Seonyoung Choi

Comparison of growth factor and cytokine expression in patients with degenerated disc disease and herniated nucleus pulposus

OBJECTIVES This study was conducted to investigate the expression of cytokines and growth factors in disc specimens obtained from patients with herniated nucleus pulposus (HNP) and degenerated disc disease (DDD). DESIGN AND METHODS MRI and Western blot analyses were performed to evaluate the levels of disc degeneration and the expression levels of cytokines and growth factors. RESULTS The levels of TNF-alpha and IL-8 were significantly greater in the DDD group than in the HNP group, but no statistical differences were observed in the expression of IL-1beta, IL-6 and IL-12 between the HNP and DDD groups. In addition, the expression of TGF beta, VEGF and NGF was significantly higher in the DDD group than in the HNP group. CONCLUSION The greater levels of cytokine and growth factor expression in the DDD group than in the HNP explain why discogenic patients usually have more severe back pain than patients with herniated discs.

Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis.

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.

Evaluation of plasma carcinogenic markers in rat hepatic tumors models induced by rat hepatoma N1-S1 cells and benzo[a]pyrene

Benzo[a]pyrene (BaP) is a known carcinogen. Grilled or smoked meat is the major source of BaP intake for human beings. Previously, we established hepatic tumor animal models by injecting rat hepatoma N1-S1 cells or concomitant injection of N1-S1 cells and BaP into healthy Sprague-Dawley rats. In this study, we performed proteomic analyses of rat plasma collected from a hepatic tumor model and compared them to controls using a 3–10 pI range and large two dimensional gel electrophoreses. Proteomic analyses of rat plasma with hepatic tumors induced by the injection of N1-S1 cells resolved 1295 protein spots. Among them, 10 proteins were identified by ESI-MS-MS; four proteins were up-regulated and six proteins were down-regulated as compared to the controls. In addition, 1295 protein spots were also resolved from rats with hepatic tumors by the injection of N1-S1 cells plus BaP; five proteins were upregulated, and seven proteins were down-regulated. Of these 12 proteins, 10 proteins were identified by ESI-MS-MS. Out of 20 identified proteins, alpha-1-inhibitor 3 and zero beta-1 globin were down-regulated in both rats with hepatic tumors by N1-S1 cell-only and rats with hepatic tumors by the injection of N1-S1 cell plus BaP as compared to the controls. In addition, the identities of four proteins, including dermcidin, serum amyloid P-component (SAP), proteasome subunit alpha type-4 and glutathione peroxidase 3 (GPX-3) were confirmed by western blot analysis. Therefore, the importance of those proteins as candidate biomarkers for the development of hepatic tumors should be further elucidated.

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two-dimensional electrophoresis.

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH4HCO3 + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.

Identification of proteins expressed differently among surgically resected stage I lung adenocarcinomas.

RATIONALE Among patients with surgically resected stage I lung adenocarcinoma, some succumb to early recurrence, while others survive for more than 5 years. Few markers to predict prognoses in these patients have been accepted. Recent advances in proteomic methodologies offer a unique chance to identify new candidate biomarkers. The aim of this study is to find differences in protein expression in resected lung cancer tissue of stage I adenocarcinoma from patients with no recurrence for more than 5 years and from those with early recurrence. METHODS Lung cancer tissues were obtained from 15 patients with pathologically confirmed stage I adenocarcinoma. The patients were divided into two groups, those with recurrence within 36 months (early recurrence group, n=9) and those that were disease-free for over 5 years (disease free group, n=6). Tissue proteins were separated by a two-dimensional electrophoresis long gel system (30 × 40 cm) with set ranges (3-10 NL) and examined by nano-LC-ESI-MS/MS. Western blot assays were performed to validate these proteins. RESULTS Twelve protein spots were up-regulated and 8 were down-regulated in the disease-free group as compared with the recurrence group. Of the 12 up-regulated proteins, haptoglubin, tau-tubulin kinase-2 (TTBK2), thymidine phosphorylase, annexin-1, PIN1, CAPG, and SEC23 were validated by Western blot. Among the 8 down-regulated proteins, serpinB6 and trangelin-2 were validated. CONCLUSIONS A total of 9 differentially expressed proteins were successfully extracted, identified, and confirmed from stage I lung adenocarcinoma tissues. The increased or decreased expression of these proteins according to prognosis may be the basis for further studies of proteomics in developing prognostic biomarkers.

Plasma proteomic analysis of patients infected with H1N1 influenza virus

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1‐infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano‐ultra performance LC‐MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1‐infected group as compared with the control group. Of 14 up‐ and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine‐rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.

Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expressions in the plasma of rats and incineration workers.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.