Comparative Proteomic Analysis of Fibrosarcoma and Skin Fibroblast
Comparative Proteomic Analysis of Fibrosarcoma and Skin Fibroblast Cell Lines Hamdi UYSAL Ankara University Faculty of Veterinary Medicine Department of Biochemistry Ankara, Turkey
Introduction n Adult fibrosarcoma (a soft tissue sarcoma) defined by the World Health Organization as a 'malignant neoplasm composed of fibroblasts with variable collagen production and, in classical cases, a "herringbone" architecture. ‘ Diagnosis of fibrosarcoma is difficult to determine and like most other types of sarcomas, fibrosarcoma causes no characteristic symptoms. Fibrosarcoma can occur in any soft tissue but mostly tumour can occur in the deep soft tissues of the lower extremities, followed by upper extremities and trunk.
Introduction n Proteomics is the opportunity to detection the biomarkers that provide diagnostic and prognostic information about pathogenesis of diseases. In recent years the large number of proteomic studies to detect the biomarkers have been done. Comparative proteomic studies using 2 -D gel electrophoresis coupled mass spectrometry has been applied in various cancers, including gastric cancer, lung cancer, prostate cancer, and breast cancer.
Aim of the Study n n n In this study different protein expressions between human skin fibroblast cell line (CCD-1135 Sk) and human fibrosarcoma cell line (HT-1080) were studied by two dimensional polyacrylamide gel electrophoresis (2 D-PAGE) and liquid chromatography coupled with tandem mass spectrometry (LCMS/MS) techniques. We compared intracellular proteins by preparing lysate samples and extracellular proteins by preparing secretome samples between fibrosarcoma and fibroblast cell lines. The aim of the present study was to understanding the molecular mechanism of fibrosarcoma and investigate potential biomarkers for fibrosarcoma.
Materials and Methods Cell Culture n n Human skin fibroblast cell line (CCD-1135 Sk) and human fibrosarcoma cell line (HT-1080) were purchased from American Type Culture Collection (ATCC). Cell lines were cultured in DMEM-F 12 medium containing 10% fetal bovine serum (FBS) and 50 mg/l gentamicin. Both cell lines were incubated at 37 °C in 5% CO 2 atmosphere.
Materials and Methods Preparation of Secretome (Supernatant - Secreted Proteins) Samples n To obtain secretome samples, cells were grown to 70% confluence and washed with PBS 3 times before incubation in serum-free medium for 24 hours. After incubation, medium was harvested and filtered through a membrane filter (0, 45 μm) to remove cell debris or dead cells. n Afterwards medium was enriched and concentrated by TCA precipitation method. For this, TCA was added to serum-free medium to a final concentration of 20%, and the solution was incubated overnight at 4°C. The precipitate was collected by centrifugation and then supernatant was carefully removed. n Pellet washed with ethanol 3 times. After washing with ethanol pellet was dried at room temperature and resuspended in lysis buffer then centrifuged. The supernatant (secreted proteins) collected and protein concentration was determined by 660 nm protein assay.
Materials and Methods Preparation of Cell Lysate Samples To obtain lysate samples, cells were grown to 70% confluence and harvested with PBS-EDTA solution. Cells were collected by centrifugation and washed with PBS and sucrose solution. Afterwards supernatant was carefully removed and cells were lysed in lysis buffer then centrifuged. The supernatant collected and protein concentration was determined by 660 nm protein assay. n
Materials and Methods n n Two-dimensional Gel Electrophoresis Equal amount of proteins diluted with rehydration buffer (and applied to a 13 cm p. H 310, immobilized p. H gradient strip. Passive rehydration was performed overnight at room temperature. After passive rehydration first dimensional separation of proteins was performed. After isoelectric focusing strips were first equilibrated with 1% DTT in equilibration buffer, and then with 2, 5% iodoacetamide in the same buffer. The equilibrated strips were placed on top of the prepared 12% SDS polyacrilamide gels and sealed in place with 0, 5% low-melt agarose. Second dimensional separation of proteins was performed. After two dimensional electrophoresis gels were stained with colloidal Coomassie G-250.
Materials and Methods n n Analysis of Gel Images Stained gels were scanned using densitometer (Bio-Rad, GS-800) and analysed by PDQuest 2 -DE analysis software (Bio-Rad).
Materials and Methods n n In-gel Trypsin Digestion Spots corresponding to the proteins of interest were washed acetonitrile in 100 m. M NH 4 HCO 3 (1: 1) until all traces of Coomassie Brilliant Blue were removed. Proteins were in-gel reduced with 10 m. M DTT (30 minutes, 80 ºC) and S-alkylated with 55 m. M iodoacetamide (20 minutes, in the dark), both in 50 m. M NH 4 HCO 3. Gel particles were washed with 50 m. M NH 4 HCO 3, and then incubated with the digestion solution (20 ng/µl of trypsin in 50 m. M NH 4 HCO 3).
Materials and Methods n n LC-MS/MS Analysis 1 μl volume of sample containing the tryptic peptides was loaded on the LC-ESIq. TOF system (nano. ACQUITY ultra presssure liquid chromatography (UPLC) and SYNAPT high definition mass spectrometer with nanolockspray ion source, Waters). Columns are equilibrated with 97% mobile phase A (H 2 O, 0. 1%FA) and 3% mobile phase B (ACN, 0. 1% FA). The column temperature was set to 35°C. Peptides were trapped on a nano. ACQUITY UPLC Symmetry C 18 Trap column (5 µm particle size, 180 µm i. d. x 20 mm length) at 5 μl/min flow rate for 5 min. Peptides were then eluted from the trap column by gradient elution onto an analytical column (nano. ACQUITY UPLC BEH C 18 Column, 1. 7 µm particle size, 75 µm i. d. x 250 mm length, Waters), at 300 nl/min flow rate with a linear gradient from 5 to 35% acetonitrile over 30 min. Parallel collusion induced dissociation (MSE) was done at positive ion V mode, applying the MS and MS/MS functions over 1. 5 sec intervals with 6 V low energy and 15 -40 V high energy collusions. Mass drift was corrected by infusing glu-fibrinopeptide (500 pmol/µl) every 45 sec through the nanolockspray ion source at 300 nl/min flow rate. Peptide signal data were collected.
Materials and Methods n n LC-MS/MS Data Processing Peptide mass measurement was performed at low collusion energy and peptide sequence data collection at higher collusion energies. Tandem mass spectra extraction, charge state deconvolution and deisotoping were processed with Protein. Lynx Global Server v 2. 4 software (PLGS) (Waters Corp. , Milford, MA). Protein sequence database from Uniprot was used. The Apex 3 D data preparation parameters were set to 0. 2 min chromatographic peak width, 10000 MS TOF resolution, 150 counts for low energy threshold, 50 counts for elevated energy threshold, and 1200 counts for the intensity threshold. Databank search query was set to minimum 3 fragment ion matches per peptide, minimum 7 fragment ion matches per protein, minimum 1 peptide matches per protein and 1 missed cleavage. Carbamidomethyl-cysteine fixed modification and Acetyl Nterminal, deamidation of asparagine and glutamine, oxidation of methionine variable modifications were set. The false positive rate of the Identity. E algorithm is around 3 -4% with a randomized database and is five times larger than the original one.
Results n 2 -D PAGE Representative images of two-dimensional electrophoresis (2 -DE) maps of fibroblast lysate (a) and fibrosarcoma lysate (b) samples Differentially expressed proteins between human fibrosarcoma lysate and human skin fibroblast lysate samples. The numbered spots represent proteins up- and downregulated in fibrosarcoma lysate samples.
Result s • 2 -D PAGE n 2 -D PAGE Representative images of two-dimensional electrophoresis (2 -DE) maps of fibroblast secretome (a) and fibrosarcoma secretome (b) samples Differentially expressed proteins between human fibrosarcoma secretome and human skin fibroblast secretome samples. The numbered spots represent proteins only expressed in fibrosarcoma secretome samples
Results Spot No Accession No Protein Name Molecular Weight (Da) Isoelectric Point (p. I) PLGS Score Protein Function 1506 Q 15084 Protein disulfide isomerase A 6 53867 5, 0 27342 Chaperone 1903 P 14625 Endoplasmin 92411 4, 6 15993 Chaperone 2605 P 10809 60 k. Da heat shock protein, mitochondrial 61016 5, 6 30952 Chaperone 3413 Q 92597 Protein NDRG 1 42807 5, 4 5928 Stress-responsive protein 3602 P 48643 T complex protein 1 subunit epsilon 59632 5, 3 14835 Chaperone 3707 P 38646 Stress-70 protein, mitochondrial (GRP-75) 73634 5, 8 14715 Chaperone 3902 Q 14764 MVP (Major vault protein) 99266 5, 2 5029 Ribonucleoprotein 4505 P 78371 T-complex protein 1 subunit beta 57452 6, 0 18799 Chaperone 4601 P 30101 Protein disulfide isomerase A 3 56746 5, 9 23115 Isomerase 5706 P 31948 Stress-induced-phosphoprotein 1 62599 6, 4 5942 Co-chaperone 6002 P 62937 Peptidyl prolyl cis trans isomerase A 18000 7, 9 10210 Isomerase 7001 P 62937 Peptidyl prolyl cis trans isomerase A 18000 7, 9 8605 Isomerase 7006 P 37802 Transgelin-2 22377 8, 4 27775 Tumor Suppressor 8209 P 22626 Heterogeneous nuclear ribonucleoproteins A 2/B 1 37406 9, 2 14281 Ribonucleoprotein Differentially expressed proteins between two lysate groups of human fibrosarcoma (HT-1080) and human skin fibroblast (CCD-1135 Sk) cell lines.
Result s Spot No Accession No Protein Name 0203 P 09486 0403 P 27797 0902 P 05067 1704 P 14625 2002 P 09382 2602 P 11021 6202 P 06733 7204 P 06733 8001 P 62937 9002 P 23284 9106 P 04406 9306 Q 5 VTE 0 9307 P 00749 SPARC Calreticulin Isoform APP 751 of Amyloid beta A 4 protein Endoplasmin Galectin-1 78 k. Da glucose-regulated protein (GRP-78) Alpha-enolase Peptidyl prolyl cis trans isomerase A Peptidyl prolyl cis trans isomerase B Glyceraldehyde-3 phosphate dehydrogenase Putative elongation factor 1 alpha like 3 Urokinase type plasminogen activator Molecular Weight (Da) Isoelectric Point PLGS Score (p. I) Protein Function 34609 4, 5 4886 48111 4, 1 5534 Extracellular Matrix Protein Chaperone 84764 4, 5 1520 Serine Protease Inhibitor 92411 4, 6 7611 14706 5, 1 2855 Chaperone Galactoside-binding Protein 72288 4, 9 6867 Chaperone /Stress Response 47139 7, 2 3430 47139 7, 2 3083 Lyase 18000 7, 9 1652 Isomerase 23727 9, 9 7594 Isomerase 36030 8, 7 13439 Oxidoreductase 50153 9, 4 3961 Elongation Factor 48475 8, 3 5618 Hydrolase Differentially expressed proteins between two secretome groups of human fibrosarcoma (HT-1080) and human skin fibroblast (CCD-1135 Sk) cell lines.
Discussio n n n The structure and function of the cancer cells are different from the normal cells that they originate, for this reason protein expression is altered. These protein expression changes can be associated with tumorigenesis and analysis of these proteins is important to understand the molecular mechanism of cancer and also essential for biomarker discovery in cancer. In this study we identified 14 proteins in lysate samples and 13 proteins in secretome samples. In lysate samples 13 identified proteins were over-expressed and 1 protein downexpressed in fibrosarcoma and in secretome samples 13 identified proteins were only expressed in fibrosarcoma.
Discussion n The heat shock proteins (Hsps) are involved in protein folding, transport and helping to reach final conformation of proteins for the ability of cells to overcome stress. Their expression is changed by environmental and pathophysiological conditions, such as increased temperature or oxidative stress. Hsps such as mortalin, endoplasmin and HSP-60 were found overexpressed in fibrosarcoma lysate samples.
Discussion n The chaperones are molecular complexes that involved correct folding of proteins which is important for the cell growth and cell survival. TCP-1 -beta and epsilon are molecular chaperones and were found over-expressed in fibrosarcoma lysate samples. Calreticullin is also molecular chaperones and was only expressed in fibrosarcoma secretome samples. Also co-chaperone STI 1 was found over-expressed in fibrosarcoma lysate samples. TCP-1 proteins were reported to be over expressed in various cancer such as colorectal cancer, human lung squamous carcinoma and adenocortical carcinoma. Besides this it is reported that calreticullin is over-expressed in adenocortical carcinoma (16) and STI 1 is over-expressed in ovarian cancer.
Discussion n Among the up-regulated proteins, heterogeneous nuclear ribonucleoproteins A 2/B 1 and NDRG 1 increased in fibrosarcoma lysate samples. It is known that heterogeneous nuclear ribonucleoproteins A 2/B 1 is strongly associated in cell proliferation and cell survival in cancer. Also NDRG 1 has potential functions such as cell differentiation and cell cycle regulation. Heterogeneous nuclear ribonucleoproteins A 2/B 1 and NDRG 1 were reported to be over-expressed in various cancer such as gastric cancer, lung cancer, breast cancer and adenocortical carcinomas.
Discussion n Galectin-1, SPARC, urokinase type plasminogen activator, and isoform APP 751 of Amyloid beta A 4 proteins were secreted proteins and compared to fibroblast secretome these proteins were only detected in fibrosarcoma secretome samples. Recent studies have shown that proteolytic cleavage of Amyloid Precursor Protein has a role in cancer development and is associated with NF-k. B pathway activation. Also galectin-1 has a role in cancer such as angiogenesis and metastasis processes. SPARC modulates cell-matrix interactions and over-expressed in many types of cancer. Urokinase type plasminogen activator involved in cell proliferation and metastasis in cancer with degradation of extracellular matrix and basement membrane. These proteins were reported that expressed in many cancer types secretomes such as nasopharyngeal carcinoma and prostate cancer.
Discussion n We detected a remarkable down-expression of MVP in fibrosarcoma cell line. It is known that MVP expressions have been showed in various cancer types and it function as a transport-associated protein that can be related with multidrug resistance.
Discussion n Among the 27 identified proteins in lysate and secretome samples, 6 metabolic enzymes such as protein disulfide isomerase A 3 and A 6, alpha-enolase and peptidyl prolyl cis trans isomerase A and B were identified. These enzymes have involved many biological process such as protein folding and glucose metabolism. In particular alpha-enolase and peptidyl prolyl cis trans isomerase A were reported that over-expressed in lung cancer.
Conclusio n n n The comparative proteomics studies are essential to understanding the molecular mechanism of cancer and biomarker discovery. Our results will benefit identification of potential tumor markers and understanding of the mechanisms of fibrosarcoma.
• Acknowledgement Öğünç Meral, Ph. D
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