Original Article Open Access
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Stem Cells. Jan 26, 2013; 5(1): 9-25
Published online Jan 26, 2013. doi: 10.4252/wjsc.v5.i1.9
Impact of the antiproliferative agent ciclopirox olamine treatment on stem cells proteome
Gry H Dihazi, Asima Bibi, Olaf Jahn, Jessica Nolte, Gerhard A Mueller, Wolfgang Engel, Hassan Dihazi
Gry H Dihazi, Asima Bibi, Gerhard A Mueller, Hassan Dihazi, Department of Nephrology and Rheumatology, Georg-August University Goettingen, D-37075 Goettingen, Germany
Olaf Jahn, Proteomics Group, Max-Planck-Institute of Experimental Medicine, D-37075 Goettingen, Germany
Olaf Jahn, Deutsche Forschungsgemeinschaft Research Center for Molecular Physiology of the Brain, D-37073 Goettingen, Germany
Jessica Nolte, Wolfgang Engel, Institute of Human Genetics, Georg-August University Goettingen, D-37073 Goettingen, Germany
Author contributions: Dihazi GH and Bibi A performed the majority of experiments, interpreted the data and wrote the article; Nolte J and Engel W provided the stem cell lines and were also involved in revising the manuscript; Jahn O was responsible for the acquisition of the mass spectrometry data and revised the article critically; Mueller GA helped by the study design and manuscript writing; Dihazi H designed and concepted the study and revised the article critically; Dihazi GH and Bibi A contributed equally to this work.
Correspondence to: Hassan Dihazi, PhD, Department of Nephrology and Rheumatology, Georg-August University Goettingen, Robert-Koch-Strasse 40, D-37075 Goettingen, Germany. dihazi@med.uni-goettingen.de
Telephone: +49-551-3991221 Fax: +49-551-3991039
Received: February 7, 2012
Revised: September 19, 2012
Accepted: December 20, 2012
Published online: January 26, 2013

Abstract

AIM: To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells.

METHODS: Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA), which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics.

RESULTS: Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We could highlight, that CPX treatment of stem cells, with subsequent proliferation inhibition, resulted in an alteration of the expression of 56 proteins compared to non-treated cells, and 54 proteins compared to RA treated cells. Bioinformatics analysis of the regulated proteins demonstrated their involvement in various biological processes. To our interest, a number of proteins have potential roles in the regulation of cell proliferation either directly or indirectly. Furthermore the classification of the altered polypeptides according to their main known/postulated functions revealed that the majority of these proteins are involved in molecular functions like nucleotide binding and metal ion binding, and biological processes like nucleotide biosynthetic processes, gene expression, embryonic development, regulation of transcription, cell cycle processes, RNA and mRNA processing. Proteins, which are involved in nucleotide biosynthetic process and proteolysis, were downregulated in CPX treated cells compared to control, as well as in RA treated cells, which may explain the cell cycle arrest. Moreover, proteins which were involved in cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis, and phosphorylation were downregulated in RA treated cells compared to control and CPX treated cells.

CONCLUSION: The CPX treatment of SCs results in downregulation of nucleotide binding proteins and leads to cell cycle stop without impairment of pluripotency.

Key Words: Stem cells, Differentiation, Hypusination, Ciclopirox olamine, Proteomics, Retinoic acid



INTRODUCTION

Stem cells (SCs) are cells, which are found in all multicellular organisms, which can continuously divide and differentiate into various specialized cell types and can also self-renew to produce more stem cells[1]. The therapeutic use of embryonic stem cells (ESCs) has been constrained by problems caused by immune rejection in the patient as well as ethical issues associated with the use of embryos[2]. Spermatogonial stem cells (SSCs) are self-renewing single cells located in the periphery of the seminiferous tubules whose continuous division maintain spermatogenesis throughout the life of a male individual[3]. SSCs were isolated from murine testis and cultured for the first time in 2006[4]. The pluripotency and plasticity of these cultured cells, named multipotent adult germline stem cells (maGSCs), were proven to be similar to ESCs. The ESC-like nature of maGSCs was confirmed on the microRNA level[5], on the transcriptome level[6] and on the proteome level[7]. In a recent study, we investigated the effects of retinoic acid (RA) treatment on the protein expression profiles of maGSCs and ESCs[8]. The study revealed the important role of Eif5a and its hypusination for stem cell differentiation and proliferation.

Eif5a is a universal translation elongation factor which is highly conserved in all cells. Eif5a has been shown to be associated with translation, viability and proliferation processes[9-12]. It is the only eukaryotic protein known to have the unusual amino acid hypusine. Hypusine is essential to the function of Eif5a and is involved in protein biosynthesis by promoting the formation of the first peptide bond and translation elongation[13]. The activation of Eif5a is controlled by a unique post-translational modification called hypusination. It occurs in two steps which are controlled by two different enzymes[14,15], which inactivation can lead to hypusination inhibition. Ciclopirox olamine (CPX), the ethanolamine salt of 6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one, is a hypusination inhibitor that controls the second step of the modification, which is catalyzed by deoxyhypusine hydroxylase[14].

CPX, a synthetic antifungal agent, has been used topically to treat fungal and yeast infection of skin or mucosa for more than 20 years[16-19]. Apart from its antimycotic activity, CPX is also effective against both gram-positive and gram-negative bacteria[20]. CPX might also serve as an alternative to recombinant vascular endothelial growth factor (VEGF) treatment or to VEGF gene therapy for therapeutic angiogenesis[21]. The effect of CPX on several Saccharomyces cerevisiae mutants has been screened and tested, and it was suggested that CPX may exert its effect by disrupting DNA repair, DNA replication, cell division signals and a defect in mitotic spindle function. Furthermore CPX can influence the regulation of many processes, including signal transduction, transcription, cell division, and development[22]. Recent studies demonstrated CPX as a potential anti-cancer agent for the treatment of malignancies, including leukemia and myeloma[23-25]. However, the mechanism of CPX as a drug in angiogenesis and tumor treatment is poorly understood. CPX works as an inhibitor of the iron-dependent enzymes due to its role as a chelator of intracellular iron[22,23]. Other studies reported the inhibition of HIV-1 gene expression by CPX[26], the importance of Eif5a in embryogenesis and cell differentiation[27], in hepatocellular carcinoma[28] and in diabetes[29]. CPX has also been used as an inhibitor of hypusination.

In a recent study, the effect of CPX on the cellular viability and proliferation of ESCs and maGSCs was investigated. CPX treatment of the stem cells resulted in an antiproliferative effect on ESCs and maGSCs in vitro, but did not affect the cell pluripotency[8]. The inhibitory effect of CPX on cell differentiation was reversible and was not associated to apoptosis. The ESCs were found to be more sensitive to CPX than the maGSCs.

The aim of this study was to investigate the proteome changes of ESCs and maGSCs accompanying the treatment with CPX and subsequent inhibition of hypusination using classical proteomic techniques like 2-DE, DIGE and MS. 2D protein maps were generated from control cells and cells treated either with RA or CPX. The resulting protein maps were compared to each other and the differentially expressed proteins were investigated using bioinformatics. We could highlight that a treatment with CPX, involving proliferation inhibition, resulted in an alteration of the expression of 56 proteins compared to non-treated cells, and 54 proteins compared to RA treated cells. The majority of these proteins are involved in nucleotide binding and nucleotide biosynthetic processes, metal binding, DNA binding, and other processes which have been linked to CPX.

MATERIALS AND METHODS
Derivation and culture of maGSC and ESC lines

The derivation and culture of maGSCs 129/Sv was described previously[4]. In brief, testes from adult mice were isolated and digested using collagenase. Single cell suspension was derived after trypsin digestion followed by the culture of the testis suspension cells on a mouse embryonic fibroblasts (MEFs) feeder layer in the presence of GDNF. After appearance of morphological ES-like cells, the colonies were picked and expanded in standard ES cell conditions. In this case, the maGSC line was generated without genetic selection, only by morphological criteria. The ESC R1 line was derived from the 129/Sv mouse[30]. To maintain maGSCs and ESCs in an undifferentiated state, the cells were cultured under standard ESC culture conditions: DMEM (PAN, Aidenbach, Germany) supplemented with 20% fetal calf serum (PAN, Aidenbach, Germany), 2 mmol/L L-glutamine (PAN, Aidenbach, Germany), 50 mmol/L β-mercaptoethanol (Gibco/Invitrogen, Eggenstein, Germany), 1 × non-essential amino acids (Gibco/Invitrogen), sodium pyruvate (Gibco/Invitrogen), and penicillin/streptomycin (PAN, Aidenbach, Germany). ESCs and maGSCs were cultured on a feeder layer of mitomycin C-inactivated MEFs in the presence of 1000 U/mL recombinant mouse leukemia inhibitory factor (LIF) (Chemicon, Temecula, United States). ESCs were isolated as described previously, and male ESC lines were identified and selected by PCR amplification of Sry gene-specific sequences[31,32]. In order to differentiate maGSCs and male ESCs, the cells were plated on gelatin-coated dishes and culture medium was supplemented with 1 μmol/L RA (Sigma-Aldrich, Steinheim, Germany) instead of LIF. Cells were cultured for 48 h before they were lysed and the proteins were extracted. For examining the effect of CPX on the proteome level, ESCs and maGSCs were treated with culture medium supplemented with 2 μmol/L CPX for 72 h.

Protein extraction

The protein extraction for 2-DE was performed as described previously[7]. Briefly, 75% confluent cultures were trypsinized and washed three times with PBS. The cells were harvested by centrifugation at 200 ×g for 10 min, the pellet was treated with 0.3-0.5 mL lysis buffer [9.5 mol/L urea, 2% CHAPS (w/v), 2% ampholytes (w/v), 1% DTT]. Ampholytes and DTT were added shortly before use. After adding the lysis buffer, the samples were incubated for 30 min at 4 °C. For removing the cell debris, sample centrifugation was carried out at 13 000 ×g and 4 °C for 45 min. The supernatant was recentrifuged at 13 000 ×g and 4 °C for an additional 45 min to get maximal purity. The resulting samples were used immediately or stored at -80 °C until use.

Protein precipitation

To reduce the salt contamination and to enrich the proteins, methanol-chloroform-precipitation according to Wessel et al[33] was performed. Briefly, 0.4 mL of methanol (100%) was added to 0.1 mL aliquots of protein samples and mixed together. 0.1 mL chloroform was added to the samples and the mixture was vortexed. Subsequently 0.3 mL water was added and the solution was vortexed and centrifuged at 13 000 ×g for 1 min. The aqueous layer was removed, and another 0.4 mL methanol (100%) was added to the rest of the chloroform and the interphase with the precipitated proteins. The sample was mixed and centrifuged for 2 min at 13 000 ×g and the supernatant was removed. The pellet was vacuum dried and dissolved in lysis buffer.

Total protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, United States) according to Bradford[34]. BSA (Sigma, Steinheim, Germany) was used as a standard.

2D gel electrophoresis (2-DE)

IPG strips (11 cm, pI 5-8) were passively rehydrated in 185 μL solution containing 150 μg protein in a rehydration buffer (8 mol/L urea, 1% CHAPS, 1% DTT, 0.2% ampholytes, and a trace of bromophenol blue) for 12 h. The IEF step was performed on the PROTEAN® IEF Cell (Bio-Rad, Hercules, CA, United States). Temperature-controlled at 20 °C, the voltage was set to 500 V for 1 h, increased to 1000 V for 1 h, 2000 V for 1 h and left at 8000 V until a total of 50 000 Vhours was reached. Prior to SDS-PAGE, the IPG strips were reduced for 20 min at room temperature in SDS equilibration buffer containing 6 mol/L urea, 30% glycerol, 2% SDS 0.05 mol/L Tris-HCl, and 2% DTT on a rocking table. The strips were subsequently alkylated in the same solution with 2.5% iodoacetamide substituted for DTT, and a trace of bromophenol blue. For the SDS-PAGE, 12% BisTris Criterion precast gels (Bio-Rad, Hercules, CA, United States) were used according to manufacturer’s instructions. The gels were run at 150 V for 10 min followed by 200 V until the bromophenol blue dye front had reached the bottom of the gel.

Gel staining

For image analysis, 2-DE gels were fixed in a solution containing 50% methanol and 12% acetic acid overnight and fluorescent stained with Flamingo fluorescent gel stain (Bio-Rad, Hercules, CA, United States) for minimum 5 h. After staining, gels were scanned at 50 μm resolution on a Fuji FLA-5100 scanner. The digitalized images were analyzed using Delta 2D 3.4 (Decodon, Braunschweig, Germany). For protein visualization, 2-DE gels were additionally stained with colloidal Coomassie blue, Roti-Blue (Roth, Karlsruhe, Germany) overnight.

2D-DIGE

Protein extraction and methanol-chloroform-precipitation were performed as described above. The resulting pellet was dissolved in labeling buffer (30 mmol/L Tris-HCl pH 8.5, 9.5 mol/L urea, 2% CHAPS), centrifuged (5 min, 13 000 ×g), and the protein concentration of the supernatant was determined as described above. The preparation of the CyDyes as well as the labeling reaction was followed according to the manufacturer’s protocol (GE Healthcare).

To avoid the dye-specific protein labeling, every pair of protein samples from two independent cell extract preparations were processed in duplicate while swapping the dyes. Thereby four replicate gels were obtained, allowing to monitor regulation factors down to twofold changes[35]. An internal standard consisting of a mixture of the samples under investigation was labeled with Cy2 and included on all gels to facilitate gel matching, thereby eliminating artifacts from experimental variation. The three differentially labeled fractions were pooled. Rehydration buffer (8 mol/L urea, 1% CHAPS, 13 mmol/L DTT and 1% ampholytes 3-10) was added to make up the volume to 185 μL prior to IEF. The 2-DE was performed as described above. The CyDye-labeled gels were scanned at 50 μm resolution on a Fuji FLA5100 scanner (Fuji Photo, Kanagawa, Japan) with laser excitation light at 473 nm and long pass emission filter 510LP (Cy2), 532 nm and long pass emission filter 575LP (Cy3), and 635 nm and long pass emission filter 665LP (Cy5). Fluorescent images were acquired in 16-bit TIFF files format. Spot matching across gels and normalization based on the internal standard was performed with Delta2D software (Decodon, Greifswald, Germany). To analyze the significance of protein regulation, a Student’s t-test was performed, and statistical significance was assumed for p values less than 0.01. For protein visualization, the 2-DE gels were post-stained with colloidal Coomassie blue (Roti-Blue) overnight. Differentially regulated proteins were excised and processed for identification by mass spectrometry.

Protein identification

Manually excised gel plugs were subjected to an automated platform for the identification of gel-separated proteins[36] as described in the framework of recent DIGE-based[37] and large-scale proteome studies[38]. An Ultraflex MALDI-TOF-TOF mass spectrometer (Bruker Daltonik) was used to acquire both PMF and fragment ion spectra, resulting in confident protein identifications based on peptide mass and sequence information. Database searches in the Swiss-Prot primary sequence database restricted to the taxonomy mus musculus were performed using the MASCOT Software 2.2 (Matrix Science). Carboxamidomethylation of Cys residues was specified as fixed and oxidation of Met as variable modifications. One trypsin missed cleavage was allowed. Mass tolerances were set to 100 ppm for PMF searches and to 100 ppm (precursor ions) and 0.7 Da (fragment ions) for MS/MS ion searches. The minimal requirement for accepting a protein as identified was at least one peptide sequence match above identity threshold in addition to at least 20 % sequence coverage in the PMF.

Bioinformatics

The classification of the identified proteins according to their main known/postulated functions was carried out using DAVID bioinformatics[39,40]. This classification together with the official gene symbol was used to investigate and categorize the gene ontology (GO)-annotations (biological processes and molecular functions).

RESULTS
Comparative analysis of differentially expressed proteins in RA and CPX treated SCs by 2-DE and ontogenic classification

To explore proteome changes caused by CPX treatment, we treated ESCs as well as maGSCs with CPX for 72 h. In parallel, both cell types, ESC and maGSC, were treated with RA for 48 h. 2-DE was performed from these four different samples, as well as from the corresponding non-treated cells (Figures 1, 2, 3 and 4). Proteins, which were found to be differentially expressed, were excised and subjected to in-gel-digestion and mass spectrometry analyses. A total of 316 spots were identified, which resulted in 125 non-redundant proteins (Table 1). For further interpretation of the data, only proteins, which were regulated in the same direction in ESCs and concurrently in maGSCs, were taken into consideration.

Table 1 Non-redundant proteins.
Protein nameGene nameSwiss-protNominal massCPIPMF-scorePMF sequence coverageMS/MS-scoreMS/MS-sequence coverage
Low molecular weight phosphotyrosine protein phosphataseAcp1PPAC_MOUSE18 6366.496658024
Actin, cytoplasmic 1ActbACTB_MOUSE42 0525.21707031215
Aminoacylase-1Acy1ACY1_MOUSE45 9805.916756445
Aldose reductaseAkr1b1ALDR_MOUSE36 0526.91284313610
Aldehyde dehydrogenase, mitochondrialAldh2ALDH2_MOUSE57 0158.6221541317
Annexin A3Anxa3ANXA3_MOUSE36 5205.2844711114
Adenine phosphoribosyltransferaseAprtAPT_MOUSE19 8836.4886721627
Rho GDP-dissociation inhibitor 1ArhgdiaGDIR1_MOUSE23 4505123546611
ATP synthase subunit α, mitochondrialAtp5a1ATPA_MOUSE59 8309.710028534
ATP synthase subunit β, mitochondrialAtp5bATPB_MOUSE56 2655.1903016710
ATP synthase subunit d, mitochondrialAtp5hATP5H_MOUSE18 7955.41227016936
F-actin-capping protein subunit α-2Capza2CAZA2_MOUSE33 1185.514869199
F-actin-capping protein subunit βCapzbCAPZB_MOUSE31 6115.4117611298
Chromobox protein homolog 3Cbx3CBX3_MOUSE21 01353836676
T-complex protein 1 subunit βCct2TCPB_MOUSE57 783624861759
T-complex protein 1 subunit epsilonCct5TCPE_MOUSE60 0425.7186601386
Cofilin-1Cfl1COF1_MOUSE18 7769.195458713
UMP-CMP kinaseCmpk1KCY_MOUSE22 3795.674522910
Coactosin-like proteinCotl1COTL1_MOUSE16 0485.1866011614
Cathepsin DCtsdCATD_MOUSE45 3816.916041954
Dihydrolipoyl dehydrogenase, mitochondrialDldDLDH_MOUSE54 751911248812
Elongation factor 1-α1Eef1a1EF1A1_MOUSE50 4249.768341158
Elongation factor 1-δEef1dEF1D_MOUSE31 3884.88654799
Elongation factor 2Eef2EF2_MOUSE96 2226.45226291
Eukaryotic translation initiation factor 3 subunit FEif3fEIF3F_MOUSE38 0905.21094510614
Eukaryotic translation initiation factor 3 subunit GEif3gEIF3G_MOUSE35 9015.65435237
Eukaryotic translation initiation factor 3 subunit IEif3iEIF3I_MOUSE36 8375.3228788916
Eukaryotic translation initiation factor 4HEif4hIF4H_MOUSE27 3817.58351658
Eukaryotic translation initiation factor 5A-1Eif5aIF5A1_MOUSE17 0494.91155817022
α-enolaseEno1ENOA_MOUSE47 4536.41836417013
Electron transfer flavoprotein subunit α, mitochondrialEtfaETFA_MOUSE35 3309.5138591009
Fatty acid-binding protein, heartFabp3FABPH_MOUSE14 8106.1867721239
Peptidyl-prolyl cis-trans isomerase FKBP4Fkbp4FKBP4_MOUSE51 9395.4122381689
FascinFscn1FSCN1_MOUSE55 2156.512945266
Glyceraldehyde-3-phosphate dehydrogenaseGapdhG3P_MOUSE36 0729.26238408
Lactoylglutathione lyaseGlo1LGUL_MOUSE20 9675.11346611420
Glyoxalase domain-containing protein 4Glod4GLOD4_MOUSE33 5815.21676911513
Glutamate dehydrogenase 1, mitochondrialGlud1DHE3_MOUSE61 6408.87037605
Guanine nucleotide-binding protein subunit β-2-like 1Gnb2l1GBLP_MOUSE35 5118.911655205
Growth factor receptor-bound protein 2Grb2GRB2_MOUSE25 3365.973543617
Histone H2B type 1-BHist1h2bbH2B1B_MOUSE13 94410.852419319
Histone H2A type 2-CHist2h2acH2A2C_MOUSE13 98011.450556712
Heterogeneous nuclear ribonucleoprotein A/BHnrnpabROAA_MOUSE30 9268.783301075
Heterogeneous nuclear ribonucleoproteins C1/C2HnrnpcHNRPC_MOUSE34 4214.85732576
Heterogeneous nuclear ribonucleoprotein FHnrnpfHNRPF_MOUSE46 0435.21635620712
Heterogeneous nuclear ribonucleoprotein HHnrnph1HNRH1_MOUSE49 4545.91666113415
Heterogeneous nuclear ribonucleoprotein KHnrnpkHNRPK_MOUSE51 2305.31444625111
Heat shock protein HSP 90-αHsp90aa1HS90A_MOUSE85 1344.8131311305
Heat shock protein HSP 90-βHsp90ab1HS90B_MOUSE83 6154.86225786
Heat shock 70 kDa protein 4Hspa4HSP74_MOUSE94 8725242541023
78 kDa glucose-regulated proteinHspa5GRP78_MOUSE72 4924.978251225
Heat shock cognate 71 kDa proteinHspa8HSP7C_MOUSE71 0555.2234581544
Stress-70 protein, mitochondrialHspa9GRP75_MOUSE73 7685.8219502727
Heat shock protein β-1Hspb1HSPB1_MOUSE23 0576.11445534424
60 kDa heat shock protein, mitochondrialHspd1CH60_MOUSE61 0885.83346923210
Isocitrate dehydrogenase (NAD) subunit α, mitochondrialIdh3aIDH3A_MOUSE40 0696.3703115812
Inosine-5'-monophosphate dehydrogenase 2Impdh2IMDH2_MOUSE56 1797173501077
Inosine triphosphate pyrophosphataseItpaITPA_MOUSE22 2255.5847212815
Keratin, type I cytoskeletal 18Krt18K1C18_MOUSE47 5095.119965589
Keratin, type II cytoskeletal 7Krt7K2C7_MOUSE50 6785.613752554
Keratin, type II cytoskeletal 8Krt8K2C8_MOUSE54 5315.6245552379
Cytosol aminopeptidaseLap3AMPL_MOUSE56 5058.712647585
L-lactate dehydrogenase B chainLdhbLDHB_MOUSE36 8345.68446306
Galectin-1Lgals1LEG1_MOUSE15 1985.21097017225
Galectin-2Lgals2LEG2_MOUSE14 9847.9120886014
Lamin-B1Lmnb1LMNB1_MOUSE66 9735265601345
α-2-macroglobulin receptor-associated proteinLrpap1AMRP_MOUSE42 1897.91394917714
S-adenosylmethionine synthase isoform type-2Mat2aMETK2_MOUSE44 00367341593
28S ribosomal protein S22, mitochondrialMrps22RT22_MOUSE41 2819.211245979
Myosin-9Myh9MYH9_MOUSE227 4295.473151032
NucleolinNclNUCL_MOUSE76 7344.5113261797
Omega-amidase NIT2Nit2NIT2_MOUSE30 8256.511259759
Nucleoside diphosphate kinase ANme1NDKA_MOUSE17 3117.71257222330
Nucleoside diphosphate kinase BNme2NDKB_MOUSE17 4667.81608428730
NucleophosminNpm1NPM_MOUSE32 7114.5533313610
Nuclear pore complex protein Nup54Nup54NUP54_MOUSE55 8126.65521233
Nuclear pore glycoprotein p62Nup62NUP62_MOUSE53 3365.113435
Ornithine aminotransferase, mitochondrialOatOAT_MOUSE48 7236.2174641259
Poly(rC)-binding protein 1Pcbp1PCBP1_MOUSE37 9876.81756911512
Protein disulfide-isomerase A3Pdia3PDIA3_MOUSE57 0995.825455905
Protein disulfide-isomerase A6Pdia6PDIA6_MOUSE48 4694.97540472
PDZ and LIM domain protein 1Pdlim1PDLI1_MOUSE36 2086.420073567
Phosphatidylethanolamine-binding protein 1Pebp1PEBP1_MOUSE20 9885.11307910711
Phosphoglycerate mutase 1Pgam1PGAM1_MOUSE28 9286.81576619221
Phosphoglycerate kinase 1Pgk1PGK1_MOUSE44 9219136521287
6-phosphogluconolactonasePgls6PGL_MOUSE27 4655.51024914817
Pyruvate kinase isozymes M1/M2Pkm2KPYM_MOUSE58 3787.9178491069
Purine nucleoside phosphorylasePnpPNPH_MOUSE32 5415.81196713813
Inorganic pyrophosphatasePpa1IPYR_MOUSE33 1025.312666267
Peroxiredoxin-2Prdx2PRDX2_MOUSE21 9365.11036228522
Peroxiredoxin-6Prdx6PRDX6_MOUSE24 9695.61566710117
Proteasome subunit α type-1Psma1PSA1_MOUSE29 8136715214017
Proteasome subunit α type-6Psma6PSA6_MOUSE27 8116.4723810810
Proteasome subunit β type-3Psmb3PSB3_MOUSE23 2356.21105118730
Proteasome subunit β type-4Psmb4PSB4_MOUSE29 2115.3604210910
26S protease regulatory subunit 7Psmc2PRS7_MOUSE49 0165.616660728
26S protease regulatory subunit 6BPsmc4PRS6B_MOUSE47 3665144551099
GTP-binding nuclear protein RanRanRAN_MOUSE24 5797.81245113911
40S ribosomal protein S12Rps12RS12_MOUSE14 8587.777629511
RuvB-like 1Ruvbl1RUVB1_MOUSE50 5246613510610
Protein S100-A11S100a11S10AB_MOUSE11 2475.13614727
Splicing factor, arginine/serine-rich 1Sfrs1SFRS1_MOUSE27 84210.8804315618
Splicing factor, arginine/serine-rich 3Sfrs3SFRS3_MOUSE19 54612.38714
Serine hydroxymethyltransferase, cytosolicShmt1GLYC_MOUSE53 0656.59843192
Superoxide dismutase [Cu-Zn]Sod1SODC_MOUSE16 1046834512631
Spermidine synthaseSrmSPEE_MOUSE34 5435.21417312915
Stress-induced-phosphoprotein 1Stip1STIP1_MOUSE63 1706.418455894
StathminStmn1STMN1_MOUSE17 2645.72824698
Stomatin-like protein 2Stoml2STML2_MOUSE38 4759.51446116515
TAR DNA-binding protein 43TardbpTADBP_MOUSE44 9186.368301077
T-complex protein 1 subunit αTcp1TCPA_MOUSE60 8675.86127284
Transcription intermediary factor 1-βTrim28TIF1B_MOUSE90 5585.4101394
Tubulin α-1B chainTuba1bTBA1B_MOUSE50 8044.8128391529
Tubulin α-1C chainTuba1cTBA1C_MOUSE50 5624.85324526
Tubulin β-2A chainTubb2aTBB2A_MOUSE50 2744.61265511111
Tubulin β-2C chainTubb2cTBB2C_MOUSE50 2554.615056498
Tubulin β-5 chainTubb5TBB5_MOUSE50 0954.6169572379
ThioredoxinTxnTHIO_MOUSE12 0104.663679222
Thioredoxin-like protein 1Txnl1TXNL1_MOUSE32 6164.714478392
Ubiquitin-conjugating enzyme E2 NUbe2nUBE2N_MOUSE17 1846.211971206
Ubiquitin carboxyl-terminal hydrolase isozyme L1Uchl1UCHL1_MOUSE25 16557764168
Cytochrome b-c1 complex subunit 1, mitochondrialUqcrc1QCR1_MOUSE53 4205.79540466
Transitional endoplasmic reticulum ATPaseVcpTERA_MOUSE89 950531061405
Voltage-dependent anion-selective channel protein 1Vdac1VDAC1_MOUSE32 5029.2159578024
VimentinVimVIME_MOUSE53 7124.921864478
Figure 1
Figure 1 Embryonic stem cells control vs ciclopirox olamine treated cells. Overlay of 2-DE gels of samples from ciclopirox olamine treated embryonic stem cells (ESCs) (orange) compared to control ESCs (blue). The identified proteins are indicated with the gene names.
Figure 2
Figure 2 Multipotent adult germline stem cells control vs ciclopirox olamine treated cells. Overlay of 2-DE gels of samples from ciclopirox olamine treated multipotent adult germline stem cells (maGSCs) (orange) compared to control maGSCs (blue). The identified proteins are indicated with the gene names.
Figure 3
Figure 3 Embryonic stem cell control vs ciclopirox olamine treated cells. Overlay of 2-DE gels of samples from ciclopirox olamine treated embryonic stem cells (ESCs) (orange) compared to retinoic acid treated ESCs (blue). The identified proteins are indicated with the gene names.
Figure 4
Figure 4 Multipotent adult germline stem cell control vs ciclopirox olamine treated cells. Overlay of 2-DE gels of samples from ciclopirox olamine treated Multipotent adult germline stem cells (maGSCs) (orange) compared to retinoic acid treated maGSCs (blue). The identified proteins are indicated with the gene names.

The identified proteins were classified using DAVID bioinformatics[39,40] focusing on its information considering the GO (Gene Ontology) annotations. The terms corresponding to the molecular function and biological process were regarded (Figures 5, 6 and 7).

Figure 5
Figure 5 Molecular function. Classification of the downregulated proteins upon treatment with ciclopirox olamine (CPX) (A) or retinoic acid (RA) (B) according to their molecular functions.
Figure 6
Figure 6 Biological process. Classification of the downregulated proteins upon treatment with ciclopirox olamine (CPX) (A) or retinoic acid (RA) (B) according to their biological processes.
Figure 7
Figure 7 Biological process. Classification of the differently regulated proteins upon treatment with ciclopirox olamine (CPX) (A) or retinoic acid (RA) (B) according to their biological processes.
Comparison of the differently expressed proteins

Examination of all of the proteins, which expression was altered either by CPX or RA treatment, was performed regarding their involvement in biological processes. We found that seven proteins are involved in regulation of transcription. Among these proteins Ube2n, Tardbp, Cbx3 and Hnrnpab were downregulated in CPX treated cells compared to control, whereas Nup62 was upregulated in CPX treated cells compared to control (Table 2). Two other proteins Trim28 and Ruvbl1 were downregulated in RA treated cells compared to control. Detailed information is given in Table 2 and the protein expression regulation folds are given in Tables 3, 4, 5 and 6.

Table 2 Gene Ontology functional annotation of proteins which were regulated in this experiment according to their involvement in different biological processes.
Biological processProteinsCPX > RACPX < RACPX > cCPX < cRA > cRA < c
Monosaccharide metabolic/catabolic processes5PglsEno1Eno1Eno1
GapdhPkm2Ldhb
Eno1Pkm2
Pkm2
Nucleobase, nucleoside, nucleotide, and nucleic acid biosynthetic processes7Atp5a1AprtAtp5a1AprtNme2Aprt
Nme2Atp5a1Nme1Atp5a1
Nme1Impdh2Atp5h
Nme2Impdh2
Nme1
Pnp
RNA and mRNA processing6Sfrs1HnrnpkHnrnpc
TardbpSfrs3
Pcbp1
Regulation of transcription7Ruvbl1Nup62Ube2nTrim28
Trim28TardbpRuvbl1
Cbx3
Hnrnpab
Embryonic development5Sfrs1Psmc4Atp5a1Eno1Myh9Eno1
Eno1Eno1Myh9Atp5a1Psmc4
Gene expression16Trim28Rps12Eif3fEef1a1Sfrs3
Sfrs1Eif3iEif5aEif3i
Ruvbl1Eef1dHnrnpc
Cbx3Ruvbl1
HnrnpkEef1d
HnrnpabTrim28
Tardbp
Pcbp1
Cell cycle processes6Ruvbl1Myh9Krt7Npm1
Myh9Tubb5
Stmn1
Ruvbl1
Cell morphogenesis involved in differentiation4Trim28Myh9Uchl1Myh9Stmn1
HnrnpabTrim28
Regulation of cell proliferation4Nup62Nme2Nme2Npm1
Pnp
Regulation of signal transduction4Nup62Ube2nNpm1
Hspa5
Table 3 Proteins which are upregulated upon ciclopirox olamine treatment compared to control.
k/CPX
ESCmaGSC
Actb0.130.19
Atp5a110.410.54
Ctsd0.970.09
Eif3f10.940.43
Eif3i10.490.95
Etfa0.630.39
Hspa90.920.31
Hspb10.630.05
Hspd10.190.21
Hspd10.360.69
Myh910.630.09
Nup6210.300.60
S100a110.21
Tubb2a1.000.21
Vdac10.580.18
Table 4 Proteins which are downregulated upon ciclopirox olamine treatment compared to control.
k/CPX
ESCmaGSC
Acp11.296.01
Acy111.382.81
Akr1b12.0713.44
Aprt14.803.60
Atp5a113.501.21
Capzb3.042.35
Cbx311.722.12
Cct2112.001.28
Cct511.062.02
Eef1a112.471.74
Eef1d11.462.03
Eif5a11.312.07
Eno113.561.60
Fscn13.311.49
Glod43.351.60
Gnb2l12.6112.92
Hist1h2bb2.312.10
Hist2h2ac17.3367.90
Hnrnpab12.413.36
Hnrnpk12.001.17
Hsp90aa111.196.79
Hsp90aa111.843.02
Hspa413.281.51
Hspa41.133.14
Hspa81.747.17
Hspd11.673.07
Impdh212.592.13
Impdh21> 10027.94
Krt1812.311.44
Lgals22.828.45
Ncl11.332.61
Nit21.242.13
Nme116.251.56
Nme214.774.51
Pcbp112.211.64
Pkm213.753.27
Pnp11.202.62
Psmb411.012.32
Ruvbl111.022.14
Srm1.643.63
Shmt11> 100> 100
Tardbp11.383.52
Tcp11.473.76
Tuba1c11.873.11
Tubb2c11.383.41
Ube2n11.316.45
Uchl112.661.66
Table 5 Proteins which are downregulated upon retinoic acid treatment compared to control.
LabelRA/k
ESCmaGSC
Acp10.610.50
Actb10.530.13
Acy110.130.70
Akr1b10.430.11
Aprt10.460.39
Atp5a110.760.38
Atp5h10.690.40
Cbx31.010.47
Cotl110.500.44
Eef1d10.700.15
Eif3i10.090.92
Eno110.240.04
Eno110.550.22
Fabp30.45
Fkbp410.900.40
Glo110.740.41
Glod40.820.30
Impdh210.760.35
Impdh210.540.20
Gnb2l110.660.15
Hnrnpc10.760.43
Hsp90aa10.750.08
Hsp90aa10.490.06
Hsp90aa10.760.12
Hspa510.320.22
Hspa810.690.50
Hspb110.360.47
Hspb110.460.88
Hspb110.900.41
Hspd110.160.67
Hspd110.340.95
Itpa0.570.07
Ldhb10.420.43
Lgals20.290.03
Ncl10.260.71
Npm110.460.04
Pebp110.890.42
Pkm210.380.15
Pkm210.320.65
Pkm210.420.76
Pkm210.210.43
Psmb410.620.43
Ruvbl110.630.22
Sfrs310.410.46
Shmt110.010.00
Srm0.680.24
Trim2810.230.11
Trim2810.400.37
Tuba1c10.270.71
Tubb510.700.25
Uqcrc110.240.22
Vdac110.300.52
Table 6 Proteins which are upregulated upon retinoic acid treatment compared to control.
RA/k
ESCmaGSC
Cct21.142.16
Hspa43.962.47
Krt711.0138.11
Krt81.971.78
Myh913.063.24
Nme112.523.81
Nme211.202.48
Pdia61.7220.48
Psmc412.172.17
Vcp8.304.13
Vim13.851.16

When we looked at the molecular function of the regulated proteins, we observed that a major part of the proteins are involved in nucleotide binding (Table 7). Approximately half of these proteins were downregulated and the other half was upregulated upon CPX treatment compared to RA treated cells. About 13 proteins are involved in metal ion binding, of these five proteins (Acy1, Uqcrc1, Sfrs1, Trim28, Glo1) are involved in transition metal ion binding, like Fe3+, which is known to be important in case of CPX, as CPX works as an inhibitor of the iron-dependent enzymes due to its role as a chelator of intracellular iron. Three of the proteins involved in transition metal ion binding (Sfrs1, Trim28 and Glo1), were up-regulated upon CPX treatment compared to RA-treated cells.

Table 7 Gene Ontology functional annotation of proteins which were regulated in this experiment according to their involvement in different molecular function.
Molecular functionProteinsCPX > RACPX < RACPX > cCPX < cRA > cRA < c
Nucleotide binding41Hsp90ab1Atp5bTubb2aCct2Cct2Ldhb
Fkbp4Cct2EtfaTardbpHspa4Fkbp4
Tubb5TardbpHspa9Hspa4Myh9Tubb5
Hspa5Hspa4ActbTuba1cNme2Hnrnpc
Tubb1bActbMyh9Hspa8VcpHspa5
GapdhHsp90aa1Vdac1HnrnpabPsmc4Tuba1c
EtfaNclAtp5a1Eef1a1Nme1Hspa8
Hspa9AprtHspd1Tcp1Actb
ActbNme2Hsp90aa1Hsp90aa1
Hsp90aa1VcpNclNcl
Sfrs1Psmc4AprtSfrs3
Vdac1Nme1Ube2nAprt
Pkm2Psmc2Nme2Vdac1
Atp5a1Cct5Pkm2
Ruvbl1Nme1Pebp1
Hspd1Pkm2Atp5a1
Atp5a1Ruvbl1
Ruvbl1Hspd1
Hspd1
GTP binding8Fkbp4Nme1Tubb2aEef1a1Nme1Fkbp4
Tubb5Nme1Tubb5
Tuba1bTuba1cTuba1c
ATPase activity8Atp5a1VcpAtp5a1Atp5a1VcpAtp5a1
Psmc4Myh9Hspa8Psmc4Atp5h
Atp5bMyh9Hspa8
Psmc2
Enzyme binding8ActbActbActbGnb2l1VimActb
Npm1Gnb2l1Hspd1Pebp1
Hspd1Hspa9Hspd1
Cotl1Cotl1
Hspa9
Cofactor binding5GapdhEtfaShmt1Ldhb
EtfaShmt1
Peptidase activity6CtsdUchl1CtsdPsmb4Psmb4
Eno1Eno1Acy1Uqcrc1
Uchl1Acy1
Eno1Eno1
Metal ion binding13Trim28Atp5bAcy1Pdia6Acy1
Sfrs1Pdia6Nme2Nme2Uqcrc1
Pkm2Nme2Nme1Nme1Trim28 Pkm2
Glo1Nme1Pkm2Glo1
Eno1Eno1Impdh2Impdh2
Eno1Eno1

Overall, it could be observed that most of the proteins of interest were downregulated in either CPX or RA treated cells compared to control.

Treated cells compared to control

About 56 of the 125 identified proteins showed different expression as a reaction to CPX treatment compared to control. Of these, 14 proteins were upregulated as a reaction to CPX treatment (Table 3), whereas 44 proteins were downregulated (Table 4). The expression of 52 proteins was found to be altered in both cell types, ESCs and maGSCs, under RA treatment compared to control (Tables 5 and 6). Of these proteins, 11 were upregulated and 41 were downregulated as a reaction to RA treatment.

In both experiments the majority of the regulated proteins were downregulated as a reaction to one of the treatments. Although mainly different proteins were regulated, bioinformatics analysis revealed that the downregulated proteins in both experiments are primarily involved in the same molecular functions (Figure 5). The downregulated proteins upon CPX treatment are mainly involved in nucleotide binding, GTP binding, peptidase activity and metal ion binding, particularly magnesium ion binding. The proteins which were downregulated upon RA treatment are involved in transition metal ion binding instead of magnesium ion binding, and furthermore involved in enzyme binding. Proteins, which were upregulated upon CPX treatment, are mainly involved in nucleotide binding, whereas proteins which were upregulated upon RA treatment are involved in nucleotide and metal ion binding.

When we look at the involvement of the regulated proteins in biological processes, more differences were observed (Figure 6). Both treatments showed downregulation of proteins involved in protein complex biogenesis, nucleotide biosynthetic process, cell death and positive regulation of biosynthetic process. Additionally, proteins involved in proteolysis and positive regulation of protein metabolic process were downregulated in SCs upon CPX treatment. Proteins which were downregulated in SCs upon RA treatment are, among others, involved in cell cycle, RNA processing, glycolysis and negative regulation of protein metabolic process.

Proteins which were upregulated in SCs upon CPX treatment are involved in nucleotide binding, regulation of cell death and protein transport, whereas proteins which were upregulated upon RA treatment are involved in nucleotide binding, metal ion binding and proteolysis.

Proteins in CPX treated cells compared to RA treated cells

When the proteins in RA treated SCs were compared to CPX treated SCs, we observed that 54 proteins are differently regulated (Tables 8 and 9). Of these proteins, 31 were upregulated and 26 downregulated upon CPX treatment. In some cases, different forms of one protein, e.g., Actb, Eno1, and Hsp90aa1 were observed and showed different regulation.

Table 8 Proteins which are upregulated in stem cells upon ciclopirox olamine treatment compared to retinoic acid treatment.
RA/CPX
ESCmaGSC
Actb*10.120.10
Actb10.140.15
Atp5a110.430.48
Cotl10.190.67
Ctsd0.950.16
Eif3i0.040.87
Eno1*10.130.03
Eno110.570.40
Etfa0.680.16
Fkbp410.460.43
Gapdh10.350.59
Glo10.310.66
Glod40.850.36
Hsp90aa10.280.38
Hsp90aa10.430.13
Hsp90ab10.410.26
Hspa5*10.170.13
Hspa9*10.500.28
Hspb1*10.290.04
Hspb1*10.200.42
Hspb110.820.50
Hspd110.490.74
Hspd110.420.33
Itpa0.210.08
Mat2a10.410.14
Npm110.280.18
Nup620.570.26
Pgls10.400.68
Pkm210.220.25
Pkm210.290.86
Prdx60.460.94
Ruvbl110.640.42
S100a11*120.17
Sfrs110.700.30
Trim2810.440.08
Trim2810.420.44
Trim2810.330.28
Tuba1b*10.420.65
Tubb510.420.54
Tubb510.430.58
Vdac1*10.170.09
Figure 8
Figure 8 Enlargement of the gel spots of some proteins of interest. ESC: Embryonic stem cell; maGSC: Multipotent adult germline stem cell; CPX: Ciclopirox olamine; RA: Retinoic acid.
Table 9 Proteins which are downregulated upon ciclopirox olamine treatment compared to retinoic acid treated stem cells.
RA/CPX
ESCmaGSC
Actb12.171.10
Aldh22.612.17
Aldh22.431.21
Aprt12.201.42
Atp5b1.152.17
Capzb2.792.07
Cct2*113.702.77
Eno112.482.02
Eno112.711.51
Fscn12.202.14
Gnb2l11.812.24
Hist1h2bb7.531.62
Hist2h2ac3.89211.81
Hnrnpk2.371.58
Hsp90aa1*12.696.36
Hsp90aa16.364.33
Hspa4*112.983.72
Hspa41.353.42
Krt7> 1001.14
Krt18*14.041.76
Ncl1.803.48
Nme112.631.57
Nme2*15.7211.15
Pdia61.746.33
Psmc212.871.20
Psmc4*13.062.26
Rps12*122.05
Tardbp1.113.85
Uchl112.021.10
Vcp18.942.57

The bioinformatics analysis of these proteins, focussing on biological processes, showed involvement of the proteins in different categories (Figure 7). Proteins which were downregulated in CPX treated cells are involved in processes like protein complex biogenesis, nucleotide biosynthetic process, proteolysis, intracellular transport and regulation of cell death. Proteins which were downregulated as a reaction to RA treatment are involved in protein complex biogenesis, cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis and phosphorylation.

To get a better focus on proteins, which may play a key role in proliferation, we also focussed on proteins, which showed contrary regulation upon CPX treatment and RA treatment compared to control. This resulted in 15 proteins, of which eight were upregulated upon CPX treatment and concurrently downregulated upon RA treatment compared to control, and seven proteins, which were downregulated upon CPX treatment and concurrently upregulated upon RA treatment compared to control (proteins marked by asterisk in Tables 8 and 9).

Bioinformatics analysis of the proteins, which were downregulated upon CPX treatment along with upregulated upon RA treatment were primarily involved in metabolic processes (Nme2, Hsp90aa1, Psmc4, Rps12, Cct2 and Eno1) like protein folding (Hsp90aa1, Cct2), whereas proteins, which were upregulated upon CPX-treatment and concurrently downregulated upon RA-treatment were additionally involved in developmental processes (Psmc4, Eno1) and transport/localization (Vdac1, Hspa9).

Analysis of the molecular function of the differently regulated proteins upon CPX and RA treatment showed their important role in nucleotide binding (Nme2, Hsp90aa1, Psmc4, Hspa4, Cct2, Actb, Pkm2, Hspa5, Vdac1 and Hspa9) and metal ion binding (Pkm2, S100a11, Eno1).

DISCUSSION

CPX is a synthetic antifungal drug, which is currently used for the treatment of superficial mycoses[41]. Since two decades CPX has also been used as an antitumor agent[42]. It has been shown that CPX can be used to treat solid tumors due to its strong antiangiogenic activity[43,23]. CPX might inhibit the cell proliferation and work as an antitumor agent due to its iron chelating function, as iron is essential for cell proliferation and function[24]. In a recent study, we investigated the effect of CPX on the cellular viability and proliferation of SCs. The study demonstrated that in contrast to RA, CPX treatment resulted in a reversible antiproliferative effect[8]. The present study was conducted to understand the anti-proliferative effect of CPX on stem cells in terms of proteins and molecular processes which are involved in its mode of action.

With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins which were differently expressed in both cell lines. Bioinformatics analysis of the regulated proteins demonstrated their involvement in various biological processes. To our interest, a number of proteins have potential roles in the regulation of cell proliferation either directly or indirectly.

One of the possible mechanisms of CPX action on cell proliferation is through controlling the progression of the cell cycle[44]. We identified a number of proteins which are involved in cell cycle processes. Ruvbl1 is one of the differentially regulated proteins which is involved in cell cycle processes, gene expression and transcription regulation. It was found to be downregulated in CPX and RA treated cells compared to control (Figure 8). Ruvbl1 is an evolutionarily highly conserved eukaryotic protein belonging to the AAA+ family of ATPase’s[45]. It plays an important role in various cell cycle processes such as chromatin remodeling[46], gene activation[47], transcriptional regulation, DNA repair and transcription factor c-Myc[48]. It also controls Wnt signaling pathway through transcription-associated protein β-catenin[49,50]. Another protein, which was higher expressed in CPX treated cells compared to RA treated cells, is Trim28. Trim28 is involved in regulation of transcription and silencing gene expression through its ability to bind to DNA through interaction with a KRAB-ZFP protein. Other proteins, like Cbx3, Tardbp, and Hnrnpab, which are important in gene expression and regulation of transcription, were downregulated due to treatment with CPX. Tardpb is a DNA and RNA-binding protein, which regulates transcription and splicing. It is also involved in the regulation of CFTR (Cystic fibrosis transmembrane conductance regulator), microRNA biogenesis, apoptosis and cell division. It can repress HIV-1 transcription by binding to the HIV-1 long terminal repeat. Cbx3 seems to be involved in transcriptional silencing in heterochromatin-like complexes. It recognizes and binds histone H3 tails methylated at K9, which leads to epigenetic repression. It is suggested that these proteins, which are involved in cell cycle processes, transcription regulation and gene expression, might be potential candidates for cell proliferation regulation and their repression through down-regulation might result in cell cycle stop without impact on stem cell pluripotency.

Proteins, which are involved in nucleotide biosynthetic process and proteolysis, were downregulated in CPX treated cells compared to control, as well as in RA treated cells (Figures 6A and 7A). Nucleoside diphosphatase kinases A and B (Nme1 and Nme2) are some of the proteins which are involved in nucleotide biosynthetic process. These proteins are known to be involved in the synthesis of nucleoside triphosphatases[51] as well as in cell proliferation[52], differentiation[53] and development[54], signal transduction, G protein-coupled receptor endocytosis and gene expression. Nme1 was downregulated in CPX treated cells compared to control and RA treated cells (Figure 8). This may explain the slowdown of the proliferation of CPX treated SCs. Impdh2 is a rate limiting enzyme in the de novo synthesis of guanine nucleotides and is therefore involved in the regulation of cell growth and differentiation[55-58]. It may have a role in the development of malignancy and the growth progression of some tumors. Impdh2 was downregulated in CPX treated cells compared to control (Figure 8).

Proteins which were involved in cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis, and phosphorylation were downregulated in RA treated cells compared to control and CPX treated cells (Figures 6B and 7B).

Analysis of the molecular function of the differently expressed proteins demonstrated a potential involvement of some of these in metal ion binding, mainly iron binding. Cazzola et al[59] in 1990 established that iron is essential for proliferation, DNA synthesis and repair and mitochondrial electron transport. Therefore, it is assumed that CPX can stop the cell proliferation by regulating the expression of iron binding proteins.

The present study could give some insights into the mode of action of CPX in terms of expression regulation of various proteins. It not only shed light on the previously discussed roles of CPX, but could also provide some further insight into their mechanism. We could identify some potential candidates which can effect the cell proliferation directly or indirectly through other cellular processes. By understanding the mode of action of CPX, this study may provide new aspect that will help in the future strategy to improve therapeutic intervention in the treatment with CPX.

ACKNOWLEDGMENTS

We thank Elke Brunst-Knoblich for technical assistance.

COMMENTS
Background

Ciclopirox olamine (CPX), a synthetic antifungal agent used in the treatment of fungal and yeast infection of skin or mucosa. Apart from its antimycotic activity, CPX is also effective against both gram-positive and gram-negative bacteria. CPX might also serve as an alternative agent for therapeutic angiogenesis. CPX was also shown to have an antiproliferative effect on stem cells without affecting their pluripotency.

Research frontiers

Although CPX is used as therapeutic for different aspect the mechanism of action is still not clear. In this study, the authors investigated the impact of CPX on stem cell proteome and identified cellular mechanisms that may explain the way of action of CPX. The authors provided evidence that CPX is involved in expression regulation of nucleotide binding proteins resulting in cell cycle arrest.

Innovations and breakthroughs

It is postulated that the CPX works as an inhibitor of the iron-dependent enzymes due to its potential role as a chelator of intracellular iron. The present study could give some insights into the mode of action of CPX in terms of expression regulation of various proteins especially nucleotide-binding proteins. It not only shed light on the previously discussed roles of CPX, but could also provide some further insight into their mechanism. We could also identify some potential candidates, which can effect the cell proliferation directly or indirectly through other cellular processes.

Applications

By understanding the mode of action of CPX, this study may provide new aspects that will be helpful in the future strategy for therapeutic intervention in the treatment with CPX.

Terminology

Multipotent adult germline stem cells (maGSCs) are spermatogonial stem cells isolated from murine testis. CPX, the ethanolamine salt of 6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one, is a synthetic antifungal agent and is a hypusination inhibitor that controls the second step of the modification, which is catalyzed by deoxyhypusine hydroxylase. The hypusine is the result of a post-translational modification catalyzed by two enzymes: deoxyhypusine synthase and deoxyhypusine hydroxylase.

Peer review

This is a descriptive study in which the authors analyzed the proteome changes of embryonic stem cells and maGSCs accompanying the treatment with CPX and subsequent inhibition of hypusination using classical proteomic techniques like 2-DE, differential in-gel electrophoresis and mass spectrometry. The results are interesting and we could highlight that a treatment with CPX resulted in an alteration of the expression of 56 proteins compared to non-treated cells, and 54 proteins compared to retinoic acid treated cells. The majority of these proteins are involved in nucleotide binding and nucleotide biosynthetic processes, metal binding, DNA binding, and other processes which have been linked to CPX.

Footnotes

P- Reviewers Marchal JA, Tanabe S, Zaminy A S- Editor Wen LL L- Editor A E- Editor Zheng XM

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