María J. Muñoz-Alonso, Laura González-Santiago, Natasha...

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Plitidepsin has a dual effect inhibiting cell cycle and inducing apoptosis via

Rac1/JNK activation in human melanoma cells

María J. Muñoz-Alonso, Laura González-Santiago, Natasha Zarich, Teresa

Martínez, Enrique Alvarez, José María Rojas, and Alberto Muñoz

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

Científicas-Universidad Autónoma de Madrid, E-28029 Madrid (M.J. M.-A., L. G.-S., T. M., A.

M.); Pharma Mar S.A., E-28770 Colmenar Viejo, Madrid (M.J. M.-A., L. G.-S., E. A.); Centro

Nacional de Microbiología, Instituto de Salud Carlos III, E-28220, Majadahonda, Madrid (N.

Z., J.M. R.), Spain

JPET Fast Forward. Published on December 18, 2007 as DOI:10.1124/jpet.107.132662

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on December 18, 2007 as DOI: 10.1124/jpet.107.132662

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Running title: Dual effect of plitidepsin in human melanoma cells

Corresponding author: Alberto Muñoz, Instituto de Investigaciones Biomédicas "Alberto

Sols", Arturo Duperier, 4, E-28029 Madrid, Spain.

Phone: #34-91-585 4451 Fax: #34-91-585 4401 e-mail: [email protected]

Number of text pages: 29

Number of tables: 0

Number of figures: 6

Number of references: 40

Number of words in the Abstract: 237

Number of words in the Introduction: 749

Number of words in the Discussion: 1189

Abbreviations used: APL, plitidepsin (Aplidin®); DTIC, dacarbazine; GTPase, guanosine-

triphosphatase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PARP,

Poly(ADP-ribose) polymerase; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen

species; GSH, glutathione reduced ethyl ester.

Journal category: Cellular and Molecular


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ABSTRACT

Melanoma is the most aggressive skin cancer and a serious health problem worldwide because

of its increasing incidence and the lack of satisfactory chemotherapy for late stages of the

disease. The marine depsipeptide plitidepsin (Aplidin) is an anti-tumoral agent under Phase II

clinical development against several neoplasias including melanoma. We report that plitidepsin

has a dual effect on the human SK-MEL-28 and UACC-257 melanoma cell lines: at low

concentrations (≤ 45 nM) it inhibits the cell cycle by inducing G1 and G2/M arrest while at

higher concentrations it induces apoptosis as assessed by PARP cleavage and the appearance of

a hypodiploid peak in flow cytometry analyses. Plitidepsin activates Rac1 GTPase and c-Jun N-

terminal kinase (JNK) and, in addition, it induces AKT and p38MAPK phosphorylation. By

using inhibitors, we found that JNK and p38MAPK activation depends on Rac1 but not on

PI3K, while AKT activation is independent of Rac1 but requires PI3K activity. Plitidepsin

cytotoxicity diminishes by Rac1 inhibition or by the blockage of JNK and p38MAPK using

SB203580, but not by PI3K inhibition using Wortmannin or LY294002. Remarkably,

plitidepsin and dacarbazine, the alkylating agent most active for treating metastatic melanoma,

show synergistic anti-proliferative effect that was paralleled at the level of JNK activation.

These results indicate that Rac1-JNK activation is critical for cell cycle arrest and apoptosis

induction by plitidepsin in melanoma cells. They also support the combined use of plitidepsin

and dacarbazine in in vivo studies.


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Introduction

Metastatic melanoma is an aggressive skin cancer whose incidence has rapidly increased

over the past five decades. Although patients with early-stage melanoma can be treated

efficiently by surgical dissection, patients with metastatic melanoma have a very poor

prognosis, with a median survival of less than 1 year (Jemal et al., 2005). Effective therapies for

advanced stages of this disease have not been defined, because malignant melanoma has proven

to be highly resistant to standard antineoplastic treatment. Dacarbazine (DTIC) is an alkylating

agent considered the most active agent for treating metastatic melanoma, and it is the only drug

approved by both the American Food and Drug Administration (FDA) and the European

Agency for the Evaluation of Medicinal Products (EMEA) for use in this disease. However,

dacarbazine has only a response rate < 20%, with complete response observed in < 5% of cases

(Lev et al., 2003). Clearly, additional therapeutic agents are needed for advanced resistant

melanoma.

Plitidepsin (Aplidin®) is a marine anti-tumor agent isolated from the Mediterranean

tunicate Aplidium albicans, which displays a potent activity against human hematological and

solid tumors cell lines. The compound has completed Phase I trials with evidence of a positive

therapeutic index. The dose limiting toxicities included asthenia, skin rash, diarrhea and

muscular toxicity; no myelotoxicity except for mild lymphopenia was reported (Jimeno et al.,

2004). Plitidepsin is currently under Phase II clinical studies against various neoplasias. A

randomized Phase I/II trial against melanoma is ongoing using plitidepsin and DTIC in

combination.

Several effects of plitidepsin have been reported. In human solid tumor cells plitidepsin

induces apoptosis through a strong, sustained activation of Jun N-terminal kinase (JNK)

(Cuadrado et al., 2004) and cells partially resistant to the drug show only weak, transient JNK


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activation (Losada et al., 2004). Recently, we have demonstrated that plitidepsin-induced JNK

activation in human breast cancer MDA-MB-231 cells depends on the early induction of

oxidative stress, activation of Rac1 small GTPase and the later down-regulation of MKP-1

phosphatase (Gonzalez-Santiago et al., 2006). We have also detected that plitidepsin induces

Rac1 translocation to cholesterol-rich membrane domains and, accordingly, plitidepsin

activation of the Rac1/JNK pathway depends on membrane cholesterol content in MDA-MB-

231 and HeLa cells (Suarez et al., 2006). Moreover, plitidepsin activates other kinases such as

the epidermal growth factor receptor (EGFR), Src, p38MAPK, extracellular signal-regulated

kinase (ERK) and protein kinase C-δ (Garcia-Fernandez et al., 2002; Cuadrado et al., 2003). In

accordance with the activation of Rac1, plitidepsin inhibits the low molecular weight protein

tyrosine phosphatase (LMW-PTP) (Taddei et al., 2006), an enzyme that is inhibited by Rac1-

induced reactive oxygen species (ROS) production (Nimnual et al., 2003). Furthermore, in

leukemic cell lines plitidepsin also activates JNK, and triggers Fas/CD95 receptor and

mitochondrial apoptotic pathway through the recruitment of signaling molecules at membrane

lipid rafts (Gajate and Mollinedo, 2005).

Plitidepsin has also anti-angiogenic properties, as it reduces the expression of several

angiogenic genes in cancer xenografts (Straight et al., 2006). Moreover, plitidepsin reduces the

secretion of VEGF and blocks the stimulatory VEGF autocrine loop in leukemic MOLT-4 cells

(Broggini et al., 2003). Likewise, it inhibits the response of endothelial cells to angiogenic

stimuli (Taraboletti et al., 2004). Therefore, the in vivo anti-angiogenic effect of plitidepsin

might result from the induction of tumor or endothelial cell apoptosis (Biscardi et al., 2005).

Another mechanism that may contribute to the anti-tumoral activity of plitidepsin is its

anti-proliferative effect. Plitidepsin causes G1 arrest and G2/M blockage in leukemia cells (Erba

et al., 2003; Biscardi et al., 2005). Recently, it has been reported that therapeutic concentrations

of plitidepsin block anaplastic thyroid carcinoma cells in the G1-to-S transition of the cell cycle


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(Bravo et al., 2005). In contrast, no cell cycle perturbation by plitidepsin has been observed in

other human solid tumor cells (Garcia-Fernandez et al., 2002; Cuadrado et al., 2003).

Based on initial clinical data indicating activity of plitidepsin as unique agent against

advanced or metastatic melanoma in patients that were previously treated with chemotherapy

(Eisen et al., 2004), we have for the first time studied its effects on cultured human melanoma

cells. Our results show that plitidepsin has a dual, concentration-dependent effect on UACC-257

and SK-MEL-28 human metastatic melanoma cells. While at low concentrations it inhibits

proliferation by inducing cell cycle arrest at G1 and G2/M phases, at higher concentrations

plitidepsin induces apoptosis through Rac1-JNK activation. Moreover, we report here that

plitidepsin has a synergistic anti-proliferative activity in combination with DTIC. These results

support the interest of further studies on the potential use of plitidepsin for melanoma therapy.


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Materials and methods

Cell culture

Human SK-MEL-28 and UACC-257 melanoma cells were obtained from the American

Type Culture Collection (Rockville, USA) and NCI-Frederick Cancer, DCTD Tumor/Cell Line

Repository, Ft. Detrick U.S. Army facility in Frederick, Maryland, respectively. They were

grown in RPMI 1640 supplemented with 10% FCS and antibiotics (all from GIBCO-Invitrogen,

Paisley, UK). Plitidepsin, (APL; Aplidin®), is a cyclic depsipeptide (for its chemical structure

see Garcia-Fernandez et al., 2002) originally isolated from the tunicate Aplidium albicans and

currently obtained by total synthesis by Pharma Mar S.A. (Madrid, Spain) (Sakai et al., 1996).

Stock solutions were freshly prepared in dimethylsulfoxide (DMSO). The final DMSO

concentration for cultures was always less than 0.05% (v/v), which did not affect either cell

proliferation or JNK activation as compared to untreated cells. Glutathione reduced ethyl ester

(GSH) and Dacarbazine (DTIC; DTIC®-Dome; 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-

carboxamide) were from Sigma Chemical Co. (St. Louis, MO); Rac1 inhibitor (NSC23766,

1,2,6,7-tetrathiacyclodecane), Wortmannin, LY294002 and SB203580 were from Calbiochem-

Merck Biosciences (Darmstadt, Germany).

Flow Cytometry Analyses

Cells were stained with propidium iodide and analyzed by flow cytometry (FACScan,

Becton Dickinson, San Jose, CA, USA, equipped with a 488 nm argon ion laser). For staining,

one million cells were harvested, washed in PBS and then fixed with 70% ethanol. After

washing, fixed cells were treated with DNase-free RNase (50 µg/ml; Sigma) for 1 h at 37 ºC in

PBS-containing propidium iodide (50 µg/ml; Sigma). Ten thousand events per sample were

acquired for data analysis using CELLQUEST software (BD Bioscience, San Jose, CA).


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Western blotting

The preparation of cell protein extracts and Western blotting analysis were as described

elsewhere (Cuadrado et al., 2004; Gonzalez-Santiago et al., 2006). Briefly, cells were lysated

with RIPA buffer and 20 µg of protein were subjected to SDS-PAGE and transferred to PVDF

membranes. Membranes were blocked at room temperature for 1 h in Tris-buffered saline (25

mM Tris-HCl pH 7.4, 136 mM NaCl, 2.6 mM KCl, 0.5% Tween-20) containing 5% BSA and

incubated overnight at 4ºC with the appropriated antibody. Antibodies used were: anti-JNK1,

anti-p38MAPK, anti-actin, anti-PARP, anti-cyclin B1 and anti-cyclin A from Santa Cruz

Biotechnology; anti-phospho-JNK, anti-phospho-p38MAPK, anti-phospho-AKT (Ser473) and

anti-AKT from New England Biolabs (Ipswich, MA)/Cell Signaling (Danvers, MA); anti-Rac1

monoclonal antibody from Transduction Laboratories/BD Biosciences (Heidelberg, Germany).

After washing, blots were incubated with HPR-secondary antibodies for 1 h at room

temperature and developed by a peroxidase reaction using the ECL detection system

(Amersham-G.E. Healthcare). Protein expression levels were quantified by densitometry using

ImageJ software (National Institutes of Health, USA).

Rac1 activity assays

Bacterial expression of fusion proteins and in vitro binding assays were as previously

described (Gonzalez-Santiago et al., 2006). The plasmid pGEX-PAK-CRIB containing the

Rac1-binding domain fused to GST was kindly provided by JG Collard (The Netherlands

Cancer Institute). For in vitro binding assay, the GST-fusion protein on gluthatione-Sepharose

beads (purified from Escherichia coli BL21 (DE3) harboring this plasmid) was incubated with

cell extracts and analysed as described (Gonzalez-Santiago et al., 2006).


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Cell proliferation and CalcuSyn analysis

Cell proliferation was studied by [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl] tetrazolium

bromide (MTT) assays that were performed following the manufacturer’s instructions (MTT

Cell Proliferation Kit I, Roche Diagnostics, Mannheim, Germany). Analysis of synergy,

additive or antagonist effect of drug combination studies were determined according to the

median-effect principle analysis of Chou and Talalay (Chou and Talalay, 1983) using the

CalcuSyn software (Biosoft, Ferguson, MO). The program returns the values of the dose

required for 50% inhibition of cell proliferation or IC50 and the combination index (CI), which

reflect the nature of the interaction between drugs. A CI value of 1 indicates an additive effect

between two drugs, whereas a CI < 1 or CI > 1 indicates synergism or antagonism, respectively.

The degree of synergism is proportional to the value of CI. Values < 0.1 represent a very strong

synergism whereas range of CI values of 0.1-0.3, 0.3-0.7, 0.7-0.85 and 0.85-0.9 represent strong

synergism, synergism, moderate synergism and slight synergism, respectively.

Statistical Analysis

Results are expressed as mean ± S.E.M. Statistical significance of differences between

values was calculated by one-way ANOVA and the Dunnett posthoc test using the GraphPad

Instat3 program. Differences were considered statistically significant when P was less than

0.05. When P >0.05, the data were considered not significant (ns). The single asterisk indicates

P <0.05, the double asterisk P <0.01, and the triple asterisk P <0.001.


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Results

To determine the effect of plitidepsin against melanoma, we first analysed its anti-

proliferative activity in two cell lines, SK-MEL-28 and UACC-257. SK-MEL-28 are non-

pigmented melanoma cells that harbor mutations in B-RAF, PTEN and TP53 genes and express

moderate levels of APAF-1 protein. In contrast, UACC-257 are melanotic cells with normal,

non-mutated PTEN and TP53 genes, whereas the presence of mutated B-RAF is controversial

(Davies et al., 2002; Shields et al., 2007), and show reduced APAF-1 expression. We chose

these two cell lines to explore whether the response to plitidepsin is cell specific and if melanin

content or the most common melanoma-associated mutations can compromise the response to

the drug.

Plitidepsin inhibited the proliferation of both SK-MEL-28 and UACC-257 cell lines in a

concentration-dependent manner and with a very similar IC50 of 12-14 nM at 48 h post-

treatment (Fig. 1A). To analyse whether similarly to what happens in other cell types plitidepsin

induced apoptosis in melanoma cells, we studied by Western blotting the expression of

Poly(ADP-ribose) polymerase (PARP), whose proteolytic cleavage by caspases is a hallmark of

the apoptotic process. PARP cleavage was found as soon as 3-6 h following drug exposure at

450 nM (Fig. 1B, upper panel) and only at high concentrations of plitidepsin in both cell types

(Fig. 1B, lower panel). Flow cytometry analysis confirmed a dual, concentration-dependent

effect of plitidepsin. At low concentrations (15-45 nM) plitidepsin arrested cells in G1 cell cycle

phase, as shown by a slight accumulation of cells in the G0/G1-phase and a marked decrease in

the percentage of cells in the S-phase (Fig. 1C). However, there was also a persistent population

of cells with a G2/M-phase DNA content, which likely represents G2/M-arrested cells as

observed in other systems (Niculescu et al., 1998). In contrast, at higher concentrations (150-

450 nM) of plitidepsin induced the formation of a hypodiploid sub-G1 peak indicative of


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apoptosis (Fig. 1C). The same results were observed in UACC-257 cells (data not shown).

Consistent with the effects on the cell cycle, Western blot analysis revealed that exposure to

plitidepsin for 24 hours resulted in a significant concentration-dependent reduction of the levels

of cyclin A and cyclin B, two cyclins whose expression are dependent of cell cycle progression

through S-phase and peak at G2 and G2/M, respectively (Fig. 1D).

Next, we examined whether plitidepsin affected JNK activity in melanoma cells. Fig. 2A

shows that plitidepsin induced a rapid, progressive and sustained activation of JNK in SK-MEL-

28 and UACC-257 melanoma cells, as measured by Western blotting analysis of

phosphorylated JNK levels consistent with changes noted in other cell types (Garcia-Fernandez

et al., 2002; Cuadrado et al., 2003; Gonzalez-Santiago et al., 2006). JNK activation was

concentration-dependent, being first detected at 15 nM (Fig. 2B and 2C). Plitidepsin also

activated Rac1 GTPase with short kinetics (Fig. 3A and 3B) and this effect was abolished by

exogenous GSH (Fig. 3C). These results parallel those recently reported for breast, renal and

cervical carcinoma cells (Gonzalez-Santiago et al., 2006; Suarez et al., 2006). In addition, pre-

treatment with NSC23766, a specific Rac1 inhibitor, prevented the cytostatic/cytotoxic effect of

plitidepsin in a concentration-dependent manner (Fig. 3D).

To get further insight the mechanism of action of plitidepsin in melanoma cells we

explored putative actions on other kinases involved in cell survival/death such as AKT and

p38MAPK. Unexpectedly from the pro-survival role of AKT, plitidepsin induced AKT

activation as seen by an increase in the phosphorylation at residue Ser473 (Fig. 4A, middle

panels). This effect was not blocked by the Rac1 inhibitor NSC23766. In contrast, p38MAPK

was also activated but in a Rac1-dependent fashion (Fig. 4A, bottom panels). As in previous

results with MDA-MB-231 or HeLa cells (Gonzalez-Santiago et al., 2006), JNK activation was

sensitive to Rac1 inhibition (Fig. 4A, top panels). The putative role of phosphatidylinositol 3-

kinase (PI3K) in the activation of these kinases by the compound was investigated by using


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Wortmannin and LY294002, two PI3K inhibitors. While neither of these inhibitors prevented

the activation of JNK or p38MAPK by plitidepsin, they abolished totally that of AKT (Fig. 4B).

In untreated cells, Wortmannin and LY294002 reduced the basal levels of phosphorylated AKT

but had no effect on those of phosphorylated JNK or p38MAPK (Fig. 4B). Likewise, neither of

these two inhibitors affected cell viability per se nor changed the cytostatic/cytotoxic effect

induced by plitidepsin (data not shown).

We also used SB203580, a compound usually considered as specific inhibitor of

p38MAPK. However, by concentration-curve analyses we found that SB203580 prevented JNK

and p38MAPK activation by plitidepsin with similar efficacy (Fig. 5A), as it has been reported

in breast and renal cancer cells (Cuadrado et al., 2003). Extending the crucial role that these

enzymes, particularly JNK, play in other cell types (Cuadrado et al., 2004), SB203580 reverted

the anti-proliferative action of plitidepsin (Fig. 5B). Likewise, SB203580 inhibited the induction

of SK-MEL-28 cell apoptosis by the drug as assessed by flow cytometry (Fig. 5C). As

SB203580 itself caused G1 arrest, no effective estimation of its effect on the inhibition of cell

cycle by plitidepsin can be done, although a moderate recovery of cells in the S-phase was

observed in cells pretreated with SB203580 before incubation with 45 nM plitidepsin (Fig. 5C).

Finally, we studied the effect of the combined treatment of melanoma cells with

plitidepsin and DTIC. As plitidepsin (Figure 1A and 6A), DTIC alone inhibited the proliferation

of SK-MEL-28 and UACC-257 cells in a concentration-dependent manner, although with

distinct potency: IC50 of 843 µg/ml and 127 µg/ml (according to CalcuSyn analysis),

respectively, at 48 h post-treatment (Figure 6A). We found that the combination of plitidepsin

and DTIC was more effective at inhibiting cell proliferation than each compound alone in both

cell lines at all tested concentrations. Data analysis by the Chou and Talalay method (Chou and

Talalay, 1983) using the CalcuSyn software defined plitidepsin and DTIC to act synergistically

(combination index, CI < 1) across a broad range of concentrations. Figure 6B illustrates the


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CI/fractional effect plots showing the CI values versus the fraction of cells affected (Fa) by

plitidepsin and DTIC in combination. In SK-MEL-28 cells, the CI values for Fa < 0.75 were

less than 0.3, indicating a strong synergism between plitidepsin and dacarbazine. Similarly, in

UACC-257 cells a synergistic effect of plitidepsin and DTIC was found, but the degree of

synergism was lower than that in SK-MEL-28 cells, with CI values ranging from 0.3 to 0.7 at

the majority of tested Fa. Remarkably, the level of JNK phosphorylation induced by suboptimal

concentrations (45 nM) of plitidepsin was higher when cells were cultured in the presence of

DTIC (Fig. 6C, top panel). This effect was specific, as no such increase was observed in the

case of AKT or p38MAPK (Fig. 6C, middle and bottom panels).


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Discussion

This is the first molecular characterization of the action of plitidepsin on human

melanoma cells. We report that plitidepsin has a potent anti-proliferative effect by inhibiting

cell cycle progression at pharmacologically relevant concentrations (IC50 around 15 nM) that are

similar to the circulating plasma concentrations of plitidepsin observed in phase II clinical trials

(Celli et al., 2004). In addition, at higher concentrations plitidepsin induces cell death by

apoptosis.

Our results demonstrate that in melanoma cells plitidepsin causes a rapid activation of

Rac1 linked to a strong activation of JNK and p38MAPK, as it happens in breast, renal and

cervical carcinoma cells, which leads to a rapid induction of apoptosis (Cuadrado et al., 2004;

Gonzalez-Santiago et al., 2006; Suarez et al., 2006). However, in contrast to these other tumor

types, in which plitidepsin does not modulate the cell cycle, at low concentrations it causes G1

arrest and G2/M blockage in SK-MEL-28 and UACC-257 melanoma cells. In agreement with

this observation, cyclin A and cyclin B, which are associated to progression of the cycle to the

G2 phase, are reduced by plitidepsin. Cell type differences in plitidepsin action are presumably

due to the specific alterations acquired by each type of cancer cells during the neoplastic

process.

Although SB203580 lacks specificity of JNK versus p38MAPK inhibition, it appears that

JNK is the main mediator of plitidepsin activity, because cells lacking p38MAPK, but no those

lacking JNK, display normal plitidepsin sensibility (Cuadrado et al., 2004). By using specific

inhibitors, we demonstrate that JNK participates in the apoptosis induced by plitidepsin, since

pre-treatment with SB203580 decreased the number of cells in the sub-G1 fraction.

Furthermore, despite it induces G1 arrest, SB203580 reverted the anti-proliferative action of low

concentrations of plitidepsin, suggesting that JNK is also involved in the plitidepsin-mediated


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cell cycle arrest. Our data also demonstrate that Rac1 acts upstream of JNK in melanoma cells,

as a specific inhibitor of this GTPase abrogates the cytotoxicity and phosphorylation of JNK by

plitidepsin. These findings are consistent with published data (Gonzalez-Santiago et al., 2006)

and studies showing that Rac1 triggers apoptosis via JNK in response to TNFα (Jin et al., 2006)

and ceramide (Brenner et al., 1997).

A wide number of anti-tumor drugs currently in clinical use cause cell death by JNK-

mediated apoptosis. In addition, the involvement of JNK in the regulation of cell cycle

progression has also been noted in previous reports: JNK mediates G2/M arrest induced by

sulforaphane (Cho et al., 2005) and diallyl trisulfide (Antosiewicz et al., 2006) in human

prostate cancer cells, and by thiazolidin compounds in human non-small-cell lung and colon

cancer cells (Teraishi et al., 2005). Interestingly, glial cell line-derived neurotropic factor

induces G2/M cell cycle delay via the Rac1/JNK pathway (Fukuda et al., 2005). Likewise, JNK

has been shown to contribute to G1 arrest mediated by a ginseng metabolite, compound K, in

human monocytic leukemia cells (Kang et al., 2005).

Our results suggest that plitidepsin induces an early oxidative stress, an upstream activator

of the Rac1/JNK pathway (Gonzalez-Santiago et al., 2006), the nature of which remains to be

characterized. One possibility is that an initial generation of ROS causes the activation of Rac1,

that in turn induces more ROS (Nimnual et al., 2003) causing a positive feedback loop that may

lead to apoptosis. Alternatively, the amplified ROS signaling might be due to JNK, similar to

that JNK-dependent ROS formation by TNF (Ventura et al., 2004) and diallyl trisulfide

(Antosiewicz et al., 2006). In addition, plitidepsin-mediated JNK activation might induce

stabilization of JNK pathway components, thereby leading to feedback up-regulation of

apoptotic signaling as described for other apoptotic stimuli (Xu et al., 2005).

The activation of AKT by plitidepsin has not been detected in other human cancer cells

that have high basal levels of this protein (Cuadrado et al., 2003), and is somehow puzzling


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giving the pro-survival role of this enzyme. Strikingly, AKT activation in response to other anti-

cancer drugs has also been described. Thus, in NIH 3T3 cells doxorubicin, etoposide and

staurosporine activate AKT preceding the onset of apoptosis (Tang et al., 2001; Kim et al.,

2006; Lee et al., 2006). Moreover, activation of AKT by daunorubicin has been observed in

human acute myeloid leukemia cell lines (Plo et al., 1999). Conceivably, the survival signal due

to AKT activation by plitidepsin signaling is overridden by its pro-apoptotic effects. Supporting

this view, the synergistic effect of plitidepsin and DTIC are paralleled by their cooperation

activating JNK but not AKT or p38MAPK.

Consistent with our observation that its anti-proliferative activity as a single agent in

melanoma cell lines, plitidepsin induces long lasting objective remissions and tumor control in a

subset of advanced resistant melanoma patients (Eisen et al., 2004). DTIC is the only approved

therapy for patients with metastatic melanoma, but the response rates are < 20% and the

complete responses rarely exceed 5%. Notably, we found a stronger synergism between

plitidepsin and DTIC in SK-MEL-28 (IC50 of DTIC ~ 843 µg/ml) than in UACC-257 (IC50 of

DTIC ~ 127 µg/ml) cells, suggesting that this combination may be promising in the treatment of

DTIC-resistant patients. The synergy between plitidepsin and DTIC may well result from their

different signaling leading to apoptosis. Thus, while DTIC induces methylation of nucleic acids

or direct DNA damage resulting in cell death (Kyrtopoulos et al., 1997), plitidepsin activates

apoptosis initially acting from the cell membrane (Suarez et al., 2006). Since JNK is crucial for

plitidepsin activity (Cuadrado et al., 2004) and its degree of phosphorylation/activation

correlates with the anti-proliferative potency of plitidepsin (Figs. 1 and 2), the increased JNK

activation by the combined treatment with plitidepsin and DTIC in comparison with plitidepsin

alone may contribute to explain the synergistic activity of these drugs. In addition, the inhibition

of VEGF secretion by plitidepsin could potentiate the therapeutic effect of DTIC, which has

been shown to up-regulate VEGF expression (Lev et al., 2003; Broggini et al., 2003). These


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results support the ongoing Phase I/II trial combining plitidepsin and DTIC in first line against

advanced melanoma.

The differential response of melanoma cells to various concentrations of plitidepsin may

have potential implications for the in vivo activity of this agent. Once plitidepsin reaches a

tumor site in vivo, it is plausible that the cellular response to the agent can fall into one of two

pathways; apoptosis at higher tissue concentrations and cell-cycle arrest in tumor areas with a

more limited exposure to plitidepsin. Since tumors are heterogeneous, and physicochemical and

physiological barriers can lead to heterogeneous accumulation of the drug in solid tumors (Jain,

1999), a compound that has the ability to affect cell proliferation at different concentrations via

distinct pathways could better allow for local tumor control.

In conclusion, this work shows that plitidepsin has a potent anti-tumoral activity against

human melanoma cells in vitro. This effect appears to be dual, with a cytostatic response at low

concentrations and a cytotoxic response at the higher concentration range. The work also

reiterates the importance of the activation of Rac1/JNK pathway by plitidepsin as a major

operating mechanism for this agent. In addition, it provides a rationale for the clinical

evaluation of plitidepsin in combination with DTIC in advanced melanoma patients.


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Acknowledgements

We are grateful to NCI-Frederick Cancer, DCTD Tumor/Cell Line Repository for providing us

with the UACC-257 cell line.


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Footnotes

This work was supported by SAF2004-01015 and SAF2007-60413 (to AM) and SAF2003-

02604 and SAF2006-04247 (to JMR) from Ministerio de Educación y Ciencia, and Intramural

ISCIII (03/ESP27) (to JMR) and RD06/002/0009 (to AM) from Instituto de Salud Carlos III of

Spain.

Alberto Muñoz, Instituto de Investigaciones Biomédicas "Alberto Sols", Arturo Duperier, 4, E-

28029 Madrid, Spain.

Phone: #34-91-585 4451 Fax: #34-91-585 4401 e-mail: [email protected]


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Legends for figures

Figure 1. Cytostatic and cytotoxic effects of plitidepsin on melanoma cells. (a) Cell

proliferation of SK-MEL-28 and UACC-257 cells incubated in the presence of the indicated

concentrations of plitidepsin for 24 h, 48 h and 72 h, as measured by the MTT assay. Data are

mean ± SEM values obtained in four independent experiments performed in quadruplicate. (b)

Western blot analysis showing the kinetic of PARP cleavage at 450 nM plitidepsin (upper

panel) and the concentration-dependent PARP cleavage in cells treated with the plitidepsin in

serum-free medium for 24 h (lower panel). (c) Flow cytometry analysis of DNA content in SK-

MEL-28 cells incubated with vehicle (control) or the indicated concentrations of plitidepsin for

24 h. Data are mean ± SEM values obtained in four independent experiments of the percentage

of cells in the different cell-cycle phases and of the hypodiploid (sub-G1) population. (d)

Western blot analysis showing the expression levels of cyclin A and cyclin B in SK-MEL-28

cells treated with the indicated concentrations of plitidepsin. As a control, the membrane was

reprobed with anti-actin antibody. Right panel shows the quantification of cyclin A (white

columns) and cyclin B (black columns) expression levels after normalization to actin levels.

Figure 2. Plitidepsin induces JNK activation in melanoma cells. (a) Kinetics of JNK

activation in cells treated with 450 nM plitidepsin in serum-free medium for the indicated times,

as measured by Western blot analysis using a specific phospho (P)-JNK antibody. (b) Western

blot analysis showing concentration-dependent JNK activation in cells treated with plitidepsin

for 1 h. (c) Quantification of JNK activation, normalized to total JNK levels, at the indicated

concentrations of plitidepsin.


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Figure 3. Plitidepsin induces activation of the Rac1 GTPase. (a) SK-MEL-28 cells were

serum-starved for 18 h and then incubated with 450 nM plitidepsin for 15 min. Rac1 activity

was estimated by binding of Rac1-GTP to immobilized GST-PAK-CRIB, as described

(Gonzalez-Santiago et al., 2006). For positive and negative controls, cell lysates from untreated

cells were incubated, respectively, with GTP or GDP to precipitate activated GTP-bound Rac1

or inactivated GDP-bound Rac1. The levels of Rac1 in whole extracts were detected by Western

blot. Values of Rac1 activation were normalized to those of total Rac protein levels. (b) Kinetics

of Rac1 activation in cells treated with 450 nM plitidepsin for the indicated times. (c) Effect of

GSH on the activation of Rac1 by plitidepsin. SK-MEL-28 cells were pretreated with 0.5 mM

GSH (30 min) before plitidepsin (450 nM) addition. (d) Effect of Rac1 inhibitor (NSC23766) in

the plitidepsin activity as measured by the MTT assay. SK-MEL-28 cells were pretreated for 6 h

with the indicated concentrations of Rac inhibitor and then for further 24 h with 45 nM (white

columns) or 150 nM (black columns) plitidepsin. Data are mean ± SEM values of two

independent experiments performed in quadruplicate. ** p < 0.01.

Figure 4. Plitidepsin induces Rac1-dependent JNK and p38MAPK activation and PI3K-

dependent AKT activation. (a) Western blot analysis showing the levels of activated JNK,

AKT and p38MAPK in SK-MEL-28 cells upon plitidepsin treatment alone or in combination

the Rac1 inhibitor. Cells were pretreated overnight with NSC23766 (100 µM) or vehicle before

plitidepsin (450 nM) addition for the indicated times. (b) Effect of PI3K inhibitors on the

activation of AKT, JNK and p38MAPK by plitidepsin. Cells were pretreated with LY294002

(LY; 10 µM), Wortmannin (W; 1 µM) or vehicle for 1 h and then with plitidepsin (450 nM) for

an additional hour. The levels of phosphorylated and total AKT, JNK and p38MAPK were

analysed by Western blot using appropriate antibodies.


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Figure 5. The inhibition of JNK/p38MAPK activation abrogates the cytostatic and

cytotoxic effects of plitidepsin in SK-MEL-28 cells. (a) SB203580 inhibits plitidepsin-

mediated JNK and p38MAPK activation, as analysed by Western blot. Cells were incubated for

1 h in the presence of the indicated concentrations of SB203580 (SB) before plitidepsin (450

nM; 1 h) addition. (b) Effect of SB203580 (SB) on the anti-proliferative activity of plitidepsin,

as measured by the MTT assay. SK-MEL-28 cells were pretreated 1 h with the indicated

concentrations of SB203580 and then for a further 48 h with 15 nM (white columns) or 45 nM

(black columns) plitidepsin. Data are mean ± SEM values of two independent experiments

performed in quadruplicate. ** p < 0.01. (c) SB203580 inhibits plitidepsin-mediated apoptosis.

Cells were pretreated for 1 h with 10 µM SB203580 before addition of the indicated

concentrations of plitidepsin. Three independent experiments were done. Flow cytometry

histograms show the percentage of cells in the different cell-cycle phases found 24 h after

plitidepsin treatment in a representative experiment.

Figure 6. Synergistic anti-tumoral activity of plitidepsin and dacarbazine. (a) Cell

proliferation of SK-MEL-28 and UACC-257 cells after 48 h incubation in the presence of the

indicated concentrations of plitidepsin alone (○) or in combination with 10 µg/ml (●), 20 µg/ml

(□), 50 µg/ml (■), 150 µg/ml (∆) or 450 µg/ml (▲) of dacarbazine was investigated by using the

MTT assay. Data are mean ± SEM values of four independent experiments performed in

quadruplicate. (b) CI/fractional effect plots showing the combination index (CI) values versus

the fraction of cells affected (Fa) by plitidepsin and DTIC in combination for SK-MEL-28 and

UACC-257 cells. Combination analyses were done using CalcuSyn software, as indicated in

Materials and Methods. The degree of synergism (CI values < 0.1 and range of CI values of 0.1-

0.3, 0.3-0.7, 0.7-0.85 and 0.85-0.9 represent a very strong synergism, strong synergism,

synergism, moderate synergism and slight synergism, respectively) is indicated by dotted lines.


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(c) Western blot analysis showing the levels of phosphorylated JNK, AKT and p38MAPK in

cells treated with plitidepsin and/or dacarbazine. Cells were pretreated for 1 h with dacarbazine

(DTIC; 400 µg/ml) or vehicle before plitidepsin (45 nM; 1 h) addition.


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