Proteomic Analysis of the Resistance to Aplidin in Human Cancer

Proteomic Analysis of the Resistance to Aplidin in Human Cancer Cells Laura Gonza´ lez-Santiago,†,‡,# Patricia Alfonso,§,# Yajaira Sua´ rez,†,‡ Antonio Nu´ n ˜ ez,§ ‡ ‡ ,† Luis F. Garcıá-Ferna´ ndez, Enrique Alvarez, Alberto Mun ˜ oz,* and J. Ignacio Casal*,§ Instituto de Investigaciones Biome´dicas “Alberto Sols”, Consejo Superior de Investigaciones Cientı´ficas, Universidad Autońoma de Madrid, Arturo Duperier, 4, E-28029 Madrid, Spain, PharmaMar S.A., E-28770 Colmenar Viejo, Madrid, Spain, and Unidad de Tecnologıá de Proteıńas, Programa de Biotecnologıá, Centro Nacional de Investigaciones Oncolo´gicas (CNIO), Melchor Fernańdez Almagro, 3, E-28229 Madrid, Spain Received August 24, 2006

Aplidin (plitidepsin) is an antitumoral agent that induces apoptosis via Rac1-JNK activation. A proteomic approach using 2D-DIGE technology found 52 cytosolic and 39 membrane proteins differentially expressed in wild-type and Aplidin-resistant HeLa cells, of which 39 and 27 were identified by MALDITOF mass spectrometry and database interrogation. A number of proteins involved in apoptosis pathways were found to be deregulated. Alterations in Rab geranylgeranyltransferase, protein disulfide isomerase (PDI), cystathionine γ-lyase, ezrin, and cyclophilin A (CypA) were confirmed by immunoblotting. Moreover, the role of PDI and CypA in Aplidin resistance was functionally confirmed by using the inhibitor bacitracin and overexpression, respectively. These deregulated proteins are candidates to mediate, at least partially, Aplidin action and might provide a route to the cells to escape the induction of apoptosis by this drug. Keywords: Aplidin • differential proteomics • 2D-DIGE • chemoresistance • apoptosis pathways

Introduction Aplidin (plitidepsin) is an antitumor compound in phase II clinical trials against a wide variety of neoplasias. Chemically, it is a macrocyclic depsipeptide originally isolated from the marine tunicate Aplidium albicans and currently obtained by total synthesis.1 Aplidin is a potent inducer of apoptosis, with IC50 in the low nanomolar range, in many tumor cell types in vitro, and it also exhibits antitumor activity in xenograft models.2-4 Aplidin induces apoptosis via caspase-dependent and -independent mechanisms, irrespective of the p53 tumor suppressor status. However, the mechanism of action of Aplidin is not fully understood. In human solid tumor cells, it induces a strong, sustained activation of Jun N-terminal kinase (JNK) that is mostly dependent on activation of the Rac1 small GTPase and potentiated by the down-regulation of the MKP1 phosphatase.5,6 In addition, Aplidin activates other kinases such as the epidermal growth factor receptor (EGFR), Src, and p38 mitogen-activated protein kinase.4 In human HeLa cervical cancer cells, Aplidin activates extracellular signal-regulated kinase (ERK) and protein kinase C-δ and rapidly induces the * Corresponding authors: J. Ignacio Casal, Biotechnology Program, Centro Nacional de Investigaciones Oncolo´gicas, Melchor Fernandez Almagro, 3, 28029 Madrid, Spain. Phone, +34 91 224 69 20; fax, + 34 91 224 69 72; e-mail, [email protected]. Alberto Mun j oz: e-mail, [email protected]. † Consejo Superior de Investigaciones Cienti´ficas-Universidad Autońoma de Madrid. ‡ PharmaMar S.A. § Centro Nacional de Investigaciones Oncolo´gicas (CNIO). # These authors contributed equally to this study.

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Journal of Proteome Research 2007, 6, 1286-1294

Published on Web 03/06/2007

apoptotic mitochondrial pathway, thus, promoting cytochrome C release from the mitochondria and caspase-3 activation.7 We have recently reported that Rac1-JNK activation, but not MKP-1 down-regulation, is dependent on glutathione depletion.6 Our data indicate that Aplidin induces an early oxidative stress linked to the disruption of glutathione homeostasis and activation of Rac1-JNK. However, the nature of the initial events that trigger Aplidin signaling and effects remains unknown. Very little is known about the mechanisms of intrinsic or acquired resistance to Aplidin. To gain insight into this crucial clinical point, Losada and colleagues generated, by continuous exposure to the drug, a subline of HeLa cells displaying partial resistance to Aplidin (HeLa-R) as a model of acquired resistance.8 HeLa-R cells do not show cross-resistance to several clinically relevant antitumoral agents such as cisplatin, etoposide, paclitaxel, camptothecin, or doxorubicin. Accordingly, resistance of HeLa-R cells was unrelated to P-glycoprotein expression.8 Confirming the important role of JNK for Aplidin action, HeLa-R cells show only weak and transient activation of Rac1 and JNK.8 Thus, these cells represent a powerful tool for the study of the mechanism of action of Aplidin in tumor cells and for the investigation and development of methods to prevent resistance to this drug in vivo. The purpose of this study was to characterize the molecular basis of resistance to Aplidin using human HeLa cervical cancer cells. We have used differential two-dimensional electrophoresis (2D-DIGE) to analyze the pattern of protein expression in the cytosolic and membrane fractions of the Aplidin-resistant HeLa cell line in comparison to parental HeLa cells (HeLa-wt). 10.1021/pr060430+ CCC: $37.00

 2007 American Chemical Society

Proteomic Analysis of Cell Resistance to Aplidin

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Figure 1. A representative 2D map of the membrane proteins obtained by DIGE analysis. Protein extracts from the membrane of wild-type (HeLa-wt) and Aplidin-resistant (HeLa-R) HeLa cells were covalently labeled with Cy3 (green) and Cy5 (red) fluorochromes, respectively. Overlapping image illustrates the changes in protein abundance after treatment of the cells with the drug for a long period of time. Those proteins whose expression varied within statistical significance (Student’s t-test, p < 0.01) and were unequivocally identified by PMF are named and indicated by arrows. Numbers correlate with those included in Table 1.

These two sets of proteins were chosen for proteomic analysis because studies carried out with 14C-labeled Aplidin showed that the molecule transiently locates to the plasma membrane and then predominantly localizes in the cytosol bound to macromolecules. In contrast, no drug distributes to the cell nucleus or remains attached to the cytoskeleton.9 Moreover, the induction of cell death by Aplidin is independent of “de novo” protein synthesis as it occurs in the presence of cycloheximide. Together, these data suggest that pre-existing cytosolic/membrane proteins are responsible for Aplidin sensitivity. Proteins that were differentially expressed (up- or downregulated) in each fraction were selected for identification. In total, 39 and 27 distinct proteins were identified in the cytosolic and membrane fractions, respectively, which are related to regulation of cell survival, transformation, apoptosis, and oxidative stress mechanisms. Such differentially expressed proteins are candidates to be directly or indirectly related to the resistance of human cancer cells to Aplidin.

Materials and Methods Materials and Cell Culture. Wild-type HeLa cells (HeLa-wt) and HeLa-R cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1 mM glutamine (all from GIBCO-Invitrogen, Paisley, U.K.). Aplidin was a kind gift of PharmaMar. Stock solutions were freshly prepared in dimethylsulfoxide (DMSO) and diluted in the cell culture to the final concentrations indicated. Rab geranylgeranyltransferase inhibitor (GGTI-298) was purchased

from Calbiochem. Bacitracin (disulfide isomerase inhibitor), DLpropargylglycine (cystathionine γ-lyase inhibitor) and human recombinant cyclophilin A were from Sigma-Aldrich (St. Louis, MO). Cells were transfected at around 50% confluence in 6-cm dishes with 5 µg of DNA using lipofectamine (Invitrogen). We used 3 µg of expression plasmid for N-Ezrin (provided by Dr. M. Quintanilla, Instituto de Investigaciones Biome´dicas, Madrid) and the pSG5 vector as carrier to adjust the total amount of DNA. After overnight incubation with lipofectamine-DNA mixtures, transfected cells were washed twice in phosphatebuffered saline (PBS) and incubated with fresh medium supplemented with 0.5% FCS. Each treatment was carried out in triplicate cultures. Protein extracts were prepared following standard procedures and analyzed using Western blotting. Subcellular Fractioning. To prepare subcellular fractions, cell monolayers were washed in PBS and harvested for 20 min in chilled hypotonic lysis buffer (20 mM Hepes, pH 7.4). Lysates were then homogenized and passed consecutively eight times through a 9G needle and 10 times through a 19G needle. Homogenates were centrifuged at 3000 rpm for 5 min to pellet intact cells and nuclei. The supernatants were then spun at 100 000g for 30 min at 4 °C in a refrigerated TL-100 ultracentrifuge to sediment particulate material. The supernatant was collected for the cytosolic fraction analysis, and the pellet was resuspended in hypotonic lysis buffer for membrane fraction analysis. Differential Two-Dimensional Electrophoresis (2D-DIGE). Protein extracts were precipitated using the 2D Clean-UP kit Journal of Proteome Research • Vol. 6, No. 4, 2007 1287

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Gonza´ lez-Santiago et al.

Table 1. Differentially Expressed Proteins between Resistant and Wild-type HeLa Cells Identified in the Membrane Fraction by 2D-DIGE and Mass Spectrometry spot no.a

protein AC (Swiss-Prot)

name

size (Da)

pI

1 2

P62937 Q9UBS4

PPIA DNJBB

18098 40774

7.8 5.8

3 4 5 6 7 8 9 10 11 12 13 14 15

P30837 P25705 P06576 Q13011 P04406 Q14697 Q92947 Q99714 P50213 P30101 Q15084 P55809 P47985

AL1B1 ATPA ATPB ECH1 G3P GANAB GCDH HCD2 IDH3 PDIA3 PDIA6 SCOT UCRI

57680 59751 56560 36136 36070 107263 48127 27003 40022 57146 48490 56650 29918

6.6 8.3 5.0 8.2 8.6 5.7 6.3 7.9 6.5 6.0 5.0 6.8 8.6

16 17

P14735 Q969N2

IDE PIGT

118614 42335

6.3 8.8

18 19

P07910 P23760

HNRPC PAX3

32393 22971

4.9 9.6

21 22

P35232 P60983

PHB GMFB

29843 16743

5.6 5.2

23 24 25 26 27

P60709 P17661 P04264 P05783 Q9UKN7

ACTB DESMIN K2CI K1C18 Myo15

41737 53404 65886 47926 397469

5.3 5.2 8.2 5.3 9.3

28

Q5E966

IF32

36878

5.4

Mascot score

coverage (%)

peptides matched/ unmatched

Chaperone Activity 27 5/4 -2.20 39 13/12 1.26 Energy and Metabolism 161 32 15/9 2.87 101 7/3 1.46 1.41 157 44 10/13 103 30 10/16 1.81 165 19 18/12 1.50 -1.49 254 81 16/8 1.90 115 32 10/13 -1.36 222 51 23/14 1.69 137 25 9/5 1.37 160 35 15/13 1.91 114 32 9/9 1.57 Protein Metabolism 91 15 16 1.78 84 20 6/5 -1.32 Regulation of Nucleic Acid Metabolism 166 40 14/10 -3.07 73 22 5/11 1.24 Signal Transduction 193 66 16/17 1.49 73 36 4/8 1.8 Structural Proteins 115 22 9/6 1.44 105 21 7/3 1.55 71 19 8/5 1.40 2.33 72 5 14/12 1.77 Translation Regulator Activity 127 32 9/7 1.57 82 153

common name

average ratiob

Peptidyl-prolyl-cys-trans isomerase A DNAJ Homologue Aldehyde dehydrogenase ATP synthase R-chain ATP synthase β-chain 5d 2,4 dienoyl-CoA isomerase Glycerald.3Phosphate dehydrogenase Neutral R-glucosidase precursor Glutaryl-CoA Dehydrogenase 3-hydroxyacil-CoA dehydrogenase Isocitrate dehydrogenase (NAD) R-subunit Protein disulphide isomerase A3 Protein disulphide isomerase A6 Succinyl CoA Ubiquinol-cyt c reductase iron-sulfur subunit Insulin protease GPI-transamidase Heterogeneous nuclear ribonucleoprotein Transcription factor activity Prohibitin Glia maturation factor β β-actina Desmin Keratin 2 Keratin 18 Myosin XV A Eukaryotic Translation Initiation Factor 3

a Spot numbers correspond to those included in the 2D image (Figure 1). b Average ratio between Aplidin-resistant and wild-type HeLa cells calculated considering 4 replica gels. Statistic analysis reveval significant ratios in all cases (Student’s t-test, p < 0.005).

(GE Healthcare) and resuspended in the appropriate 2D-DIGE lysis buffer (7 M urea, 2 M thiourea, 25 mM Tris-HCl, 4% CHAPS, pH 8.5). Fifty micrograms of each protein extract was labeled with 400 pmol CyDyes on ice for 30 min in the dark as previously described.10 HeLa-wt and HeLa-R protein extracts were labeled with Cy3 and Cy5, respectively. An internal pool was generated by combining equal amounts of extracts from each cell type; this pool was labeled with Cy2 dye and was included in all gel runs. For membrane fractions, 50% trifluoroethanol (TFE) was added to the rehydration buffer to increase the solubility of these hydrophobic proteins and improve the isoelectrofocusing (IEF).11 Samples were run using commercial IPG strips for the IEF (pH: 3-10, 24 cm length) and standard continuous 12% SDS-PAGE for the second dimension. Finally, a preparative gel containing 500 µg of each cytosolic or membrane fraction was run and stained with Sypro Ruby (Molecular Probes) for protein visualization and spot picking, after matching against the analytical gels. Analysis of Gel Images. Proteins were visualized with a fluorescence scanner (Typhoon 9400, GE Healthcare). The images were processed using DeCyder software v5.1. The DeCyder differential in-gel analysis (DIA) module was used for pairwise comparisons of HeLa-wt and HeLa-R cells to the mixed standard present in each gel and for the calculation of normalized spot volumes/protein abundance. The matching between gels was performed using the internal pool included 1288

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in each gel. The 24 spot maps (12 for cytosolic and 12 for membrane fraction) corresponding to the eight gels were used to calculate average abundance changes and paired Student’s t-test p-values for each protein across the different gels. This was done using the DeCyder biological variation analysis (BVA) module and the Cy3/Cy2 and Cy5/Cy2 ratios for each individual protein. The protein spots that demonstrated a significant change (p < 0.01) in abundance between the protein fractions of HeLa-wt and HeLa-R cells were selected for further characterization using mass spectrometry (MS). Identification of Proteins by MALDI-TOF Peptide Mass Fingerprinting. Spots of interest were excised from the gel automatically using an Ettan-Picker robot (GE Healthcare) and subjected to tryptic digestion according to a previous protocol12 with minor variations.13 Proteins were first reduced (10 mM DTT) and then alkylated (50 mM iodoacetic acid). Following vacuum-drying, the gel pieces were incubated with modified porcine trypsin (Promega) at a final concentration of 10 ng/µL in 50 mM ammonium bicarbonate for 16 h at 37 °C. Supernatants were collected, vacuum-dried, redissolved in 0.5 µL of 0.1% TFA, and added onto a matrix consisting of 0.5 µL of 5 mg/mL 2-5-dihydroxybenzoic acid in water/acetonitrile (2:1) with 0.1% TFA. MALDI-TOF MS analysis of the samples was carried on a mass spectrometer Autoflex (Bruker Daltonics) in positive ion reflector mode. The ion acceleration voltage was 20 kV. Each spectrum was internally calibrated with the masses

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Figure 2. A typical 2D map of the cytosolic proteins obtained by DIGE analysis. Cytosolic protein extracts from wild-type (HeLa-wt) and Aplidin-resistant (HeLa-R) HeLa cells were covalently labeled with Cy3 (green) and Cy5 (red) fluorochromes, respectively. Overlapping image illustrates the changes in protein abundance when wild-type and resistant expression profiles were compared. Arrows indicate those protein whose expression varies within the 99th confidence level (Student’s t-test, p < 0.01) and were unequivocally identified by PMF. Numbers correlate with those included in Table 2.

of two trypsin autolysis products. Smoothing and signal-tonoise criteria were applied following the default Flex Control 1.1 and XMASS 5.1.1 parameters. For PMF identification, the tryptic peptide mass maps were transferred using MS BioTools 2.1 program (Bruker Daltonics) as input to search Swiss-Prot using Mascot software (Matrix Science). Carbamidomethylation (C) and oxidation (M) as fixed and variable modifications, respectively, were taken into account for database searching. Up to one missed cleavage was considered, and a mass accuracy between 50 and 100 ppm was used for all trypticmass searches. Reporting of protein identifications was made according to standardized guidelines.14 Western Blotting. Cell protein extracts were prepared following standard procedures.5 Protein extracts were electrophoresed in polyacrylamide gels and transferred to PVDF (Pall Corporation) membranes. The filters were washed, blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (25 mM Tris, pH 7.4, 136 mM NaCl, 2.6 mM KCl, and 0.5% Tween-20), and incubated overnight at 4 °C with the appropriate antibody. Blots were washed three times for 10 min in PBS and 0.1% Tween-20 and incubated with horseradish peroxidase (HRP) secondary antibodies for 1 h at room temperature. Blots were developed using a peroxidase reaction with the ECL detection system (Amersham Biosciences). The following antibodies were used: anti-JNK1 (rabbit polyclonal, 1:1000 dilution; Santa Cruz, California), anti-phospho-JNK1 (rabbit polyclonal, 1:1000 dilution, Cell Signaling, Beverly, MA), anticyclophilin (rabbit polyclonal, 1:1000 dilution; Upstate, Lake

Placid, UT), anti-PDI (rabbit polyclonal, 1:1000 dilution; Stressgen, Victoria, BC, Canada), anti-ezrin (mouse monoclonal, 1:1000 dilution; Sigma), anti-RabGGTase (rabbit polyclonal, 1:1000 dilution), and anti-cystathionine γ-lyase (rabbit polyclonal, 1:2000 dilution) antibodies, kindly provided by Dr. Seabra (Imperial College London) and Dr. Nishi (Kagawa University), respectively. HPR-conjugated anti-mouse IgG (H+L) was purchased from Promega and HPR-conjugated anti-rabbit IgG (H+L) was from MP Biomedicals. Cell Survival: MTT Assays. [3-(4,5-Dimethythiazol-2-yl)-2,5diphenyl] tetrazolium bromide (MTT) assays were performed following the manufacturer’s instructions (MTT cell proliferation kit I, Roche Diagnostics, Mannheim, Germany).

Results Differential Protein Expression Analysis of Membrane Fractions of Aplidin-Sensitive and -Resistant HeLa Cells. The expression patterns of proteins in the membrane fractions of HeLa-wt and HeLa-R cells were compared using the DeCyder software. This allowed pairwise comparisons to mixed standard for the calculation of normalized spot volume/protein abundance. Four replica gels were used to compare the protein profiles and to calculate average changes and paired Student’s t-test p-values for the detection of significant abundance changes. All these spots were excised from a preparative gel, digested with trypsin, and identified using MALDI-TOF MS. Figure 1 shows the 2D-map of a representative gel. Arrows Journal of Proteome Research • Vol. 6, No. 4, 2007 1289

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Table 2. Differentially Expressed Proteins between Resistant and Wild-type HeLa Cells Identified in the Cytosolic Fraction by 2D-DIGE and Mass Spectrometry spot no.a

protein AC (Swiss-Prot)

name

size (Da)

pI

Mascot score

coverage (%)

peptides matched/ unmatched

Anchor Protein 25 16/12 Chaperone Activity 23 14/11 14 10/6 28 18/12 24 4/4 Energy and Metabolism 55 25/10 26 15/10 57 15/8 30 12/11 5 1c 35 13/10 19 6/5 64 36/12 35 15/18 49 8/4 45 18/6

average ratiob

1

P15311

EZRI

69268

5.95

113

2 3 2 4

P11021 P34932 P11142 P04792

GRP78 HSP74 HSP7C HSPB1

72333 94300 70898 22783

5.01 5.16 5.37 5.76

96 78 175 75

5 6 7 8 9 10 11 12 13 14 15

Q02790 P49915 Q15181 P14618 Q6ZMR3 P07195 Q15274 P07237 P30101 P18669 P00558

FKBP4 GUAA IPYR KPYM LDH6A LDHB NADC PDIA1 PDIA3 PGAM PGK1

51673 76715 33095 57806 36507 36507 31138 57116 56782 28673 44483

5.35 6.42 5.54 7.95 6.51 5.72 5.81 4.69 5.61 6.75 8.3

279 133 194 103 3.48 170 68 431 140 80 218

16 17 18 19

P36871 Q92696 P31939 P60174

PGM1 PGTA PUR9 TPIS

61318 65072 64616 26538

6.32 5.45 6.27 10.7

20

P13765

2DOB

31088

6.31

21 22 23 24

P32929 Q13618 P02679 Q96FW1

CTH CUL-3 FIBG OTUB1

44508 88930 51512 31493

6.21 8.68 5.27 4.85

9

Q06323

PSME1

28723

5.78

23 8 25

Q92522 P61978 O60506 Q01105

HIX HNRPK HNRPQ SET

22474 50976 69603 33489

10.7 5.39 8.68 4.23

26 27

P31948 Q9Y3F4

STIP1 STRAP

62639 38438

6.4 4.98

92 146

Signal Transduction 15 16/10 39 14/9

-1.67 -1.43

Stress induced phosphoprotein 1 Serine-Threonine kinase recep-associated protein

28 29 30 5 31

O43707 P04264 P05787 Q9BQE3 P06753

ACTN4 K2C1 K2C8 TBA6 TPM3

105246 65886 53573 50548 32819

5.22 8.16 5.52 4.96 4.75

76 92 248 82 338

Structural Proteins 12 12/9 17 11/12 41 20/17 20 30/15 69 28/10

1.95 -2.07 1.76 -1.98 1.39

Actinin R-4, cytoskeletal Keratin type II, citokeratin 1 Keratin type II, citokeratin 8 Tubulin R-6 Tropomyosin R-3 chain, cytoskeletal associated

32

P60842

IF41

46154

5.32

68

Translation Regulator Activity 19 7/3

4.053d 78 99 246

3 1c 26 11/9 18 7/6 78 16/9 Immune Response 80 31 7/11 Protein Metabolism 2,355d/2,912d 7 2c 2.525d 2 1c 69 16 8/6 127 40 8/6

1.45

common name

Ezrin, cytoskeletal anchoring activity

3.66 1.61 3.39 -2.02

Heat shock 70 kDa

-2.16 -1.71 -1.56 -1.61 2.14 -1.49 -1.58 3.79 1.98 -1.55 -2.96 -2.09 1.58 -1.51 -1.49

FK506 binding protein, isomerase activity GMP Synthase Inorganic pyrophosphatase Pyruvate kinase L-Lactate dehydrogenase A-like 6A L-lactate dehydrogenese β-chain Nicotinate-nucleotide pyrophosphorylase Protein disulfide isomerase A1 Protein disulfide isomerase A3 Phosphoglyceratomutase Phosphoglycerate kinase 1, polymerase R cofactor Phosphoglucomutase1 RAB geranylgeranyltransferase Bifunctional purine, biosynthesis protein Triosephosphate isomerase

-1.46

MHC receptor activity

-2.84 -2.09 1.50 -1.53

Cystathionine γ-lyase Ubiquitin-specific protease fibrinogen, coagulation factor Otubain 1, Ubiquitin proteasome system protein Proteosome activator

151 48 13/11 Regulation of Nucleic Acid Metabolism 71 30 5/4 -1.49 134 38 15/9 1.50 104 24 7/2 -1.61 112 22 8/6 -1.59

1.44

Heat shock 71 kDa Heat-shock protein β

Histone H1X heterogeneous nuclear riboprotein K heterogeneous nuclear riboprotein Q MHC receptor activity, Phosphatase2A inhibitor

Eukaryotic initiation factor

a

Spot numbers correspond to those included in the 2D image (Figure 2). b Average ratio between Aplidin-resistant and wild-type HeLa cells calculated considering 4 replica gels. Statistic analysis reveval significant ratios in all cases (Student’s t-test, p < 0.005). c The protein identification was comfirmed by LC-MS/MS (data not shown). Only peptides considered for MS/MS are included. d Sequest score.

indicate those proteins identified whose expression varies within the 99th confidence level, based on the variance of the mean change within the cohort. In some cases, the same protein was identified in different spots across the 2D gel, suggesting the occurrence of post-translational modifications. Moreover, in a few spots, several individual proteins were clearly identified together in the same spots. This was probably due to the broad pH gradient used for the isoelectrofocusing. In these particular cases, the average ratio observed corresponds to the combination of all the proteins present in this 1290


spot and individual changes must be confirmed by additional approaches. Because of post-translational modifications, degradation, and alternative splicing isoforms, a total of 39 proteins corresponding to 27 distinct proteins was unequivocally identified by mass spectrometry in the membrane fraction. These proteins are summarized in Table 1. Differential Protein Expression Analysis of Cytosolic Fractions of Aplidin-Sensitive and -Resistant HeLa Cells. Previous studies using 14C-labeled Aplidin have shown that the drug transiently locates to the plasma membrane and then pre-

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Table 3. Relative Levels of Expression of Five Selected Proteins in HeLa-wt and HeLa-R Cells

Figure 3. Validation of proteins differentially expressed in HeLawt and HeLa-R cells. Representative Western blot analysis of membrane (A) and cytosolic (B) proteins using the indicated antibodies. Focal adhesion kinase (FAK) and extracellularregulated kinase (ERK) were used as loading controls, respectively.

dominantly localizes in the cytosol bound to macromolecules. Thus, it was our interest to further analyze changes of protein expression in this subcellular compartment. After protein extraction and fractionation, cytoplasmic extracts of HeLa-R were compared to those of HeLa-wt cells using the same approach described above. The protein profiles obtained by 2D-DIGE in four replica gels were compared computationally and allowed to calculate average changes and paired Student’s t-test p-values for the detection of significant abundance changes. All these spots were excised from a preparative gel and further digested with trypsin. Figure 2 shows a representative 2D-DIGE image of the protein profile of the cytosolic extract. Arrows indicate those 52 proteins whose expression varied within the 99th confidence level and were definitely identified by MS analysis. They correspond to 39 different proteins, which are summarized in Table 2. Confirmation of Differential Expression by Western Blot Analysis. We selected, for further study and validation, two membrane proteins, disulfide isomerase (PDI) and cyclophilin, and three cytosolic proteins, ezrin, cystathionine γ-lyase, and Rab geranylgeranyltransferase (RabGGT) due to their relation to tumoral processes such as glutathione metabolism, apoptosis, and cell transformation. Western blot analyses confirmed the results of the proteomic study: PDI, ezrin, cystathionine γ-lyase, and RabGGTase R were up-regulated, while cyclophilin was down-regulated in HeLa-R cells as compared to HeLa-wt cells (Figure 3). The ratios of the different expression levels were quantified in three independent experiments by densitometric analysis of immunoblots and shown in Table 3. Major differences were found for cystathionine γ-lyase and RabGGTaseR. Focal adhesion kinase (FAK) and extracellularly regulated kinase (ERK) were used as controls for membrane and cytosolic proteins, respectively. Functional Validation. For a further, functional confirmation of the implication of PDI, RabGGT, and cystathionine γ-lyase in the cellular response to Aplidin, we took advantage of the existence of inhibitors for these enzymes: bacitracin, GGTI298, and DL-propargylglycine, respectively. Pretreatment of

protein name

HeLa-R/HeLa-wt

Disulfide isomerase Cyclophilin A Ezrin Cystathionine lyase RabGGTase R

1.8 0.7 1.6 2.1 2.3

HeLa-R cells with bacitracin at doses that caused only limited toxicity had a small but consistent effect increasing JNK phosphorylation that is crucial for apoptosis induction by Aplidin5 (Figure 4A) and abrogating the relative resistance to the drug of HeLa-R cells (Figure 4B). In contrast, no effect was observed for GGTI-298 and DL-propargylglycine (data not shown). In addition, we overexpressed cyclophilin A (CypA) in both HeLa-wt and HeLa-R cells. To this end, we added human recombinant CypA protein to the culture medium, a procedure commonly used in the literature.15,16 CypA overexpression did not change the activation of JNK or the sensitivity of HeLa-wt cells to Aplidin (Figure 5A,B). In line with its down-regulation in HeLa-R cells, overexpression of CypA abrogated the partial resistance to Aplidin of HeLa-R cells, although it did not affect JNK activation (Figure 5A,B). To examine the role of ezrin up-regulation, we transfected HeLa-wt and HeLa-R cells with a dominant negative ezrin mutant (N-ezrin)17 or with an empty vector as control. No changes were found in Aplidin sensitivity or JNK activation following N-ezrin expression in either HeLa-wt or HeLa-R cells (Figure 5C,D). Collectively, these results indicate that PDI and cyclophilin A, but perhaps not ezrin, contribute to the resistance to Aplidin at least in HeLa cells.

Discussion Proteomic analysis, utilizing high-resolution 2-DE coupled with MS has successfully identified protein changes in drug resistance studies.18-21 The introduction of CyDye fluorophores (2D-DIGE) for prelabeling of protein samples, which adds a quantitative component to conventional 2-D analysis, allows the comparison of protein expression changes across multiple samples simultaneously with statistical confidence.22,23 In this study, we have used proteomic techniques to compare proteins expressed in the cytosol and membrane fractions of Aplidinsensitive and -resistant HeLa cells. We have detected 91 proteins differentially expressed, of which 66 were identified. These proteins are good candidates to mediate the action and/ or sensitivity of human cancer cells to this antitumoral drug. Historically, comparative studies of drug-resistant cells to their nonresistant counterparts have focused on single specific proteins, such as P-glycoprotein involved in drug efflux,24 glutathione-S-transferases involved in drug detoxification,25,26 metallothionein involved in drug sequestration,27 and topoisomerase II involved in DNA metabolism.28 However, the mechanism by which cancer cells develop resistance toward an anticancer drug is complex and quite probably implies the combination of several factors. Thus, the global protein picture in drug-resistant cancer cells is of interest. In this study, we observed a general increase in several heatshock proteins like HSP7C, HSP5A, HSPA8, HSP4A, and GRP58, which are mainly involved in protein folding, assembly, and trafficking. These chaperones are known to increase resistance Journal of Proteome Research • Vol. 6, No. 4, 2007 1291

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Figure 4. Effect of protein disulfide isomerase inhibition on cellular resistance to Aplidin. HeLa-wt and HeLa-R cells were incubated for 16 h with the indicated doses of bacitracin (Bac) and then treated with either vehicle (control) or Aplidin. (A) Western blot analysis of phosphorylated and total JNK in cells treated with 450 nM Aplidin for the indicated times. Numbers refer to fold-increase JNK phosphorylation after normalization to total JNK level. (B) Effect of bacitracin on Aplidin-induced cytotoxicity. HeLa-wt (white columns) and HeLa-R (black columns) cells were incubated for 16 h with the indicated doses of bacitracin or vehicle (-) and then treated with increasing doses of Aplidin alone or in combination with bacitracin for 24 or 48 h. Cell survival was estimated by the MTT method as described in Materials and Methods. The percentage of surviving cells was calculated in each case with respect to cultures treated with vehicle and the corresponding bacitracin concentration. Forty-eight hours treatment using high (3 mM) bacitracin dose was cytotoxic. Three experiments were performed in triplicate, *p < 0.05.

to cell death induced by a variety of stimuli.29,30 In particular, GRP78 has been demonstrated to play a role in protection against citotoxicity and apoptosis induced by environmental factors. As Aplidin is a potent apoptosis inducer, it is interesting that a number of up-regulated proteins in the extracts of HeLa-R cells have been previously described as molecules able to prevent apoptosis. In this sense, not only chaperones, but also other proteins included in Tables 1 and 2, have been linked to apoptosis. HNRPC (Heterogeneous nuclear ribonucleoprotein), which is thought to be involved in RNA splicing, belongs to a critical set of protein substrates that are cleaved by ICElike proteases during apoptosis. Interestingly, the expression of this protein is decreased in the HeLa-R cells, supporting the idea that apoptosis pathways are mainly associated to Aplidin resistance. Among other proteins identified there are two members of the immunophilin family, FKBP4 and PPIA/cyclophilin. These cis-trans isomerases are involved in protein folding and trafficking. Moreover, FKBP4 and cyclophilin bind to rapamycin and cyclosporin, respectively, and play a role in the immunosuppression mediated by these molecules. The expression of both proteins is decreased in HeLa-R cells. The results obtained in experiments overexpressing CypA support a role of this protein in the sensitivity to Aplidin but at a level distint to JNK, difficult to propose in view of the numerous effects attributed to Cyp proteins.31,32 The ERM proteins (ezrin, radixin, moesin) together with merlin compose a subgroup of the band 4.1 superfamily. These proteins act as membrane cytoskeletal linker proteins, mediating the interactions between the cytoplasmic domains of transmembrane proteins and actin. Ezrin interacts with p85, the regulatory subunit of phoshatidylinositol 3-kinase and activates the protein kinase Akt that protects cells against apoptosis. Overexpression of ezrin in the HeLa-R cells may 1292


activate the Akt pathway, inhibiting the induction of apoptosis by the drug. In addition, a role of Fas in Aplidin-induced apoptosis has been shown in leukemic cells.33 Ezrin links Fas to the actin cytoskeleton, and it is an essential requirement for the susceptibility to Fas-mediated apoptosis.34 The lack of effects of a transfected dominant negative Ezrin mutant does not support a major role of this protein in Aplidin sensitivity, and does not definitively exclude a certain contribution. Rab geranylgeranyltransferase is up-regulated in HeLa-R cells. Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C-termini that mediate vesicle transport in the secretory and endocytotic pathways. It has been proposed that Rab proteins cycle between soluble and membrane bound forms as part of their regulatory role in protein trafficking. They differ from other geranylgeranylated small GTPases in several important respects: (i) most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs, -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. It has been suggested that the absence of cleavage in this enzyme indicates a lower level of apoptosis in cells.35 Therefore, its overexpression should contribute to enhance the resistance to apoptosis in the resistant cells. Cystathionine γ-lyase is significantly repressed in HeLa-R cells. It is a key cytoplasmic enzyme in the trans-sulfuration pathway that converts cystathionine derived from methionine into cysteine. Glutathione synthesis in the liver is dependent upon the availability of cysteine; low synthesis of cysteine should reduce the levels of gluthatione. Alternative splicing of this gene results in two transcript variants encoding different

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isoforms. It has been reported that cystathionine γ-lyase overexpression inhibits cell proliferation via an H2S-dependent modulation of ERK1/2 phosphorylation and p21Cip/WAF-1.36 Since cystathionine γ-lyase is repressed in HeLa-R cells, they should have reduced levels of glutathione. As Aplidin alters glutathione homeostasis, increasing the ratio of oxidized to reduced forms,6 low levels of GSH are expected to render the cells more sensitive to oxidative stress. Our results using the inhibitor did not however change the relative resistance to the drug of HeLa-R cells. As a whole, the data regarding the role of cystathionine γ-lyase in Aplidin action remain difficult to explain. Protein disulfide isomerases (EC 5.3.4.1), such as PDI, are endoplasmic reticulum resident proteins that catalyze protein folding and thiol-disulfide interchange reactions.37 PDI has a role in conferring resistance to apoptosis under hypoxia and a potential role in the oxygen-sensing apparatus. PDI has also been reported to be a negative regulator of nuclear factor kappa B, a major inductor of cell survival by regulating numerous genes.38 Our results showing PDI up-regulation in HeLa-R cells and abrogation of drug resistance of these cells by means of a specific disulfide isomerase inhibitor (bacitracin) indicate that PDI plays a role in Aplidin action and/or resistance. In summary, we describe here, using a powerful proteomic analysis, a number of changes in the level of expression of membrane and cytosolic proteins that should contribute to Aplidin action. Some of these alterations provide a major evasion route for the cells to escape the induction of apoptosis by Aplidin and will help to design customized models for therapeutic intervention in cancer patients. Further work is needed to elucidate the contributions of other individual proteins. Sensitive proteomic technologies, such as 2D-DIGE, are a unique way to gain deeper insight into these resistanceassociated mechanisms.

Figure 5. Effect of cyclophilin A overexpression and of ezrin inhibition on cellular resistance to Aplidin. (A) HeLa-wt and HeLa-R cells were co-incubated with human recombinant cyclophilin A (CypA; 5 nM) and either vehicle (control) or Aplidin (450 nM) for the indicated times. Western blot analysis of phosphorylated and total JNK was performed. Numbers refer to fold-increase JNK phosphorylation after normalization to total JNK level. (B) Effect of CypA on Aplidin-induced cytotoxicity. HeLa-wt (white columns) and HeLa-R (black columns) cells were incubated with the indicated concentrations of Aplidin alone or in combination with CypA or vehicle (-) for 48 h. Cell survival was estimated by the MTT method as described in Materials and Methods. The percentage of surviving cells was calculated in each case with respect to cultures treated with vehicle and the corresponding CypA concentration. Two experiments were performed in triplicate, *p < 0.05. (C) HeLa-wt and HeLa-R cells were transfected with a plasmid encoding the N-ezrin dominant negative mutant or an empty vector as control. One day later, cells were treated with 450 nM Aplidin for the indicated times. Western blot analysis of phosphorylated and total JNK was performed. Numbers refer to foldincrease JNK phosphorylation after normalization to total JNK level. (D) Effect of N-ezrin on Aplidin-induced cytotoxicity. HeLa-wt (white columns) and HeLa-R (black columns) cells transfected with N-ezrin or empty vector were incubated for 24 h with Aplidin (450 nM) or vehicle (-). Cell survival was estimated by the MTT method. The percentages of surviving cells are shown. Two experiments were performed in triplicate, *p < 0.05.

Acknowledgment. We thank Teresa Martıńez for her excellent technical assistance. This work has been partially supported by Grant SAF2004-01015 from the “Ministerio de Educacioń y Ciencia” of Spain. The CNIO and the Instituto de Investigaciones Biome´dicas were partially supported by the RTICC (Red de Centros de Cańcer, FIS RD06/0020/009 and RD06/0020/0083) and the Comunidad Autońoma de Madrid (S-GEN-0266/2006). References (1) Sakai, R.; Rinehart, K. L.; Kishore, V.; Kundu, B.; Faircloth, G.; Gloer, J. B.; Carney, J. R.; Namikoshi, M.; Sun, F.; Hughes, R. G., Jr.; Garcia Gravalos, D.; de Quesada, T. G.; Wilson, G. R.; Heid, R. M. Structure-activity relationships of the didemnins. J. Med. Chem. 1996, 39 (14), 2819-2834. (2) Lobo, C.; Garcia-Pozo, S. G.; Nun j ez de Castro, I.; Alonso, F. J. Effect of dehydrodidemnin B on human colon carcinoma cell lines. Anticancer Res. 1997, 17 (1A), 333-336. (3) Depenbrock, H.; Peter, R.; Faircloth, G. T.; Manzanares, I.; Jimeno, J.; Hanauske, A. R. In vitro activity of aplidine, a new marinederived anti-cancer compound, on freshly explanted clonogenic human tumour cells and haematopoietic precursor cells. Br. J. Cancer 1998, 78 (6), 739-744. (4) Cuadrado, A.; Garcia-Fernandez, L. F.; Gonzalez, L.; Suarez, Y.; Losada, A.; Alcaide, V.; Martinez, T.; Fernandez-Sousa, J. M.; Sanchez-Puelles, J. M.; Mun j oz, A. Aplidin induces apoptosis in human cancer cells via glutathione depletion and sustained activation of the epidermal growth factor receptor, Src, JNK, and p38 MAPK. J. Biol. Chem. 2003, 278 (1), 241-250. (5) Cuadrado, A.; Gonzalez, L.; Suarez, Y.; Martinez, T.; Mun j oz, A. JNK activation is critical for Aplidin-induced apoptosis. Oncogene 2004, 23 (27), 4673-4680.

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Proteomic Analysis of the Resistance to Aplidin in Human Cancer

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