and transferred to nylon (Immobilon P, Millipore) membranes. The

filters were washed, blocked with 5% BSA in Tris-buffered saline (25 mM Tris pH 7.4, 136

mM NaCl, 2.6 mM KCl, 0.5% Tween-20), and incubated overnight at 4ºC with the appropriate

antibody (1:1000 dilution). Blots were washed three times for 10 min in PBS + 0.1% Tween-

20 and incubated with HRP-conjugated anti-rabbit, anti-mouse, or anti-goat antibodies for 1 h

at room temperature. Blots were developed by a peroxidase reaction using the ECL detection

system (Amersham-Pharmacia Biotech).


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RESULTS

Aplidin induces apoptosis of MDA-MB-231 breast cancer cells. Aplidin showed

high activity against MDA-MB-231 cells (Fig. 1A), with an antiproliferative IC50 of 5 nM at 48

h post-treatment (not shown). This concentration is pharmacologically relevant as the circulating

plasma levels of Aplidin in patients exposed to the maximum tolerated dose in phase I clinical

studies was 7.1 nM (26). Dose-dependent inhibition of DNA synthesis was detected 1-3 h after

drug addition (Fig. 1B). By flow cytometry analysis Aplidin was found to induce the

subdiploid profile which is a hallmark of apoptosis (Fig. 1C). At 24 h, 20-25% apoptotic cells

were found in cultures growing in normal medium treated with 50-500 nM Aplidin, and

around 10% with 5 nM. In line with this, Aplidin-treated cells showed time- and dose-

dependent nuclei condensation and fragmentation in comparison to the homogeneous staining of

untreated cells as assessed by DAPI staining (Fig. 1D top) and the generation of the active form

of caspase 3 (Fig. 1D bottom). When added simultaneously to the drug, Z-VAD-fmk, a pan-

caspase inhibitor, decreased the cytotoxic activity of Aplidin by around 50% as assessed by

crystal violet staining (not shown). This indicates that Aplidin–induced apoptosis is mediated

by caspase-dependent and –independent mechanisms. The cytotoxic action of Aplidin is

triggered rapidly, as a pulse treatment of five minutes (using 500 nM) gave maximal

antiproliferative effect at 48 h (not shown).

Aplidin -induced apoptosis relies on a sustained JNK and p38MAPK activation.

Doses 10-100-fold higher than the antiproliferative IC50 were used to identify the primary

targets and early mechanism of action of Aplidin. To examine the role of MAPKs in

Aplidin-induced apoptosis, we studied the effects of Aplidin on the activity of ERK1/2,

JNK, and p38MAPK using antibodies recognizing specifically the active phosphorylated


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forms. Aplidin caused rapid, strong, and sustained activation of p38MAPK and JNK without

altering their concentrations (Fig. 2A). The same result was obtained in immune complex kinase

assays (not shown). Addition of Aplidin in vitro to immune complexes from untreated cells

did not affect kinase activity (not shown), suggesting that p38MAPK/JNK activation in vivo is

due to effects on their at least eleven upstream regulators (22). In agreement with the expression

of an activated K-ras oncogene (27) MDA-MB-231 cells displayed a high basal level of

phosphorylated ERK1/2 in unstimulated conditions, that was mostly unaffected by Aplidin

treatment (Fig. 2A). Likewise, Aplidin did not change the cellular content of phosphorylated

Akt, an enzyme that prevents cell death by multiple apoptosis inducers (28, review) (Fig. 2A).

To examine the importance of these effects in the induction of apoptosis we used SB

203580, a pyridinyl imidazol that inhibits some p38MAPK isoforms (29, 30). However,

control experiments showed that in MDA-MB-231 cells SB 203580 inhibits both, JNK and

p38MAPK, at 5-30 µM (Fig. 2B and not shown). This correlated with the inhibition of

Aplidin-induced apoptosis: by flow cytometry SB 203580 (30 µM) decreased the number of

apoptotic cells from 41% to 16% after 24 h of Aplidin treatment in serum-free medium (Fig.

2C), an effect clearly visible by phase-contrast microscopy (Fig. 2D). These data indicated that

the activation of JNK and/or p38MAPK plays a critical role in the induction of MDA-MB-231

cell apoptosis by Aplidin. Moreover, activation of these kinases was unaffected by Z-VAD-

fmk (not shown), suggesting that these events lie upstream of caspase activation in the

apoptosis cascade.

Aplidin induces Src-dependent EGFR phosphorylation. Since JNK activation by

some stimuli involves Src-mediated EGFR phosphorylation (31), we studied whether Aplidin

could have this mode of action. We found that Aplidin caused EGFR phosphorylation, which

was partially blocked by pretreatment with PP2, a specific inhibitor of Src (32, 33) (Fig. 3A).


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Aplidin induced a more sustained EGFR phosphorylation than EGF (Fig. 3B top): it peaked

at 1-4 h and remained above the baseline for 24 h (Fig. 3B middle). Accordingly, the activation-

linked down-regulation of EGFR content was delayed in Aplidin-treated cells with respect to

EGF-treated cells (Fig. 3B middle). Combined treatment of MDA-MB-231 cells with EGF and

Aplidin resulted in cellular toxicity and persistent EGFR phosphorylation indicating a dominant

and probably aberrant effect of Aplidin (Fig. 3B bottom). This was further suggested by the

analysis of the binding of the adaptor proteins Grb2 and Shc to EGFR. In contrast to what

happened upon EGF addition, EGFR activation by Aplidin did not cause binding of Grb2 or

Shc to the receptor (Fig. 3C). Moreover, in cells treated with both agents Grb2 binding was

also inhibited, as was binding of Shc at early times (2 and 30 min post-treatment) although it

strongly increased later (60 min) (Fig. 3C).

EGFR is subjected to inhibitory and activating phosphorylation of specific residues by

several kinases. Five sites of in vivo autophosphorylation have been identified in the EGFR:

Tyr992, Tyr1068, Tyr1086, Tyr1148 and Tyr1173 (34-36). To make sure that Aplidin has a

stimulatory effect on EGFR we used residue-specific anti-phosphotyrosine antibodies. At least

four out of these five aminoacids became phosphorylated upon Aplidin addition (Fig. 3D). To

assess the specificity of EGFR activation, the effect of Aplidin on platelet-derived growth

factor receptor (PDGFR) was studied in NIH 3T3 mouse fibroblasts. Aplidin did not induce

phosphorylation of this receptor (Fig. 3E). These cells were sensitive to Aplidin although they

lack EGFR expression, indicating that EGFR transactivation is not required for Aplidin-induced

apoptosis. Additionally, given that PP2 significantly reduces Aplidin-induced EGFR

phosphorylation and that Src transactivates EGFR in some cell systems (31, 37, 38), we

studied the effect of Aplidin on Src expression and activity. By means of an antibody which

specifically recognizes the activation-linked phospho-Tyr418 of Src, Aplidin rapidly activated


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Src without altering the amount of protein (Fig. 3F). Since MDA-MB-231 cells express Src and

Fyn, but not Yes (not shown), which are the three ubiquitously expressed members of the Src-

kinase family that have at least partially overlapping functions (39), the possibility that Aplidin

may also affect Fyn cannot be ruled out.

The role of EGFR activation in Aplidin action was studied in embryonic fibroblasts

from EGFR-deficient mice (egfr-/- mef). Both in the presence and in the absence of serum, egfr-

/- mef underwent apoptosis with a delayed kinetics in comparison to wild-type MEF (Fig. 4A

and not shown). At late times after treatment, however, no differences were found in cell

proliferation or viability (not shown). Consistent with this, activation of p38MAPK and JNK

egfr-/- mef was reduced and delayed, respectively, as compared to wild-type mef (Fig. 4B,

upper panels). To examine the contribution of EGFR and Src to the activation of

p38MAPK/JNK by Aplidin we used PP2. Pretreatment with PP2 and also with GSH caused

only partial reduction of p38MAPK and JNK activation in egfr-/- and wild-type mef. SB

203580 had a stronger effect than PP2 and GSH in egfr-/- cells. In addition, we studied the

contribution of the activation of Src and p38MAPK/JNK to the cytotoxic action of Aplidin by

measuring the viability of MDA-MB-231 cells and wild-type and egfr-/- mef treated with the

drug in the presence of PP2 or SB 203580. In all three cell types SB 203580 significantly

inhibited Aplidin action. In contrast, PP2 was only effective in MDA-MB-231 cells but not in

mef (Fig. 4C). In agreement with the differential activation of p38MAPK/JNK, these

experiments also confirmed that egfr-/- mef are less sensitive to the drug than wild-type mef.

Together, these results indicate that activation of EGFR, Src and p38MAPK/JNK is involved in

Aplidin action in MDA-MB-231 cells, whereas only EGFR and p38MAPK/JNK, but not Src,

seem to be involved in the cytotoxic effect of Aplidin in mouse embryo fibroblasts. EGFR

activation participates in, but is not sufficient for the induction of apoptosis by Aplidin.


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Glutathione depletion is critical for the activation of Src, EGFR, JNK and p38MAPK by

Aplidin. Reactive oxygen species (ROS) mediate JNK activation by pro-inflammatory

cytokines (interleukin-1, tumor necrosis factor-α) (40), an effect that in the case of H2O2

requires Src-dependent EGFR transactivation (31). These data led us to investigate the putative

role of ROS in Aplidin action. As shown in Fig. 5A and B (and data not shown), pretreatment

of MDA-MB-231 cells with various antioxidants such as ascorbic acid, DPI (an inhibitor of

flavin-containing enzymes such as NAD(P)H oxidase), Tiron (a scavenger of superoxide ions)

and vitamin E, or the addition of catalase (H2O2 degrading enzyme) did not affect the induction

of EGFR phosphorylation by Aplidin. In contrast, two compounds that raised the cellular

level of GSH: NAC (a GSH precursor) and EBS (a GSH peroxidase mimetic) reduced EGFR

phosphorylation by Aplidin (Fig. 5C). Confirming the involvement of GSH in Aplidin

action, treatment of cells with exogenous GSH diminished, while BSO (a specific inhibitor of

γ-glutamylcysteine synthetase) increased Aplidin-induced EGFR phosphorylation (Fig. 5C).

None of the agents tested (NAC, EBS, DPI, Tiron) modified EGFR activation by its ligand

EGF (Fig. 5D).

Next, we analyzed the possible link between GSH content, Src and EGFR and the

activation of JNK and p38MAPK. Exogenously added GSH and NAC, but not BSO, reduced

JNK and p38MAPK activation by Aplidin (Fig. 6A). Likewise, GSH also blocked Src

activation by Aplidin, while BSO had no effect (Fig. 6B). In addition, PP2 and tyrphostin

AG1478 (an EGFR inhibitor) also inhibited the activation of these kinases by Aplidin (Fig.

6C). Together, these data indicate that the depletion of GSH activates Src, and that activation of

Src and EGFR is partially responsible for the activation of JNK and p38MAPK. EGF alone did

not induce p38MAPK and it induced JNK only weakly, and did not affect the activation of

these two kinases by Aplidin (Fig. 6D).

Aplidin induces cytotoxicity in renal cancer cells. To examine whether the


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mechanism of action of Aplidin in MDA-MB-231 cells could be extrapolated to human renal

cancer cells we used the A-498 and ACHN cell lines. Both were sensitive to the

antiproliferative (IC50= 5 and 15 nM, respectively) and cytotoxic action of Aplidin (Fig.

7A). As in MDA-MB-231, we found that Aplidin induced EGFR phosphorylation in A-498

cells (Fig. 7B). The effect on Src was also investigated. In contrast to breast cancer cells,

both A-498 and ACHN cells showed a high level of Src activity which was not affected by

Aplidin (Fig. 7C). Since the drug activated JNK and p38MAPK in A-498 and ACHN cells

(Fig. 7D), together these results indicate that in these two renal cancer cell lines the activation

of these kinases by Aplidin does not require further Src activation. SB 203580 reduced the

activation of both kinases (Fig. 7D). The contribution of EGFR, p38MAPK and JNK

activation, and also of Src to the cytotoxic effect of Aplidin in A-498 and ACHN cells was

studied by flow cytometry using AG1478, PP2 and SB 203580. In ACHN cells all three

inhibitors reduced the population of hypodiploid cells, with the following range of potency:

SB 203580>AG1478>PP2 (Table I). Double AG1478 + SB 203580 treatment was more

effective than either agent alone, while combined treatments including PP2 inhibited also

Aplidin but showed high toxicity in untreated cells. A possible explanation may be that the

inhibition of the basal activity of more than one of the target enzymes of these agents is

deleterious, whereas cells treated with Aplidin and combinations of inhibitors may display

kinase activities similar to untreated cells. A-498 cells were very sensitive to the absence of

serum (not shown) and, unexpectedly, in low serum neither of the inhibitors reduced the

induction of apoptosis by Aplidin (Table I). In these cells only the double AG1478 + SB

203580 treatment had a small inhibitory effect, while the triple AG1478 + PP2 + SB203580

treatment had a higher effect though it was toxic in the absence of Aplidin.


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DISCUSSION

We have examined the effect of the novel antitumor agent Aplidin in human breast and

renal cancer cells. Aplidin shows cytotoxicity at pharmacologically relevant concentrations

linked to both inhibition of cell proliferation and apoptosis. It induces apoptosis via caspase-

dependent and –independent mechanisms irrespective of the cellular p53 status through the

sustained activation of the serine/threonine kinases JNK and p38MAPK. These events are

triggered rapidly, in part by the induction of GSH depletion and Src tyrosine kinase.

Aplidin activates EGFR by a mechanism that is partly dependent on Src activation.

Several findings indicate that EGFR activation by Aplidin is aberrant: first, it is weaker and

more prolonged than that induced by EGF; second, it is accompanied by a delay in the down-

regulation of the receptor; and third, it does not induce binding to the receptor of adaptor

proteins such as Grb2 and Shc, suggesting differences in signaling with respect to EGF. Our

data obtained in human MDA-MB-231 and renal ACHN cancer cells and in mouse fibroblasts

indicate that EGFR activation is involved in but not sufficient for Aplidin-induced apoptosis.

Apart from its common physiological ligands EGF and TGF-α, multiple physiological and non-

physiological stimuli and agents transactivate EGFR, including redox stress, activation of

various G-protein coupled receptors and voltage-gated Ca2+ channels, cytokine receptors,

osmotic stress, and UV- and γ-radiation (41, 42). The intensity and duration of EGFR

transactivation, in addition to substrate availability and other signals, is critical for the cellular

response, either proliferation or alternative pathways. Remarkably, Aplidin has a dominant

effect on EGF, altering the binding of adaptor proteins and inducing stronger activation of JNK

and p38MAPK and apoptosis also in EGF-treated MDA-MB-231 cells. Moreover, these cells

harbour mutated p53 and ras genes and high levels of activated ERK and Akt, two enzymes that


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promote cell survival and proliferation. Together, these data emphasize the potent cytotoxic

activity of Aplidin.

Src has been proposed to transactivate EGFR upon several stimuli such as oxidative

stress and G-protein-coupled receptor activation, in the latter case by forming a complex with

Pyk2 that directly phosphorylates EGFR (38). In other systems EGFR phosphorylation by Src

results in hyperactivation of receptor kinase activity (37, 43) and in the enhancement of the

mitogenic response of cells to EGF (44). Since Src binds to and is substrate of EGFR (45), an

activation loop resulting from this mutual catalytic regulation between both kinases (46) can

amplify Aplidin signal causing the abnormally long activation and half-life of EGFR. To what

extent this activation loop is responsible for the sustained activation of JNK and p38MAPK is

unknown, but the partial inhibition by tyrphostin AG1478 supports this idea. Though direct

binding of Aplidin to EGFR or indirect induction of receptor dimerization cannot be ruled out,

our data support that Aplidin causes EGFR activation through the previous activation of Src.

In line with our data showing the activation of Src by Aplidin, deregulation of Src activity is

involved in the caspase-independent apoptosis induced by the adenoviral early region 4 open

reading frame 4 protein (47). Thus, like c-Myc (48, review), c-Src may transduce either

proliferative or apoptotic signals.

Aplidin action is inhibited by NAC, a potent antioxidant which can be a source of SH

metabolites, stimulate GSH synthesis, enhance glutathione-S-transferase activity, promote

detoxification and act directly on ROS (49). Together with the inhibitory effects of exogenous

GSH and the stimulation by BSO of EGFR and Src activation by Aplidin, and the activation

of Src by ROS in many cell systems, these results strongly suggest that the activation of Src by

Aplidin depends on the depletion of cellular GSH. NAC has been found to inhibit receptor

kinase signaling in many cell systems, suppressing the activation of ERK by H2O2 (50), of

ERK and JNK by interleukin-1 (51), and of ERK, JNK and p38MAPK by arsenite (52).

Therefore, Aplidin might act by inducing oxidative stress at the plasma membrane, leading to


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a reduction in GSH level and Src activation. Alternatively, Aplidin may directly modulate

GSH synthesis and/or metabolism. Early GSH depletion is suggestive of an oxidative process

and renders the cells more sensitive or precedes apoptotic cell death induced by various agents

(53 and refs. therein), though this is not the case for others such as camptothecin or etoposide

(54). GSH regulates JNK and p38MAPK (55). Similarly to our results, the level of intracellular

GSH is a critical regulator for the induction of JNK and p38MAPK by alkylating agents such as

methyl methanesulfonate and N-methyl-N´-nitro-N-nitrosoguanidine (56).

JNK is involved in many cellular responses such as proliferation, differentiation and

apoptosis. It is thought that the duration of its activation may be crucial in the signaling

decision: sustained but not transient JNK activation participates in the apoptosis induced by

several stress stimuli (12, 57, 58). This supports that the sustained JNK activation reported in

this work is crucial for the pro-apoptotic effect of Aplidin. Like JNK, p38MAPK is also

involved in the apoptotic response of many cells to cytotoxic agents (16, 59). Our results show

that activation of both p38MAPK and JNK by Aplidin depends in part on Src activation, as

does that caused by changes in the redox status in some systems (33).

Data obtained using inhibitors indicate that in human breast cancer cells activation of

EGFR, Src and p38MAPK/JNK are involved in Aplidin action, while Src activation is

dispensable in mouse embryo fibroblasts. Compared to breast cells, the inhibitory effect of

AG1478, PP2 and SB203580 is variable in human renal cancer cells: similar in ACHN but

lower in A-498 cells, suggesting that other signaling pathways may be implicated in Aplidin

action in this cell line. Therefore, Aplidin-induced apoptosis in carcinoma cells is partly the

result of Src activation, perhaps in combination with additional effects of GSH depletion, which

leads to the sustained activation of JNK and p38MAPK. Since NAC and PP2 inhibit but do not

completely block JNK/p38MAPK activation by Aplidin and are cytotoxic in long incubations,

the contribution of GSH depletion and Src activation cannot be precisely determined, and other

upstream regulators of JNK/p38MAPK might be involved in Aplidin action. The lack of


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appropriate, specific inhibitors of JNK or p38MAPK has hampered the elucidation of the role

of each individual kinase.

Several anticancer drugs in clinical use modulate JNK activity. JNK mediates the

apoptosis induced by DNA-damaging drugs such as etoposide (VP-16) and camptothecin in

human myeloid leukemia cells (14) and of vinblastine in KB3 carcinoma cells (18). In MDA-

MB-231 cells, paclitaxel-induced apoptosis is mediated by the induction of JNK, which causes

inactivation by phosphorylation of the antiapoptotic Bcl-2 protein (17). Taxol has also been

shown to increase p38MAPK, ERK, and to lesser extent JNK activity in human breast cancer

cells (59). Strikingly, tamoxifen, a partial agonist/antagonist of the estrogen receptor which has

a cytostatic action by inhibiting estrogen action, also has a cytotoxic effect in the estrogen

receptor-negative MDA-MB-231 and BT-20 cell lines via the induction of apoptosis (61). This

effect is mediated by JNK activation, and inhibited by vitamin E but not by NAC or GSH (60).

In HepG2 hepatoma cells, 5-fluorouracil-induced apoptosis is mediated by JNK activation (61).

Also, cisplatin induces apoptosis through both JNK and p38MAPK (16).

In summary, we report that Aplidin induces apoptosis of human breast and renal

cancer cells via the sustained activation of the signaling kinases JNK and p38MAPK. We show

that in MDA-MB-231 cells this is in part a consequence of Src activation, which is preceded by

cellular GSH depletion. In addition, Aplidin causes aberrant EGFR activation, which is also

involved in the activation of p38MAPK and JNK in MDA-MB-231 cells and mouse fibroblasts,

and in the cytotoxic effect in the latter and in renal ACHN cells. To establish the contribution of

the abnormal EGFR activation to the anticancer activity of Aplidin will require to correlate

EGFR expression and tumor sensitivity in patients. Our results indicate that there must be

additional Aplidin targets leading to JNK/p38MAPK activation. In view of the promising

clinical results of Aplidin, the identification of these targets and also the elucidation of the

signaling from GSH, EGFR and Src to JNK and p38MAPK merit further investigation.


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Acknowledgements. We thank Dr. E. Wagner for providing us with the wild-type and

egfr-/- mouse embryonic fibroblasts, Dr. J. Martín for his help with the Src experiments and

Dr. J. Brugge for the anti-Src antibody. L.G. and V.A. were in part supported respectively

by a postdoctoral fellowship from Consejo Superior de Investigaciones Científicas and a

predoctoral fellowship from Ministerio de Educación y Cultura of Spain. We are also grateful

to Robin Rycroft for his valuable assistance in the preparation of the English manuscript.

This work was supported by Grant SAF2001-2291 from Plan Nacional de Investigación y

Desarrollo of Spain.

The abbreviations used are: APL: Aplidin; BSA: Bovine Serum Albumin; BSO: L-

buthionine,S,R-sulfoximine; DAPI: 4´,6-diamino-2-phenylindole; DPI: diphenelene

iodinium; EBS: ebselen; EGF: epidermal growth factor; EGFR: epidermal growth factor

receptor; ERK: extracellular signal-regulated kinases; GSH: glutathione; H2O2: hydrogen

peroxide; JNK: Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; NAC: N-

acetylcysteine; PDGF: platelet-derived growth factor; PDGFR: platelet-derived growth factor

receptor; PI, propidium iodide; PP2: 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-

D]pyrimidine


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FIGURE LEGENDS

FIG. 1. Effect of Aplidin on MDA-MB-231 cell growth and viability. A, Phase-

contrast micrographs of cells treated with 50 nM Aplidin (APL) or vehicle (Control) for 24 h.

Scale bar, 25 µM; B, Dose-dependent kinetics of the inhibition of DNA synthesis by Aplidin.

DNA synthesis was calculated by measuring thymidine incorporation as described in

Experimental Procedures at the indicated times after treatment with 5 nM (circles), 50 nM

(triangles), or 500 nM (squares) Aplidin; C, Flow cytometry analysis of DNA content in

cultures incubated in the absence (Control) or presence of the indicated doses of Aplidin in

normal growth medium for 24 h. Percentages of apoptotic cells corresponding to the subdiploid

population are shown, D top, Fluorescence microscope image of cultures treated with vehicle

(Control) or Aplidin (500 nM) for 24 h after nuclear staining with DAPI. Arrows indicate

cells at an advanced stage of nuclear condensation and degradation. Bottom, western blot

analysis showing the activation of caspase 3 upon Aplidin treatment. An antibody which

specifically recognizes the active, truncated caspase 3 polypeptide was used. As control, filters

were reprobed with an anti-β-actin antibody.

FIG. 2. Aplidin induces apoptosis of MDA-MB-231 cells through the sustained

activation of p38MAPK and JNK. A, Levels of activated p38MAPK, JNK, ERK, or Akt

in cells treated with Aplidin (APL, 500 nM) for the indicated times or left untreated (C) were

measured by western blot using phospho-specific antibodies (P-p38, P-JNK, P-ERK, P-Akt).

Antibodies against total protein (p38, JNK) were used to rule out effects at the expression level.

B, SB 203580 inhibits p38MAPK and JNK activation by Aplidin. Cells were pretreated with

SB 203580 (30 µM) or vehicle (C) for 1 h before Aplidin addition as indicated. Western blot


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analysis was as above. C, Flow cytometry analysis showing that Aplidin-induced apoptosis is

inhibited by SB 203580. Cells were treated with vehicle (Control), Aplidin (500 nM), SB

203580 (30 µm) or Aplidin plus SB 203580 (same doses) for 24 h in serum-free medium. D,

Phase-contrast micrographs of cultures treated for 48 h with vehicle, Aplidin, SB 203580 or

Aplidin plus SB 203580 as in C. Scale bar, 35 µM. Values shown correspond to a

representative experiment. Those shown in A and B were performed three times and those in C

and D twice.

FIG. 3. Aplidin induces Src-dependent EGFR phosphorylation. A, MDA-MB-231

cells were either pretreated or not for 1 h with PP2 (25 µM) and then treated with EGF (20

ng/ml, 5 min) or Aplidin (500 nM, 1 h) as indicated. Phosphorylated EGFR levels were

analyzed by immunoprecipitation with anti-EGFR antibody followed by western blotting using

anti-phosphotyrosine antibody. Total EGFR levels were measured by reprobing the filters with

anti-EGFR antibody. B, Time course of induction of EGFR phosphorylation by EGF (20

ng/ml; top), Aplidin (500 nM; middle) or their combination (same doses; bottom) analyzed as

above. C, Time-course of Grb2 and Shc binding to EGFR upon treatment with EGF (20

ng/ml), Aplidin (500 nM), or their combination. Binding was analyzed by

immunoprecipitation with anti-EGFR antibody followed by western blotting using anti-Grb2 or

anti-Shc antibodies. Western blotting using these antibodies was done to estimate total Grb2

and Shc content. D, Cells were treated for the indicated times with either EGF (20 ng/ml) or

Aplidin (500 nM). Western blots (20 µg/lane) were performed using each of the four residue

specific anti-phosphotyrosine antibodies shown. E, PDGFR is not phosphorylated upon

Aplidin treatment. NIH 3T3 cells were treated with Aplidin (500 nM) or platelet-derived

growth factor (PDGF, 10 ng/ml) for the indicated times. Phosphorylated and total PDGFR


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were measured by immunoprecipitation and western blot using anti-PDGFR and anti-

phosphotyrosine antibodies. F, Aplidin induces a rapid and sustained Src phosphorylation.

Cells were treated with vehicle (Control) or Aplidin (500 nM) for the indicated times, and the

amount of activation-linked phosphorylated Src was measured by immunoprecipitation with an

anti-Src antibody followed by western blotting using an specific anti-phospho-Tyr418 antibody

as described in Experimental Procedures. The amount of total Src in the immunoprecipitates is

shown below. Results shown correspond to a representative experiment. Those shown in A

and B were performed three times and those in C-F twice.

FIG. 4. Involvement of EGFR, Src and p38MAPK/JNK in Aplidin action. A,

EGFR is involved in Aplidin-induced apoptosis. Dot-plot diagrams of Annexin V Alexa Fluor

488 conjugate/propidium iodide (PI) flow cytometry of egfr-/- and wild-type mef after treatment

with Aplidin. Cells were incubated for 5 h in the absence (C) or presence of 500 nM

Aplidin (APL) in serum-free medium. The cells were labeled with annexin V- Alexa Fluor

488 conjugate and PI and analyzed by flow cytometry. The lower left quadrants of each panel

show the viable cells (Alexa Fluor 488-/PI-), the upper right quadrants contain the non-viable,

necrotic and/or late apoptotic cells (Alexa Fluor 488+/PI+), and the lower right quadrants

represent the early apoptotic cells (Alexa Fluor 488+/PI-). The inserts in each quadrant represent

the percentage of cells. One representative experiment out of two, which gave similar results, is

shown. B top, Levels of activated p38MAPK and JNK in egfr-/- and wild-type mef treated with

Aplidin (500 nM) for the indicated times or left untreated (C) were measured by western blot

using phospho-specific antibodies as described in legend to Fig. 2. Antibodies against total

p38MAPK or JNK were used for control. To reduce basal enzyme activities cells were

incubated overnight in serum-free medium. Three independent experiments were performed.

Values from a representative experiment are shown. Bottom, Effect of SB 203580, PP2 and

GSH on p38MAPK and JNK activation by Aplidin. Cells were pretreated with SB 203580


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(30 µM), PP2 (25 µM), GSH (15 mM) or vehicle (C) for 1 h before Aplidin addition (500

nM, 1 h) as indicated. Conditions were as reported above. C, Inhibition of Aplidin

cytotoxicity in human MDA-MB-231 cells and wild-type and egfr-/- mef by SB 203580 (30 µM)

or PP2 (25 µM). Cultures were pretreated with the inhibitors for 2 h before Aplidin (500 nM)

addition, and the total cell mass in the cultures was measured 16 h (MDA-MB-231 and egfr-/-

fibroblasts) or 4 h (wild-type fibroblasts) later by the crystal violet staining method. Mean

values and standard deviations referred to those obtained in Aplidin-untreated controls are

shown; ***, p<0.001.

FIG. 5. Aplidin -induced EGFR phosphorylation relies on cellular GSH

depletion. Phosphorylated- and total-EGFR were measured by immunoprecipitation and

western blotting as described in legend to Fig. 3. A, Some antioxidants (NAC, 10 mM; EBS,

40 µM) but not others (DPI, 10 µM; Tiron, 10 mM) inhibit Aplidin-induced EGFR

phosphorylation. Where indicated, MDA-MB-231 cells were pretreated with the antioxidants 1

h before addition of Aplidin, and extracts were prepared 1 h later. B, Exogenous catalase does

not affect EGFR phosphorylation by Aplidin. Cells were incubated with catalase (1,000

U/ml) for 1 h as indicated before Aplidin treatment. As control we used cells treated with

H2O2 (10 mM) plus or minus catalase pretreatment. C, Cellular GSH content modulates EGFR

phosphorylation by Aplidin. Treatment of cells with exogenous GSH (15 mM) or BSO (1

mM) 1 h or 24 h respectively before Aplidin had opposite effects on the induction of EGFR

phosphorylation. In A-C Aplidin was used at 500 nM for 1 h. D, Antioxidants did not affect

EGF-induced EGFR phosphorylation. Cells were pretreated with DPI, Tir, NAC, or EBS for

1 h at the doses indicated above before addition of EGF (20 ng/ml, 10 min). Results shown


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correspond to a representative experiment. Those shown in A and C were performed three times

and those in B and D twice.

FIG. 6. Sustained activation of p38MAPK and JNK by Aplidin is partially

dependent on GSH depletion and Src and EGFR activation. A, Intracellular GSH

level modulates the activation of p38MAPK and JNK by Aplidin. MDA-MB-231 cells were

treated with the indicated agents that modulate GSH levels at the doses indicated in legend to

Fig. 4 for 1 h (GSH, NAC) or 24 h (BSO), and thereafter were treated with Aplidin (500 nM)

or left untreated. Levels of activated p38MAPK or JNK were measured by western blot using

phosphospecific antibodies as described in legend to Fig. 2. Antibodies against total p38MAPK

or JNK were used for control. Three independent experiments were done. Values from a

representative experiment are shown. B, Src activation by Aplidin depends on GSH

depletion. Cells were treated with exogenous GSH (15 mM) or BSO (1 mM) for 1 h or 24 h

respectively and thereafter treated with Aplidin (500 nM). Activated Src was measured 45 min

later by western blot using a specific anti-phospho-Tyr418 antibody. Antibody against total Src

was used for control. C, Inhibition of Src and EGFR reduces p38MAPK and JNK activation

by Aplidin. Cells were pretreated with PP2 (25 µM) or AG1478 (20 µM) before addition of

Aplidin (500 nM). Phospho-p38MAPK and phospho-JNK were measured as above in

extracts prepared 1 h after Aplidin treatment. The experiment was performed three times.

Values from a representative experiment are shown. D, Aplidin has a dominant effect over

EGF on p38MAPK and JNK activity. Cells were treated either with EGF (20 ng/ml), Aplidin

(500 nM), or their combination for 1 h, and the levels of phospho-p38MAPK and phospho-

JNK were measured as above. Two independent experiments gave the same result.


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FIG. 7. Human renal cancer cells are sensitive to Aplidin . A, Phase-contrast

micrographs of A-498 and ACHN cell cultures treated with vehicle (Control) or Aplidin

(APL) for 24 h in normal growth medium. Scale bar, 40 µM. B, Time-course of the induction

of EGFR phosphorylation by Aplidin in A-498 cells. Total and activated EGFR were

measured by immunoprecipitation and western blot as described in legend to Fig. 3. C, Effect

of Aplidin on Src activity in A-498 (top) and ACHN (bottom) cells. Total and activated Src

were measured by western blot using respectively the anti-Src antibody and the activation-

linked anti-phospho-Tyr418 antibody described in Experimental Procedures. D, Activation of

p38MAPK (top) and JNK (bottom) by Aplidin in A-498 (left) and ACHN (right) cells. The

inhibitory effect of SB 203580 (30 µM) is shown. Total and activated forms of both kinases

were measured by western blotting using appropriate antibodies as in legend to Fig. 2. In all

experiments Aplidin was used at 500 nM. Results shown correspond to a representative

experiment. Those shown in A-C were performed three times and those in D twice.


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Table I

Role of EGFR, Src and p38MAPK/JNK in Aplidin -induced apoptosis in renal

ACHN and A-498 cells. Flow cytometry analysis of DNA content using PI in cultures

incubated in the absence or presence of Aplidin (500 nM) in serum-free medium (ACHN) or

in medium supplemented with 1% serum (A-498) for 16 h. Percentages of apoptotic cells

corresponding to the subdiploid population are shown. Cells were treated with the indicated

inhibitor(s) from 2 h before Aplidin addition to the end of the experiment, or left untreated

(control). One representative experiment out of two, which gave similar results, is shown.

Treatment Cells

Single ACHN A-498Control 16.8 10.0Aplidin 50.9 33.9AG1478 17.2 14.3

SB 203580 15.2 15.9PP2 19.5 13.0

AG1478 + Aplidin 35.0 38.9SB 203580 + Aplidin 22.8 37.8

PP2 + Aplidin 40.3 46.0Combined

Control 9.0 1.5Aplidin 51.1 32.1

AG1478 + SB 203580 12.7 10.5AG1478 + PP2 48.2 13.7

SB 203580 + PP2 38.9 31.6AG1478 + SB 203580 + PP2 39.6 29.5

AG1478 + SB 203580 + Aplidin 15.6 26.6AG 1478 + PP2 + Aplidin 25.7 44.8

SB 203580 + PP2 + Aplidin 12.0 39.0AG1478 + SB 203580 + PP2 + Aplidin 11.1 22.8


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Sánchez-Puelles and Alberto MuñozLosada, Victoria Alcaide, Teresa Martínez, José Maria Fernández-Sousa, José Maria Ana Cuadrado, Luis F. García-Fernández, Laura González, Yajaira Suárez, Alejandro

kinase and p38 kinasesustained activation of epidermal growth factor receptor, Src, Jun N-terminal

AplidinTM induces apoptosis in human cancer cells via glutathione depletion and

published online October 31, 2002J. Biol. Chem.

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aplidintm induces apoptosis in human cancer cells via glutathione depletion and sustained activation...

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