Article pubs.acs.org/jpr
Proteomic Profiling of Germ Cell Cancer Cells Treated with Aaptamine, a Marine Alkaloid with Antiproliferative Activity Sergey A. Dyshlovoy,†,‡ Ina Naeth,† Simone Venz,§,∥ Michael Preukschas,† Henning Sievert,† Christine Jacobsen,† Larisa K. Shubina,‡ Manuela Gesell Salazar,∥ Christian Scharf,⊥ Reinhard Walther,§ Marcel Krepstakies,¶ Poornima Priyadarshini,¶ Joachim Hauber,¶ Sergey N. Fedorov,‡ Carsten Bokemeyer,† Valentin A. Stonik,‡ Stefan Balabanov,*,†,# and Friedemann Honecker*,†,# †
Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany ‡ Laboratory of Marine Natural Products Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, Russian Federation § Department of Medical Biochemistry and Molecular Biology, ∥Interfacultary Institute of Genetics and Functional Genomics, Department of Functional Genomics, and ⊥Department of Otorhinolaryngology, Head and Neck Surgery, University of Greifswald, Greifswald, Germany ¶ Heinrich Pette Institute−Leibniz Institute for Experimental Virology, Hamburg, Germany S Supporting Information *
ABSTRACT: Aaptamine is a marine compound isolated from the sponge Aaptos aaptos showing antiproliferative properties via an undefined mode of action. We analyzed the effects of aaptamine treatment on the proliferation and protein expression of the pluripotent human embryonal carcinoma cell line NT2. Effects on proliferation, cell cycle distribution, and induction of apoptosis were analyzed. At lower concentrations, including the IC50 of 50 μM, aaptamine treatment resulted in a G2/M phase cell cycle arrest, whereas at higher concentrations, induction of apoptosis was seen. Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of the most significantly up- and down-regulated proteins. Aaptamine treatment at the IC50 for 48 h resulted in alteration of 10 proteins, of which five each showed upand down-regulation. Changes in the 2D map were frequently noticed as a result of post-transcriptional modifications, e.g., of the hypusine modification of the eukaryotic initiation factor 5A (eIF5A). Observed alterations such as increased expression of CRABP2 and hypusination of eIF5A have previously been identified during differentiation of pluripotent cells. For the first time, we describe changes in protein expression caused by aaptamine, providing valuable information regarding the mode of action of this compound. KEYWORDS: aaptamine, marine alkaloids, antiproliferative activity, germ cell cancer, proteome analysis, eIF5A, hypusination, differentiation
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INTRODUCTION Aaptamine (8,9-dimethoxy-1H-benzo[de][1,6]naphtyridine) is a natural compound, initially isolated in 1982 by Nakamura et al. from the marine sponge Aaptos aaptos.1 The substance has been previously shown to regulate a variety of biological processes, including smooth muscle activity and antioxidative capacity. Furthermore, aaptamine derivatives possess antimicrobial, antifungal, and antiretroviral as well as cancer-preventive activity.2−6 In addition, aaptamine inhibits various enzymes, particularly the activity of monoamine oxidase (MAO) and protein kinase C (PKC). Aaptamine-related compounds have been shown to inhibit sortase A and exhibit antifouling activities as well as potent antiproliferative properties (for review, see ref 7). However, the exact molecular mode of action of aaptamine and its derivatives is © 2012 American Chemical Society
still unknown. In recent studies, it has been shown that aaptamine intercalates into DNA with relatively weak binding affinity8 and induces apoptosis at relatively high concentrations.9 Furthermore, this compound shows inhibitory activity on the proteasome by mechanisms unrelated to cytotoxicity.10 In addition, Akoi et al. recently reported that aaptamine induces the promoter of p21 in the human osteosarcoma cell line MG63 in a p53-independent way. This effect was followed by a G2/M phase cell cycle arrest.11 Despite these recent advances in our understanding of the effects of aaptamine, knowledge of the molecular targets and the mode of action of this promising substance are still elusive. Received: October 28, 2011 Published: March 13, 2012 2316
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Coulter Vi-CELL (Beckman Coulter, Krefeld, Germany). In brief, 8 × 104 cells/well were seeded in 12-well plates and incubated overnight. The medium was replaced with fresh medium containing aaptamine at concentrations of 0, 1, 5, 10, 25, or 50 μM in a total volume of 2 mL/well, and cells were incubated for 48 h. Drug-containing medium was removed, and cells were washed with 0.5 mL of PBS and trypsinized with 0.5 mL of trypsin-EDTA solution (Invitrogen). The number of viable (trypan blue excluding) and non-viable cells in both the medium and after trypsination was evaluated with the Beckman Coulter Vi-CELL counting trypan blue positive and negative cells. Assays were performed in triplicates, and Student’s t test was used to determine statistical significance.
Here, we present a combinational approach of a global proteome screening and subsequent functional analyses to explore the mode of action of aaptamine. In contrast to data generated by gene array analysis, using a proteome screening approach offers the advantage to identify altered proteins as the directly involved effector molecules. Thereby, we identified aaptamine induced changes in the protein expression pattern of the human germ cell cancer cell line NT2. Interestingly, further analyses revealed protein alterations induced by post-transcriptional modifications as a main consequence of aaptamine treatment. In particular, our studies demonstrate the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF5A) as a prominent target of aaptamine action. Building on our previous aaptamine alkaloids bioactivity research,12 this work represents the first approach based on global proteome screening and provides insights into the molecular mechanisms behind aaptamine bioactivity.
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Protein Preparation
Preparation of protein extracts for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blotting was performed as described previously.15,16 In brief, 2 × 106 cells/well were seeded in culture flasks, incubated overnight, and treated with aaptamine for 48 h. Cells were harvested with medium flow, pelleted by centrifugation for 5 min at 453g, and washed 3 times with PBS, followed by pelleting using the same conditions. For 2D-PAGE cells were lysed with 450 μL of 2DPAGE lysis buffer (9 M urea, 4% [w/v] CHAPS, 1% [v/v] Pharmalyte, 1% [w/v] DTT, 0.01% [w/v] bromophenol blue) at room temperature for 20 min. For Western blotting, cells were lysed with 100 μL of lysis buffer (0.88% [w/v] NaCl, 50 mM Tris-HCl (pH 7.6), 1% NP-40 [v/v], 0.25% [w/v] NaClO2, 1 mM PMSF, 1 mM Na3VO4) on ice for 20 min. Lysates were frozen overnight at −20 °C and centrifuged at 11170g for 10 min. Protein concentration in the supernatants was determined by Bradford assay.17
EXPERIMENTAL SECTION
Reagents and Antibodies
Aaptamine (8,9-dimethoxy-1H-benzo[de][1,6]naphtyridine) was isolated from the marine sponge Aaptos sp. as described before.9 GC7 (N1-guanyl-1,7-diaminoheptane), 3H-spermidine, CNI-1493 (N,N′-bis[3,5-bis[N-(diaminomethylideneamino)-Cmethylcarbonimidoyl]phenyl]decanediamide tetrahydrochloride), puromycin (3′-deoxy-N,N-dimethyl-3′-[(O-methyl-L-tyrosyl)amino]adenosine), and cisplatin (cis-diamminedichloroplatinum(II), 1 mg/mL in dH2O) were purchased from NeoCorp, Weilheim, Germany. Coomassie Brilliant Blue G 250 was purchased from Carl Roth, Karlsruhe, Germany. Primary and secondary antibodies used are listed in the Supporting Information (Supplementary Table S1).
Western Blotting
Cell Lines and Culture Conditions
Protein extracts were diluted with lysis buffer and loading dye up to a total protein concentration of 1−1.5 μg/μL, boiled 5 min at 99 °C, and subjected to electrophoresis in 12.5−15% SDS-PAGE at 120 V. Proteins were transferred from gel to 0.2 μm pore PVDF membrane (Millipore, Bedford, MA, USA) at 20 V for 1 h. The membrane was blocked with 5% [w/v] nonfat dry milk in 0.05% Tween-20/TBS and treated with primary antibody solution according to the manufacturer’s protocol (for antibodies used, see Supporting Information). After washing, the membranes were incubated with appropriate secondary antibodies during 1 h at RT. The signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol.
NT2 cells13 were obtained from DSMZ (Braunschweig, Germany), and 293T cells were obtained from ATCC. NT2 and 293T cells were cultured in DMEM medium supplemented with Glutamax-I (Invitrogen, Paisley, U.K.) containing 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Cells were incubated at 37 °C in a humidified atmosphere with 5% (v/v) CO2. Cytotoxicity Assay
In vitro cytotoxicity of aaptamine was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which was performed as previously described.14 In brief, 6 × 103 cells/well were seeded in 96-well plates and incubated overnight. The medium was replaced with medium containing aaptamine at concentrations of 25, 50, 100, or 200 μM in a total volume of 0.1 mL/well, and cells were incubated for 48 h. After addition of 0.5 mg/mL MTT (Sigma, Taufkirchen, Germany), the cells were further incubated for 2 h. The medium was removed, and 0.05 mL/well of DMSO (Sigma) was added. The plates were agitated for 10 min, and optical density was read with a photometer (Victor2 Wallac 1420, PerkinElmer, Wellesley, MA, USA) at 570 nm. The background was read at 620 nm. The experiment was performed in triplicate, and the result was expressed as the drug concentration that inhibits viable cells by 50% (inhibitory concentration, IC50), compared to cells treated with the solvent (ethanol, 0.5% [v/v]) alone.
Two-Dimensional Gel Electrophoresis (2D-PAGE)
2D-PAGE was performed as previously described.15 In brief, aliquots of 450 μL of solution containing 1 mg of protein extract were loaded on a linear gradient Immobiline Dry Strip (IPG Strip pH 4−7, 24 cm, Amersham Biosciences, Uppsala, Sweden), followed by rehydration of strips overnight and isoelectric focusing (IEF) using the Protean IEF cell (Bio-Rad, Hercules, CA, USA). The first dimension was performed at 10000 V for 80 kVh. After IEF, IPG strips were equilibrated with 1% DTT in urea buffer (6 M urea, 4% SDS and 50 mM of 1.5 M Tris-HCl (pH 8.8)) for 15 min at RT and then alkylated with 4.8% iodoacetamide in urea buffer for 15 min at RT. The equilibrated strips were directly loaded onto 15% SDS-polyacrylamide gels (27 cm × 21 cm × 1.5 mm) and overlaid with 0.6% [w/v] agarose in dH2O. Second dimension electrophoresis was conducted in SDS-running buffer
Cell Proliferation Assay
Evaluation of cell growth inhibition by aaptamine was performed with the trypan blue method using the Beckman 2317
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50; S/N threshold 3; noise window width (m/z) 250. The fragmentation was proven by an implemented ion fragmentation calculator with mass tolerance of 0.2 m/z and the use of monoisotopic masses.
(1.44% [w/v] glycine, 0.3% [w/v] Tris base, 0.1% [w/v] SDS at 20 °C) for 14 h. Gels were fixed and stained with colloidal Coomassie Blue (400 mL/gel, containing 0.13% [w/v] Coomassie Brilliant Blue G 250, 3.6% [v/v] H2SO4, 1.44 M NaOH, 20.3% [w/v] CCl3COOH) overnight and destained for 48 h with dH2O. All experiments were performed in triplicates.
Mini-2D Western Blotting Analysis (2D-WB)
2D-WB analysis was used to confirm the results of 2D-PAGE. Protein samples were prepared as described for 2D-PAGE. Aliquots of 125 μL of solution containing 30 μg of protein were loaded on a linear gradient Immobiline Dry Strip (IPG Strip pH 4−7, 7 cm Amersham Biosciences), followed by rehydration of strips overnight and isoelectric focusing (IEF) using the Protean IEF cell. The first dimension was carried out at 5000 V for 5 kVh. Consecutive equilibration and alkylation of the strips were carried out as described for the 2D-PAGE experiment. The equilibrated strips were directly loaded onto 15% SDS-polyacrylamide gels, overlaid with 0.6% [w/v] agarose in dH2O, and run 3 h at 65 V. Consecutive transfer, blocking, incubation with antibodies and detection steps were carried out as described for the Western blotting experiments; see above. Relative optical density of the signal intensity of the spots was quantified with Bio 1D 15.01 software (Vilber Lourmat, France). After the transfer step, polyacrylamide gels were stained with Coomassie Blue as described for the 2DPAGE experiment and scanned using the GS-800 Calibrated Densitometer. Images of the Coomassie Blue stained gels are provided in the Supporting Information section (Figures S1−4).
2D Gel Image Analysis
The 2D-gels were scanned using the GS-800 Calibrated Densitometer (BioRad). Spot matching, normalization of the digital images (based on total optical density), and gel image analysis was performed using Delta 2D 4.0 software (Decodon, Greifswald, Germany). Quantitative analysis was performed using fold change value and Student’s t test comparing treated and nontreated cells. Assumed difference in spot intensity of relevant spots was ≥2 times, and assumed confidence level for p-values was ≤0.05. Protein Identification by Mass Spectrometry
Protein spots of interest were excised from the gel manually and transferred to 96-well plates. Digestion with trypsin and subsequent spotting of peptide solutions onto the MALDI targets were performed automatically in the Ettan Spot Handling Workstation (Amersham-Biosciences) using a modified standard protocol. In-gel protein digestion was performed as previously described.18 The MALDI-MS measurement of spotted peptide solutions was carried out on a 4800 MALDIToF/ToF Analyzer (Applied Biosystems, Foster City, CA, USA). The spectra were recorded in reflector mode in a mass range from 800 to 4000 Da with a focus mass of 2000 Da. The instrument was calibrated with the autolytical fragment of trypsin, an internal calibration was automatically performed using this peak for one-point-calibration. Additionally, MALDIMS/MS analysis was performed for the five strongest peaks of the MS spectrum after subtraction of peaks corresponding to background or trypsin fragments. Information regarding the number of peptides that resulted in a positive MS/MS signal are given in Table 1. After calibration, a combined database search of MS and MS/MS measurements was performed using the GPS Explorer software (Ver. 3.6, Applied Biosystems, Foster City, CA, USA). Peak lists were compared with the SwissProt database v56.1 human taxonomy. Peptide mixtures that yielded at least twice a mowse score of 56 (p < 0.05) for database results were regarded as positive identifications. The default significance threshold was p < 0.05.
Detection of Apoptosis
To examine induction of apoptosis by aaptamine, FACSbased analysis with Annexin-V-FLUOS (Roche, Mannheim, Germany) and propidium iodide (PI) (Sigma) double staining was performed as previously described.19 In brief, NT2 cells were preincubated overnight in 6-well plates (2 × 105 cells/ well). The medium was changed with medium containing different concentrations of aaptamine. After 48 h of treatment, cells were harvested with trypsin-EDTA solution, washed with PBS, and incubated with 0.1 mL of Annexin-V-FLUOS and PI containing labeling buffer for 30 min in the dark at RT. Cells were analyzed using the FACS Calibur (BD Bioscience, Bedford, MA, USA). The results were analyzed with BD Bioscience Cell Quest Pro software. Cell Cycle Analysis
The cell cycle distribution was analyzed by flow cytometry using PI staining. NT2 cells were treated as described above (see Detection of Apoptosis),20 and then each sample was washed with ice-cold PBS and fixed with 1 mL of 100% ethanol at −20 °C overnight. After addition of 0.5 mL of PBS and pelleting (5 min at 220g), cells were resuspended in 0.2 mL of buffer containing RNase (0.2 mg/mL, Roth) and PI (0.02 mg/mL) and incubated for 30 min in the dark on ice. Then 0.2 mL of PBS was added, and cells were analyzed using the FACS Calibur. The results were quantitatively analyzed using the BD Bioscience Cell Quest Pro software.
Identification of Hypusine Modification by Mass Spectrometry
For identification of the post-translational modification of lysine to hypusine (KHyp) in the protein eIF5A, we performed a manual in-gel digest similar as described18 with LysC protease (200 ng per gel spot) instead of trypsin. The measurements were also performed using a 4800 MALDI-ToF/ToF analyzer. The spectra were recorded in reflector mode in a mass range from 300 to 1000 Da with a focus mass of 700 Da and in a mass range from 875 to 4000 Da with a focus mass of 2000 Da. The instrument was calibrated with an external calibration using matrix dimers at m/z 379.0930 and des-Arg1-Bradykinin at m/z 904.4681 for the mass range of 300 to 1000 or using Mass Standards Kit for Calibration of AB SCIEX TOF/TOF instruments as default calibration. MALDI-MS/MS analysis was performed for m/z 549.31 and for m/z 922.67. The MS and MS/MS spectra were annotated using the Data Explorer Software Vers. 4.9 (build115). Peak detection was executed using the following parameters: valley to baseline; % centroid
Quantitative Real-Time PCR (qRT-PCR)
RNA was isolated from treated and nontreated NT2 cells using TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol. cDNA was prepared by reverse transcription of 1 μg total RNA using oligo(dT)18 primer and RevertAid M-MuLV reverse transcriptase (Fermentas, St. Leon-Rot, Germany). The DyNAmo SYBR Green qPCR Kit (Finnzymes, Vantaa, Finland) was used for quantitative PCR, and 20 μL reactions were set up according to the 2318
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ENO1
CSDE1
ACL6A
SRSF3
PEA15
TPT1
CTSD
CRABP2
CFL1
2
3
4
5
6
7
8
9
10
Cellular retinoic acid-binding protein 2 (CRABP2) Cofilin-1
Splicing factor, arginine/serinerich 3 Astrocytic phosphoprotein PEA15 Translationally- controlled tumor protein Cathepsin D
Cold shock domain- containing protein E1 Actin-like protein 6A
Eukaryotic translation initiation factor 5A-1 Alpha-enolase
protein name
P23528
P29373 18491
15683
44524
19583
P13693 P07339
15031
19318
47430
88829
47139
16821
theoretical Mw (kDa)
Q15121
P84103
O96019
O75534
P06733
P63241
Swiss-Prot accession no
Spot numbers are equal to the spot numbers in Figure 2.
EIF5A
1
a
gene name
spot no.
318
392
449
254
94
358
123
213
236
208
protein score
11
11
19
11
8
11
9
15
13
15
no. of peptides assigned to the protein
2
3
4
3
1
3
2
2
1
1
no. of sequenced peptides
65
68
46
55
50
53
29
26
42
74
sequence coverage (%)
8.2
5.4
6.1
4.8
4.9
11.6
5.4
5.9
7.0
5.1
theoretical pI
Table 1. Differentially Expressed Proteins of Untreated versus Aaptamine-Treated NT2 Cells after Identification with MALDI-TOF-MSa
3.49
2.60
2.20
2.12
2.03
0.46
0.41
0.39
0.33
0.16
fold change protein
cytoskeleton and cell movement
cytoskeleton and cell movement metabolism (intracellular protein breakdown) cell signaling/differentiation
DNA binding and transcriptional regulation translational regulation (RNAbinding protein) DNA binding and transcriptional regulation translational regulation (RNA processing) cell signaling
protein biosynthesis
protein function
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manufacturer’s instructions. One microliter template cDNA was used per reaction. Expression of human eIF5A, cofilin-1, CRABP2, and α-enolase were analyzed using the appropriate quantitect qRT-PCR-primers (Qiagen, Hilden, Germany; eIF-5A: QT00099911, cofilin-1: QT00201348, CRABP2: QT00063434, and α-enolase: QT01681722). For normalization, expression of RPLP0 was used (QT01839887). The qPCR was performed on a Mx3000P cycler (Stratagene, Amsterdam, The Netherlands) with the following cycling conditions: 95 °C for 7 min and 35 cycles of 95 °C for 10 s and 60 °C for 15 s. Relative expression was calculated using the 2−ΔΔCT method. To test statistical significance, data were analyzed by unpaired Student’s t tests, and a p-value of
