Giman Jung

Secreted CXCL1 Is a Potential Mediator and Marker of the Tumor Invasion of Bladder Cancer

Purpose: The purpose of this study was to identify proteins that are potentially involved in the tumor invasion of bladder cancer. Experimental Design: We searched for the candidate proteins by comparing the profiles of secreted proteins among the poorly invasive human bladder carcinoma cell line RT112 and the highly invasive cell line T24. The proteins isolated from cell culture supernatants were identified by shotgun proteomics. We found that CXCL1 is related to the tumor invasion of bladder cancer cells. We also evaluated whether the amount of the chemokine CXCL1 in the urine would be a potential marker for predicting the existence of invasive bladder tumors. Results: Higher amount of CXCL1 was secreted from highly invasive bladder carcinoma cell lines and this chemokine modulated the invasive ability of those cells in vitro. It was revealed that CXCL1 regulated the expression of matrix metalloproteinase-13 in vitro and higher expression of CXCL1 was associated with higher pathologic stages in bladder cancer in vivo. We also showed that urinary CXCL1 levels were significantly higher in patients with invasive bladder cancer (pT1-4) than those with noninvasive pTa tumors (P = 0.0028) and normal control (P < 0.0001). Finally, it was shown that CXCL1 was an independent factor for predicting the bladder cancer with invasive phenotype. Conclusions: Our results suggest that CXCL1 modulates the invasive abilities of bladder cancer cells and this chemokine may be a potential candidate of urinary biomarker for invasive bladder cancer and a possible therapeutic target for preventing tumor invasion.

Reduced Plasma Level of CXC Chemokine Ligand 7 in Patients with Pancreatic Cancer

Background: Early detection is essential to improve the outcome of patients with pancreatic cancer. A noninvasive and cost-effective diagnostic test using plasma/serum biomarkers would facilitate the detection of pancreatic cancer at the early stage. Methods: Using a novel combination of hollow fiber membrane–based low-molecular-weight protein enrichment and LC-MS-based quantitative shotgun proteomics, we compared the plasma proteome between 24 patients with pancreatic cancer and 21 healthy controls (training cohort). An identified biomarker candidate was then subjected to a large blinded independent validation (n = 237, validation cohort) using a high-density reverse-phase protein microarray. Results: Among a total of 53,009 MS peaks, we identified a peptide derived from CXC chemokine ligand 7 (CXCL7) that was significantly reduced in pancreatic cancer patients, showing an area under curve (AUC) value of 0.84 and a P value of 0.00005 (Mann–Whitney U test). Reduction of the CXCL7 protein was consistently observed in pancreatic cancer patients including those with stage I and II disease in the validation cohort (P < 0.0001). The plasma level of CXCL7 was independent from that of CA19-9 (Pearsons r = 0.289), and combination with CXCL7 significantly improved the AUC value of CA19-9 to 0.961 (P = 0.002). Conclusions: We identified a significant decrease of the plasma CXCL7 level in patients with pancreatic cancer, and combination of CA19-9 with CXCL7 improved the discriminatory power of the former for pancreatic cancer. Impact: The present findings may provide a new diagnostic option for pancreatic cancer and facilitate early detection of the disease. Cancer Epidemiol Biomarkers Prev; 20(1); 160–71. ©2011 AACR.

Nanocapsules incorporating IgG Fc-binding domain derived from Staphylococcus aureus protein A for displaying IgGs on immunosensor chips.

To enhance the sensitivities and antigen-binding capacities of immunosensors, oriented immobilization of antibodies on the surface of the sensor chip is critical, but to date, this has not been adequately achieved. We describe a way of adsorbing immunoglobulin (Ig) proteins onto 32-nm bio-nanocapsules (BNCs) through IgG Fc-binding domains derived from Staphylococcus aureus protein A (ZZ-BNC). This arrangement permits approximately 60 molecules of mouse total IgG bind to ZZ-BNC and all the IgG Fv regions to be displayed outwardly for the effective binding of antigens. ZZ-BNCs adsorbed onto the gold surface of the sensor chip of the quartz crystal microbalance (QCM) could markedly enhance the sensitivity and antigen-binding capacity of the chip. On the sensor chip of surface plasmon resonance (SPR), antibodies on the ZZ-BNCs showed higher affinities to each antigen than those on protein A. The BNC-coated sensor chip is very stable, and should prove useful for various immunosensor applications due to oriented immobilization of antibodies.

Identification of Adipophilin as a Potential Plasma Biomarker for Colorectal Cancer Using Label-Free Quantitative Mass Spectrometry and Protein Microarray

Background: The aim of this study was to identify a new plasma biomarker for use in early detection of colorectal cancer. Methods: Using the combination of hollow fiber membrane (HFM)-based low-molecular weight protein enrichment and two-dimensional image converted analysis of liquid chromatography and mass spectrometry (2DICAL), we compared the plasma proteome of 22 colorectal cancer patients with those of 21 healthy controls. An identified biomarker candidate was then validated in two larger cohorts [validation-1 (n = 210) and validation-2 (n = 113)] using a high-density reverse-phase protein microarray. Results: From a total of 53,009 mass peaks, we identified 103 with an area under curve (AUC) value of 0.80 or higher that could distinguish cancer patients from healthy controls. A peak that increased in colorectal cancer patients, with an AUC of 0.81 and P value of 0.0004 (Mann–Whitney U test), was identified as a product of the PLIN2 gene [also known as perilipin-2, adipose differentiation-related protein (ADRP), or adipophilin]. An increase in plasma adipophilin was consistently observed in colorectal cancer patients, including those with stage I or stage II disease (P < 0.0001, Welchs t test). Immunohistochemical analysis revealed that adipophilin is expressed primarily in the basal sides of colorectal cancer cells forming polarized tubular structures, and that it is absent from adjacent normal intestinal mucosae. Conclusions: Adipophilin is a plasma biomarker potentially useful for the detection of early-stage colorectal cancer. Impact: The combination of HFM and 2DICAL enables the comprehensive analysis of plasma proteins and is ideal for use in all biomarker discovery studies. Cancer Epidemiol Biomarkers Prev; 20(10); 2195–203. ©2011 AACR.

Bionanocapsule-based enzyme–antibody conjugates for enzyme-linked immunosorbent assay

Macromolecules that can assemble a large number of enzyme and antibody molecules have been used frequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We generated bionanocapsules (BNCs) of approximately 30nm displaying immunoglobulin G (IgG) Fc-binding ZZ domains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin-biotin complex system, biotinylated ZZ-BNCs showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays.

Carbonic Anhydrase I as a New Plasma Biomarker for Prostate Cancer

Serum prostate-specific antigen (PSA) levels ranging from 4 to 10 ng/mL is considered a diagnostic gray zone for detecting prostate cancer because biopsies reveal no evidence of cancer in 75% of these subjects. Our goal was to discover a new highly specific biomarker for prostate cancer by analyzing plasma proteins using a proteomic technique. Enriched plasma proteins from 25 prostate cancer patients and 15 healthy controls were analyzed using a label-free quantitative shotgun proteomics platform called 2DICAL (2-dimensional image converted analysis of liquid chromatography and mass spectrometry) and candidate biomarkers were searched. Among the 40,678 identified mass spectrum (MS) peaks, 117 peaks significantly differed between prostate cancer patients and healthy controls. Ten peaks matched carbonic anhydrase I (CAI) by tandem MS. Independent immunological assays revealed that plasma CAI levels in 54 prostate cancer patients were significantly higher than those in 60 healthy controls (P = 0.022, Mann-Whitney U test). In the PSA gray-zone group, the discrimination rate of prostate cancer patients increased by considering plasma CAI levels. CAI can potentially serve as a valuable plasma biomarker and the combination of PSA and CAI may have great advantages for diagnosing prostate cancer in patients with gray-zone PSA level.

Plasma biomarker for detection of early stage pancreatic cancer and risk factors for pancreatic malignancy using antibodies for apolipoprotein-AII isoforms.

We recently reported that circulating apolipoprotein AII (apoAII) isoforms apoAII-ATQ/AT (C-terminal truncations of the apoAII homo-dimer) decline significantly in pancreatic cancer and thus might serve as plasma biomarkers for the early detection of this disease. We report here the development of novel enzyme-linked immunosorbent assays (ELISAs) for measurement of apoAII-ATQ/AT and their clinical applicability for early detection of pancreatic cancer. Plasma and serum concentrations of apoAII-ATQ/AT were measured in three independent cohorts, which comprised healthy control subjects and patients with pancreatic cancer and gastroenterologic diseases (n = 1156). These cohorts included 151 cases of stage I/II pancreatic cancer. ApoAII-ATQ/AT not only distinguished the early stages of pancreatic cancer from healthy controls but also identified patients at high risk for pancreatic malignancy. AUC values of apoAII-ATQ/AT to detect early stage pancreatic cancer were higher than those of CA19–9 in all independent cohorts. ApoAII-ATQ/AT is a potential biomarker for screening patients for the early stage of pancreatic cancer and identifying patients at risk for pancreatic malignancy (161 words).

A rapid enzyme-linked immunosorbent assay with two modes of detection for measuring cytokine concentration.

Interleukin (IL)‐6 and IL‐8 were measured in 101 serum samples collected from eight intensive‐care unit patients using a polystyrene‐based stick enzyme‐linked immunosorbent assay (STICKELISA) system. This system consisted of an immobilized‐antibody ELISA stick and a noncontact spectrophotometer. Cytokine concentration was detected by two ways: first, rapidly and semi‐quantitatively by naked‐eye observation of the color change and second, quantitatively using the spectrophotometer for accurate concentration determination. The spectrophotometric assay enabled the quantitation of as little as 100 pg/mL cytokine and took only 45 min to complete. There was a good agreement between the STICKELISA observations and data ob‐tained using a plate ELISA system. The agreement between STICKELISA naked‐eye observation and plate ELISA determination was 94 and 85% for IL‐6 and IL‐8, respectively. The correlation coefficients between the STICKELISA spectrophotometric determination and plate ELISA determination were 0.88 and 0.91 for IL‐6 and IL‐8, respectively, in a 0.1–5 ng/mL cytokine concentration range. These results demonstrate that the STICKELISA system is a simple, rapid, and quantitative method for bedside cytokine measurement in critical‐care settings. J. Clin. Lab. Anal. 23:40–44, 2009.

Serum proteome analysis in patients with rheumatoid arthritis receiving therapy with etanercept, a chimeric tumor necrosis factor-alpha receptor.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder of the synovium resulting in the destruction of affected joint cartilage and bone structures. Etanercept is a biological agent that blocks the tumor necrosis factor‐α (TNF‐α)‐mediated inflammatory processes in RA patients, and has a regenerative effect on cartilage. In order to identify novel disease‐related proteins and candidate biomarkers, we performed proteomic profiling of the serum in patients with RA who were treated with etanercept.

An original patient-derived xenograft of prostate cancer with cyst formation.

The high rate of failure of new agents in oncology clinical trials indicates a weak understanding of the complexity of human cancer. Recent understanding of the mechanisms underlying castration resistance in prostate cancer led to the development of new agents targeting the androgen receptor pathway; however, their effectiveness is limited. Hence, there is a need for experimental systems that are able to better reproduce the biological diversity of prostate cancer in preclinical settings. In this study, we established a unique patient‐derived xenograft (PDX) model to identify biomarkers for treatment efficacy and resistance and better understand prostate cancer biology.