Proteomic Analysis of Human Leukemic U937 Cells Incubated with

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Proteomic Analysis of Human Leukemic U937 Cells Incubated with Conditioned Medium of Mononuclear Cells Stimulated by Proteins from Dietary Mushroom of Agrocybe aegerita Yuh-Tai Wang,† Zhu-Jun Huang,‡ and Hung-Min Chang*,‡ Life Science Center, Hsing Wu College, No. 11-2, Fen-Liao Road, Lin-Kou, Taipei 112-44, Taiwan and Graduate Institute of Food Science and Technology, National Taiwan University, Taipei 106-17, Taiwan Received April 12, 2004

Proteomic studies on the differentiation induction of human myelocytic lymphoma U937 cells into macrophages by the mononuclear cell-conditioned media (MNC-CM) prepared with fraction 2 proteins of Agrocybe aegerita (AA-f2-MNC-CM) were conducted for the first time. Spotted proteins in the 2-dimensional electrophoresis (2-DE) gels were digested and analyzed simultaneously with ImageMaster 2D Elite analysis software. Treatment of U937 cells with AA-f2-MNC-CM for 5 days resulted in a significant change of the 2-DE protein maps. It was found that 87 and 54 spots were different in quantity by more than 50% in cells which were treated with MNC-CM prepared with 5 and 25 µg AA-f2/mL, respectively. Among these, 53 individual proteins were successfully identified and grouped into seven categories based on cellular functions. The down-regulation of these proteins in each concentration of AA-f2 treatment might be associated with the physiological changes, which resulted in the growth inhibition and differentiation of U937 cells. Moreover, it gave a sign into the cancer therapy mechanism of U937 cells treated with dietary mushroom AA-f2. Keywords: Agrocybe aegerita • fungi • U937 cell • differentiation • proteomics

Introduction Proteomic analysis has been proven to be the most powerful technique for the identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions.1 The strategy in proteomics is the combination of the high resolving power of 2-dimensional electrophoresis (2-DE) with the highly sensitive matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS), whereas 2-DE allows us to simultaneously separate and identify hundreds to thousands of proteins for changes in molecular size, isoelectric point, phosphorylation, and the identification of specific protein spots. This technique of differentiating and identifying the enzymes secreted from different metabolic pathways is a powerful screening method for detecting unexpected changes in protein expression that may be missed by conventional biochemical techniques and may lead to the discovery of novel proteins and/or to the development of new biocatalysts.2,3 There are numerous applications in proteomics; the potential approach is in the clinical and biomedical fields.4,5 Diseaseassociated proteins can be identified by comparing the protein profiles of normal versus diseased tissues or biological fluids * To whom correspondence should be addressed. Telephone: +886-22363-0231 ext 2776. Fax: +886-2-2362-0849. E-mail: Changhm@ ccms.ntu.edu.tw. † Life Science Center, Hsing Wu College. ‡ Graduate Institute of Food Science and Technology, National Taiwan University.

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using protein expression proteomics.6 Hence, these proteins are potential diagnostic tools for drugs discovery; that is, proteomics can provide major opportunities to elucidate disease mechanisms, identify new diagnostic markers and therapeutic targets.7,8 Basidiomycetes mushrooms contain many bioactivity materials, including terpenoids, steroids, phenols, nucleotides, polysaccharides, and proteins etc.,9 that express promising antitumor, immune modulating, cardiovascular and hypercholesterolemia, antiviral, antibacterial, and antiparasitic effects.10,11 Many species of mushrooms such as Flammulina velutipes,12-14 Hypsizygus marmoreus,15 Pleurotus ostreatus,16 Lentinus edodes,17 and Agrocybe aegerita18 have been found to markedly inhibit the growth of different tumors. However, the roles of bioactive ingredients in antitumor function are still unclear. The human leukemia U937 cells have been well characterized as an in vitro model for cell proliferation and differentiation.19-21 Previously, we found that the proliferation of human leukemic U937 cells was remarkably reduced by about 80% when cells were incubated for 5 days with conditioned media (CM) of human peripheral blood mononuclear cells (MNC) stimulated with 100 mg/mL of cold water extracts (CWE) or proteins from Hypsizigus mamoreus or Agrocybe aegerita (AA) (MNC-CM method).21 Moreover, the differentiation of the thus harvested U937 cells into mature monocytes/macrophages was about 80%, determined by a nitroblue tetrazolium (NBT) test, and high levels of cytokines such as tumor necrosis factor (TNF)-R and interleukine (IL)-1β were assayed in the CM of 10.1021/pr049922h CCC: $27.50

 2004 American Chemical Society

Proteomic Analysis of U937 Cells Incubated with MNC-CM

MNC.21

AA protein-stimulated However, the antitumor mechanisms of those proteins, in terms of protein expression, remain unclear. In an attempt to replace the toxic chemicals for chemoprevention by food ingredients in remedying leukemia, the antitumor functions of dietary mushroom of A. aegerita on the inhibition of the proliferation and differentiation of human leukemia U937 cells were further investigated. Proteomics approaches were used to investigate the global changes in cellular proteins of U937 cells after treatment with proteins from A. aegerita by MNC-CM method. It is reported for the first time on 2-DE protein maps about the stimulation of leukemic U937 cells by proteins from an edible mushroom and provides information of clinical treatment for leukemia disease research.

Materials and Methods Isolation of Fraction 2 Proteins from Agrocybe aegerita (AAf2). Fresh fruiting bodies of A. aegerita, purchased from a local supermarket, were washed, air-dried and homogenized in 2-fold volume (v/w) cold (4 °C) water, followed by stirring at 4 °C for 24 h and centrifugation at 12 000 × g for 30 min. For partial purification, the supernatant was collected, precipitated by 100% ammonium sulfate, re-suspended, and then dialyzed against cold distilled water. The supernatant thus obtained was passed through a Sephacryl S-300 column (1.6 × 96 cm) in 0.3 M NaCl (eluent) and the eluate was monitored by a spectrophotometer at absorbance 280 nm. The second fraction (f2) (fraction number 19-22, 5 mL/tube), termed as AA-f2, was pooled and dialyzed against cold water, followed by lyophilization and storage at -20 °C before use. Preparation of Conditioned Media. Mononuclear cells from human peripheral blood, which were obtained from three healthy adults with informed consent, were separated by centrifugation on a density gradient (Ficoll-Hypaque, 1.077 gm/mL, Pharmacia Fine Chemicals, Uppsala, Sweden) and a concentration of 1.6 × 106 cells/mL was incubated in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 1% glutamine/ 10% heat-inactivated fetal calf serum (FCS) (Hyclone, Logan, UT). The mononuclear cell-conditioned medium (MNC-CM) was prepared by incubating the cells in the presence (5 or 25 µg/mL) of AA-f2 at 37 °C in a humidified 5% CO2 incubator for 24 h.19-21 The cell-free supernatant was then collected, filtered and stored at -70 °C until use. The CM collected from AA-f2treated MNC cultures was designated as AA-f2-MNC-CM. PBS was also used to stimulate MNC to prepare CM (PBS-MNCCM) (control) for the following experiment. Incubation of U937 Cells with AA-f2-MNC-CM. The human myeloid leukemic U937 cells, obtained from the American Type Culture Collection (Rockville, MD), were cultured in RPMI 1640 medium containing 10% FCS and 2 mM glutamine and maintained in an exponential growth status at an initial concentration of 1 × l05/mL in the presence of 20% (v/v) of AA-f2-MNC-CM or of PBS-MNC-CM at 37 °C in a humidified 5% CO2 incubator for 5 days.19,21 The cells were then collected, washed, filtered, freeze-dried, and stored at -20 °C until use. Three separate cell samples were each prepared in duplicate. Assay for Superoxide Production. Nitroblue tetrazolium (NBT) test was applied to detect the production of cytoplasmic superoxide by the differentiated myeloid cells. Cells collected from day 5 cultures were suspended in RPMI medium at a concentration of 1 × 106 cells/mL and then mixed with an equal volume of NBT test stock solution (2 mg NBT/1 µM phorbol

letters myristate acetate/mL PBS). After incubation at 37 °C for 30 min, the suspended cells (80 µL) were cytocentrifuged onto glass slides with the aid of a Cytospin (1200 rpm, 5 min) and then counter-stained with 0.5% Safranin solution. The percentage of formazan-containing cells was assessed out of 200 cells. Three separate experiments were each tested in duplicate. Sample Preparation for Proteomic Analysis. AA-f2-MNCCM-treated U937 cells were lysed and sonicated in lysis buffer (8 M urea/4% CHAPS/2% ampholine). The proteins from U937 cells were centrifuged at 8000 × g for 30 min and the upper 80% supernatant was collected for protein concentration determination using Bio-Rad protein assay kit and stored at -70 °C. Samples equal to 1.0 mg protein/350 µL were loaded on Immobline Drystrips (pH 3-10 NL, 18 cm) for 2-DE. 2-DE and Image Processing. Gel electrophoresis was performed with pH 3-10 nonlinear strips and linear gradient 1018% sodium dodecyl sulfate (SDS) polyacylamide gel and proteomic analysis was performed as described previously22. Briefly, the first dimensional isoelectric focusing (IEF) was conducted using an IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England. The gels were rehydrated (20 °C; 50 µA/gel; 30 V for 12 h) previously overnight by placing the strips gel-sidedown in sample-containing rehydration solution (8 M urea/2% CHAPS/2% ampholines) in IPGphor strip holder and covered with Drystrips cover fluid, followed by desalting (20 °C; 50 µA/ gel; 30 V for 6 h). IEF was conducted according to the following running conditions: 30 V, 12 h; 100 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 4000 V, 1 h; 6000 V, 65 kVh. For the second dimension, 10-18% polyacrylamide gradient gels using Ettan DALTsix Electrophoresis System (Amersham Pharmacia Biotech) were used to obtain an optimal separation (45 mA/gel, 8 °C, 5 h). The gels were stained with a fluorescence dye Sypro Ruby protein stain (Molecular Probes, Eugene, Oregon, USA), as reported by Lopez et al.23, and scanned with a laser scanning system (Molecular Image FX, Bio-Rad, Hercules, CA). The images were analyzed with ImageMaster software (Amersham Pharmacia Biotech, CA). All gel spots were detected and quantified automatically, using default spot detection parameters of 2-DE gel from AA-f2 untreated sample, and checked manually in agreement with the visual inspection of the gels to eliminate any artifacts. The relative volume was calculated in order to correct any differences in protein loading and gel staining. Three separate experiments were each tested in duplicate. Mass Spectrometric Identification. Protein spots of interest were excised from the gels and in-gel digested with sequencing grade modified trypsin (10 µg/mL) (Promega, Medison, WIS, USA) at 37 °C for 16 h. The resultant peptides were extracted with 50% acetonitrile/5% trifluoroacetic acid TFA), desalted, vacuum-dried, and resolved in 10 µL of 0.1% TFA for Mass spectrometry analysis. The peptide mixtures with equal volumes of matrix solution (R-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile/ 0.1% TFA) were analyzed in a MALDI-TOF mass spectrometer (Micromass, Wythenshawe, UK). The peptide masses were matched with the theoretical peptide masses of all proteins from all species of the SWISS-PROT databases. Peptide standard (Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-AlaArg) with a molecular mass of 1569.6696 (Amersham Bioscience) was used as MALTI-TOF mass standard.24 The protein search was performed using World Wide Web search programs MS-Fit provided by UCSF (http://prospector.ucsf.edu/ucsfhtml3.2/msfit.htm) with NCBI database. Journal of Proteome Research • Vol. 3, No. 4, 2004 891

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Figure 1. 2-D electrophoretograms of U937 cells incubated for 5 days with various preparations of AA-f2-MNC-CM. A (control), 0 µg AA-f2/mL; B, 25 µgAA-f2/mL; C, 50 µg AAf2/mL. Fraction 2 protein from dietary mushrooms of A. aegerita on a Sephacryl-S 300 gel permeation chromatography was used to stimulate MNC to collect conditioned medium, which was added (20%, v/v) to the culturing medium to harvest U937 cells. Table 1. Characteristics and Functions of the Identified Proteins in U937 Cells Incubated for 5 days with AA-f2-MNC-CM Prepared with 5 µG/ML spot

SWISS-PROT accession no.a

predicted pI/Mr

protein

1 2 3 4 5 6 7 8 9 10 11

P27797 P05217 Q9NY65 P02570 P08865 P06748 Q9Y3F4 P28066 P15374 P13693 Q13185

4.29/48112 4.79/49799 4.94/50062 5.29/41710 4.79/32833 4.64/32555 4.98/38414 4.69/26452 4.84/26166 4.84/19583 5.23/20810

calreticulin precursor tubulin beta-2 chain tubulin alpha-6 chain actin, cytoplasmic 1 40S ribosomal protein SA nucleophosmin UNR-interacting protein proteasome subunit alpha type 5 ubiquitin carboxyl-ferminal hydrolase isozyme L3 translationally controlled tumor protein chromobox protein homologue 3

12 13 14 15 16 17 18 19 20 21

P38646 P48643 P30101 P31943 P31150 P00813 Q13148 P40121 P51570 P11177

5.87/73635 5.45/59633 5.98/56747 5.89/49198 5.00/50550 5.63/40739 5.85/44711 5.88/38494 6.04/42246 6.20/39194

22 23 24 25 26 27 28 29 30 31 32 33 34 35

P35232 P78417 O95336 Q13162 P28070 P09211 P32119 P12268 P14618 P00558 P30040 P04406 P05092 P07737

5.57/29786 6.23/27548 5.70/27530 5.86/30521 5.72/29173 5.44/23210 5.66/21878 6.44/55770 7.95/57769 8.30/44568 6.77/28975 8.58/35899 7.82/17870 8.48/14914

stress-70 protein mitochondrial precursor T-complex protein 1 epsilon subunit protein disulfide isomerase A3 precursor heterogeneous nuclear ribonucleo protein rab GDP dissociation inhibitor alpha adenosine deaminase TAR DNA-binding protein-43 macrophage capping protein galactokinase pyruvate dehydrogenase E1 component beta subunit mitochondrial precursor prohibitin glutathione transferase omega 1 6-phosphogluconolactonase peroxiredoxin 4 proteasome subunit beta type 4 precursor glutathions S-transferase P peroxiredoxin 2 inosine-5′-monophosphate dehydrogenase 2 pyruvate kinase, M1 isozyme phosphoglycerate kinase 1 endoplasmic reticulum protein Erp29 precursor glyceraldedyde 3-phosphate dehydrogenase peptidyl-prolyl cis-trans isomerase A profiling I

a

endoplasmic reticulum lumen cytoplasmic cytoplasmic nuclear cytoplasmic and nuclear cytoplasmic cytoplasmic nuclear; associates with euchromatin and is largely excluded from constitutive heterochromatin (by similarity). mitochondrial cytoplasmic endoplasmic reticulum lumen nuclear; nucleoplasm cytoplasmic nuclear nuclear and cytoplasmic mitochondrial matrix cytoplasmic cytoplasmic cytoplasmic cytoplasmic and nuclear cytoplasmic

endoplasmic reticulum lumen cytoplasmic cytoplasmic

Accession no.: the purpose of accession number is to provide a stable way of identifying entries from release to release by SWISS-PROT.

Results and Discussion 2-DE Protein Maps of U937 Cells Associated with AA-f2MNC-CM Treatment. Previously, Ou et al.21 indicated that high positive percentages of NBT test and nonspecific esterase (NSE) 892

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test of myeloid leukemic U937 cells were observed when cells were incubated for 5 days with MNC-CM prepared with AA proteins. It suggests that the apparent maturation of cells that produce superoxide to present bactericidal effect,25 is induced by mushroom proteins through the activation of MNC. For

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Table 2. Characteristics and Functions of the Identified Proteins in U937 Cells Incubated for 5 days with AA-f2-MNC-CM Prepared with 25 µG/ML SWISS-PROT accession no.a

predicted pI/Mr

1 2 3 4 5 6 7

P07237 O00505 Q9BQE3 P10809 P02570 P02571 P11177

4.76/57081 4.8/57775 4.96/49863 5.70/61016 5.29/41710 5.31/41766 6.20/39194

8 9 10

Q99795 P52565 Q9Y535

4.85/35609 5.02/23193 4.47/22903

11 12 13 14

P10109 P30049 P31943 Q99542

5.51/19381 5.38/17479 5.89/49198 7.22/57321

protein disulfide isomerase precursor importin alpha-3 subunit tubulin alpha-6 chain 60 kDa heat shock protein actin, cytoplasmic 1 actin, cytoplasmic 2 pyruvate dehydrogenase E1 component beta subunit mitochondrial precursor cell surface A33 antigen precursor rho GDP-dissociation inhibitor 1 DNA directed RNA polymerase III subunit 22.9 kDa polypeptide adrenodoxin mitochondrila precursor ATP synthase delta chain heterogeneous nuclear ribonucleoprotein H matrix metalloproteinase-19 precursor

15 16 17 18 19 20 21 22 23 24 25

P26038 P40121 P47756 P78417 P07339 P30040 P28070 P07741 P14618 Q15654 P04406

6.09/67647 5.88/38484 5.69/30609 6.23/27548 6.10/44552 6.77/28975 5.72/29173 5.79/19464 7.95/57769 7.76/52187 8.58/35899

moesin macrophage capping protein F-actin capping protein beta subunit glutathione transferase omega 1 cathepsin D precursor endoplasmic reticulum protein ERp 29 precursor proteasome subunit beta type 4 precursor adenine phosphoribosyltransferase pyruvate kinase, M1 isozyme thyroid receptor interacting protein 6 glyceraldehyde 3-phosphate dehydrogenase

spot

a

protein

subcellular location

endoplasmic reticulum lumen cytoplasmic and nuclear (by similarity) enoplasmic reticulum lumen cytoplasmic cytoplasmic mitochondrial matrix type I membrane protein cytoplasmic nuclear mitochondrial matrix mitochondrial nuclear; nucleoplasm secreted. associated with the extracellular matrix (by similarity) nuclear and cytoplasmic cytoplasmic lysosomal endoplasmic reticulum lumen cytoplasmic and nuclear (by similarity) cytoplasmic cytoplasmic

Accession no.: the purpose of accession number is to provide a stable way of identifying entries from release to release by SWISS-PROT.

Figure 2. 2-D electrophoretogram of U937 cells incubated for 5 days with AA-f2-MNC-CM prepared with 5 mg/mL. Protein spots marked on the maps were relatively different in quantity (g 50%) and were expressed and identified by MS. All the protein spots identified were down-regulated proteins.

Figure 3. 2-D electrophoretogram of U937 cells incubated for 5 days with AA-f2-MNC-CM prepared with 25 µg/mL. Protein spots marked on the maps were relatively different in quantity (g50%) and were expressed and identified by MS. All the protein spots identified were down-regulated proteins.

further studies, AA proteins were fractionated by a Sephadex S-300 gel permeation chromatography and the pooled protein factions were subjected to MNC-CM preparation in a preliminary test. It was found that protein in pooled fraction 2 was most potent in inhibiting growth (25 µg/mL, 69%; 50 µg/mL, 91%) and inducing differentiation (25 µg/mL, 68% NBT positive; 50 µg/mL, 78% NBT positive) of U937 cells, and thus, AA-f2MNC-CM was prepared for the following experiments. Protein analysis of the U937 cells treated with or without AA-f2-MNC-CM was done using 2-DE to resolve several hun-

dred U937 cell proteins. The 2-DE gels were digitized and analyzed simultaneously with ImageMaster 2D Elite analysis software and a typical analytical 2-DE gel from U937 cells was shown in Figure 1A, which revealed the 523 protein spots detected in undifferentiated U937 cells (control). However, significant changes were observed as the 2-DE protein maps of AA-f2-MNC-CM treated U937 cells (Figure 1B,C) were compared with that of control. It suggests that proteome is strongly influenced by cell differentiation.26 For further investigations, the spot volume for each protein was quantified using Journal of Proteome Research • Vol. 3, No. 4, 2004 893

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Table 3. Proteins with Enzymatic Function and Their Catalytic Activity in U937 Cells Incubated for 5 days with AA-f2-MNC-CM Prepared with 5 µG/ML spot

accession no.

protein

8 9

P28066 P15374

proteasome subunit alpha type 5 ubiquitin carboxyl terminal hydrolase isozyme L3 protein disulfide isomerase A3 precursor

14

P30101

17 20 21

P00813 P51570 P11177

23 24

P78417 O95336

adenosine deaminase galactokinase pyruvate dehydrogenase E1 component beta subunit mitochondrial precursor glutathione transferase omega 1 6-phosphogluconolactonase

26 27 29

P28070 P09211 P12268

proteasome subunit beta type 4 precursor glutathions S-transferase P inosine-5′-monophosphate dehydrogenate 2

30 31

P14618 P00558

pyruvate kinase, M1 isozyme phosphoglycerate kinase 1

33

P04406

glyceraldehyde 3-phosphate dehydrogenase

34

P05092

peptidyl-prolyl cis-trans isomerase A

ImageMaster 2D Elite analysis software and found that 87 protein spots in Figure 1B (5 µg AA-f2/mL) and 54 protein spots in Figure 1C (25 µg AA-f2/mL) were greater than 50% change in spot volume after AA-f2-MNC-CM treatment, as compared to the corresponding protein spot in the protein map of control, and were chose for identification. Of note, these protein spots of U937 cells were expressed differently when AA-f2 was shifted 5 to 25 µg/mL, which was in accordance with the dose-dependent manner of AA proteins in inhibiting the cell growth and inducing differentiation of U937 cells by the MNC-CM method. Protein Identification and Comparison of Protein Profiles Between AA-f2 Untreated and Treated U937 Cells. These spots were excised and subsequently identified by peptide mass fingerprinting using MALDI-TOF and from SWISS-PROT database searches using Masslynx 3.5 software. As shown in Tables 1 and 2, proteins associated with 35 spots of 87 protein spots (5 µg/mL) and 25 spots of 54 protein spots (25 µg/mL) were successfully identified by this method. Among the total 60 protein spots, 53 individual proteins were confirmed. The other protein spots could not be identified by MALDI-TOF MS, indicating these proteins might be novel proteins or have not been sequenced.3 Of note, the levels of each identified protein were found to significantly decrease after image matching of 2-DE gels. That is, all identified proteins were down-regulated after AA-f2-MNC-CM treatment (Figures 2 and 3). Subsequently, the identified proteins of 5 µg AA-f2/mL treatment were classified by subcellular locations into seven groups (Table 1), including endoplasmic reticulum lumen, cytoplasmic, nuclear, mitochondrial, and others. Meanwhile, the proteins from 25 µg AA-f2 treatment (Table 2) were also grouped into endoplasmic reticulum lumen, cytoplasmic, nuclear, mitochondrial, secreted, lysosomal, and others. Among the 53 individual identified proteins, seven proteins (heterogeneous nuclear ribonucleo protein, macrophage capping protein, pyruvate dehydrogenase E1 component beta subunit mitochondrial precursor, glutathione transferase omega 1, pyruvate kinase M1 isozyme, endoplasmic reticulum protein Erp29 precursor, glyceraldedyde 3-phosphate dehydrogenase) were found to appear and down-regulate in each concentration of AA-f2 treatment. 894

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catalytic activity

cleavage at peptide bonds with very broad specificity. ubiquitin C-terminal thiolester + H2O ) ubiquitin + a thiol rearrangement of both intrachain and interchain disulfide bonds in proteins to form the native structures adenosine + H2O ) inosine + NH3 ATP + D-galactose ) ADP + D-galactose 1-phosphate pyruvate + lipoamide ) S-acetyldihydrolipoamide + CO2 RX + glutathione ) HX + R-S-glutathione 6-phospho-D-glucono-1,5-lactone + H2O ) 6-phospho-D-gluconate cleavage at peptide bonds with very broad specificity RX + glutathione ) HX + R-S-glutathione inosine 5′-phosphate + NAD+ + H2O ) xanthosine 5′-phosphate + NADH ATP + pyruvate ) ADP + phosphoenolpyruvate ATP + 3-phospho-D-glycerate ) ADP + 3-phospho-D-glyceroylphosphate D-glyceraldehyde 3-phosphate + phosphate + NAD+ ) 3-phospho-D-glyceroyl phosphate + NADH peptidylproline (omega ) 180) ) peptidylproline (omega ) 0)

Cellular Functions of Identified Enzymes. The identified enzymes of U937 cells after AA-f2-MNC-CM treatment may be grouped into several categories based on assumed cellular function (Tables 3 and 4). Among them, protein disulfide isomerase family and peptidyl-prolyl cis-trans isomerase are enzymes involved in protein structure construction; glutathione transferase family appear to be involved in catabolism of toxic substances; inosine-5′-monophosphate dehydrogenase is cell growth associated; pyruvate kinase, alpha enolase, phosphoglycerate kinase, fructose-bisphosphate aldola, and glyceraldehyde 3-phosphate dehydrogenase are enzymes involved in glycolysis pathway;27 cytidine deaminase is pyrimidine nucleotide metabolism associated;27 pyruvate dehydrogenase and malate dehydrogenase involve in pyruvate oxidation and citric acid cycle;27 cathepsin D precursor, matrix metalloproteinase19 precursor, proteasome and ubiquitin carboxyl terminal hydrolase isozyme are enzymes involved in intracellular protein breakdown and protein turnover; adenosine deaminase is purine nucleotide catabolism associated;27 galactokinase is galactose catabolism associated; 6-phosphogluconolactonase is pentose phosphate pathway associated;27 DNA directed RNA polymerase is RNA synthesis associated, ATP synthase delta chain is ATP synthesis associated. These proteins indicated that treatment of AA-f2-MNC-CM induced physiological changes of U937 cells. The area affected including synthesis of DNA and RNA, catabolism pathway (such as glycolysis pathway, pentose phosphate pathway, citric acid cycle and uric acid synthesis) and protein conformation. Since the levels of all identified proteins decreased after AA-f2-MNCCM treatment, the down-regulation of these proteins significantly affected functions of U937 cells. Hence, the proliferation inhibition and differentiation of U937 cells by AA-f2-MNC-CM are associated with the inhibition of disulfide bond formation and proteolysis of cellular protein, interruption of some catabolism pathways that cause reaction time extended, accumulation of intermediates or toxic substance, and replication inhibition of DNA or RNA that inhibits the biosynthesis of U937 cells. In preliminary tests, we demonstrated that treatment of U937 cells with AA-f2-MNC-CM resulted in a marked inhibition of

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Table 4. Proteins with Enzymatic Function and Their Catalytic Activity in U937 Cells Incubated for 5 days with AA-f2-MNC-CM Prepared with 25 µG/ML spot

accession no.

protein

1

P07237

protein disulfide isomerase precursor

7

P11177

10

Q9Y535

12 14 18 19

P30049 Q99542 P78417 P07339

pyruvate dehydrogenase E1 component beta subunit mitochondrial precursor DNA directed RNA polymerase III subunit 22.9 kDa polypeptide ATP synthase delta chain matrix metalloproteinase-19 precursor glutathione transferase omega 1 cathepsin D precursor

21 22 23 25

P28070 P07741 P14618 P04406

proteasome subunit beta type 4 precursor adenine phosphoribosyltransferase pyruvate kinase, M1 isozyme glyceraldehyde 3-phosphate dehydrogenase

proliferation and maturation of monocytes/macrophages. It revealed that the antitumor activity induced by AA-f2 was due to the stimulated secretion of differentiation-inducing factors from immunocompetent cells.28 There may be mediators which enable us to inhibit the growth of leukemic cells and induce them differentiating into mature monocytes/macrophages and functional cells.29,20,21 Thereby, the difference of 2-DE protein profiles between AA-f2 untreated and indirectly treated U937 cells maybe due to the mediators. In this study, all of the 53 identified proteins were down-regulated and only 7 of the 53 proteins appeared repeatedly in both 5 and 25 µg/mL treatments. It is possible that down-regulations of these 53 proteins are controlled by at least three types of mediators, found in the MNC-CM,19-21 that individually or synergistically trigger the differential induction of U937 cells into mature monocytes/ macrophages. Thus, the present studies not only provided a 2-DE protein database of AA-f2-MNC-CM treated cells, which gave a further analysis on the complete complement of proteins expressed and the differential mediators, but also give a sign into the cancer therapy mechanism of edible mushroom A. aegerita. It has been reported that IL-4 selectively up-regulated surfactant protein-D expression, which has a potent antiinflammatory function, and acted at the level of mRNA in isolated pulmonary epithelial cells.30 The functional imbalance of T helper cells, which play an important role in the pathogenesis of endometriosis, in endometriosis patients was due to the changes of IFN-γ and IL-4 contents.31 TNF-R treatment of articular cartilage resulted in the increased synthesis and activation of metalloproteinases in articular cartilage, increased release of proteoglycan, and increased cell death.32 Previous studies showed that there were high contents of IL-1β, TNF-R, and GM-CSF in AA-f2-MNC-CM.21,33 It is worthy of further studies on the stimulation mechanisms of cytokines on the protein expression patterns of U937 and searches of specific proteins for therapeutic application. Abbreviations: MNC-CM, mononuclear cell-conditioned media; AA-f2-MNC-CM, fraction 2 proteins of Agrocybe aegerita; MALDI-TOF MS, matrix assisted laser desorption/ionization time-of-flight mass spectrometry; NBT, nitroblue tetrazolium; EP, endoprotease; 2-DE, 2-dimensional electrophoresis; CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; SDS, sodium dodecyl sulfate.

catalytic activity

rearrangement of both intrachain and interchain disulfide bonds in proteins to form the native structures pyruvate + lipoamide ) S-acetyldihydrolipoamide + CO2 N nucleoside triphosphate ) N diphosphate + {RNA}(N) ATP + H2O + H+(In) ) ADP + phosphate + H+(Out) cleaves aggrecan at the 360-Ser-|-Phe-361 site RX + glutathione ) HX + R-S-glutathione specificity similar to, but narrower than, that of pepsin A, does not cleave the 4-Gln-|-His-5 bond in B chain of insulin cleavage at peptide bonds with very broad specificity. hydrolyzes one Arg-|-Ile bond in factor X to form factor Xa. ATP + pyruvate ) ADP + phosphoenolpyruvate D-glyceraldehyde 3-phosphate + phosphate + NAD+ ) 3-phospho-D-glyceroyl phosphate + NADH

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