Identification of differentially expressed proteins during human urinary bladder cancer progression

https://doi.org/10.1016/j.cdp.2005.01.002Get rights and content

Abstract

Comparative proteome analysis was performed between RT4 (grade-1) and T24 (grade-3) bladder cancer cell lines, in an attempt to identify differentially expressed proteins during bladder cancer progression. Among those relatively abundant proteins, seven spots changed more than two-fold reproducibly and identified by peptide mass fingerprinting using mass spectrometry and database search. We found most extensive and reproducible down-regulation of NADP dependent isocitrate dehydrogenase cytoplasmic (IDPc) and peroxiredoxin-II (Prx-II), in poorly differentiated T24 compared to well-differentiated RT4 bladder cancer cell line. Subsequent Western blotting analysis of human biopsy samples from bladder cancer patient revealed significant loss of IDPc and Prx-II in more advance tumor samples, in agreement with data on cell lines. These results suggest that loss of IDPc and Prx-II during tumor development may involve in tumor progression and metastasis. However, additional investigations are needed on large number of human samples to further verify these findings.

Introduction

Proteomics studies have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression [1], [2], [3], [4]. Such studies could lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling and developmental stages [5], [6], [7].

Recent advances in expression profiling of cancer cells by proteomic technologies, high-resolution two-dimensional electrophoresis (2DE) and mass spectrometry (MS) have made it possible to identify candidate proteins as tumor markers in various cancers. These efforts typically strive to provide a global analysis of either protein expression or protein function. More and more medical and clinical applications of tumor proteomics promised to identify different type of cancers, such as leukemia, lung cancer, liver cancer, bladder cancer and colon cancer, etc. [8]. However, only a few studies have been involved in proteomic analysis of tumor progression and metastasis in bladder cancer.

Bladder cancer is the fifth most common cancer in men and ninth in women [9]. At presentation, approximately 25–30% of bladder tumors are classified as muscle-invasive tumors, which, by definition, have already demonstrated the ability to invade and are associated with significant risk of subsequent metastasis (30–60%) [10]. About 70% of the urinary bladder TCCs are diagnosed at presentation as well-differentiated superficial lesions that are confined either to mucosa or to underlying connective tissue [11]. The rest corresponds to highly invasive stages (T2–T4) and poorly differentiated tumors [12]. Thus, a major challenge is to identify the biological properties that drive tumor behavior and progression in bladder cancer, and to identify biomarkers that can be used to inform therapeutic choice for the individual patient.

In the present study, we addressed the question whether there were protein clusters associated with progression and metastasis of bladder cancer. On the basis of these considerations, a comparative proteomic strategy, combined with two-dimensional electrophoresis separation and mass spectrometry identification, was used to search proteome alterations between early and late stages of bladder cancer. Comparative proteomics analysis was performed between RT4 and T24 bladder cancer cell lines representing well to poorly differentiated phenotypes, respectively [13], [14], [15], [16].

Section snippets

Reagents

Anti-peroxiredoxin-II (Prx-II) antibody [17] was kindly provided by Prof. H.Z. Chae, at Chunnam National University, Kwangju, Korea. Anti-cytosolic NADP isocitrate dehydrogenase antibody [18], [19], [20] was a gift from Prof. T.L. Huh at Kyungpook National University Hospital. Dithiothreitol (DTT), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), urea, iodoacetamide (IAA), silver nitrate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acrylamide is from Amresco

Differentially expressed proteins in bladder cancer cell lines

In order to identify differentially expressed proteins during bladder cancer progression, comparative proteome analysis between two-bladder cancer cell lines, RT4 (derived from superficial non-invasive) and T24 (derived from invasive cancer) were performed, using two-dimensional polyacrylamide gel electrophoresis. Seven spots were changed reproducibly more than two-folds in five independent experiments. Among them five were down-regulated and two were up-regulated in T24 compared to RT4 (Fig. 1

Discussion

In this study, we used the 2D-PAGE technique to investigate the differentially expressed proteins in bladder cancer. Because of the heterogeneous nature of human biopsy samples, our choice was to compare two bladder cancer cell lines RT4 and T24. These cell lines have been used as models of non-invasive and invasive bladder cancer, respectively [13], [14], [15], [16]. T24 cells were derived from an invasive bladder tumor of grade 3 [27] and RT4 cells were established from a well-differentiated

Acknowledgments

We thank Prof. H.Z. Chae, at Chunnam National University (Gwangju, Korea) for providing anti-Prx-II and Prof. T.L. Huh at Kyungpook National University Hospital for providing anti-IDPc antibody. We also thank Jong Shin Yoo at Korea Basic Science Institute, Daejun, South Korea for performing mass spectrometry.

This work was supported by a grant (M1-0309-11-004) from the Systems Biology Program of the Korea Ministry of Science and Technology, by a GIST internal grant, and by the Brain Korea 21

References (38)

  • L. Liotta et al.

    Molecular profiling of human cancer

    Nat Rev Genet

    (2000)
  • L. Prasannan et al.

    Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma

    Clin Cancer Res

    (2000)
  • V.E. Bichsel et al.

    Cancer proteomics: from biomarker discovery to signal pathway profiling

    Cancer J

    (2001)
  • M.A. Reymond et al.

    Standardized characterization of gene expression in human colorectal epithelium by two-dimensional electrophoresis

    Electrophoresis

    (1997)
  • P.C. Herrmann et al.

    Cancer proteomics: the state of the art

    Dis Markers

    (2001)
  • M.J. Droller

    Bladder cancer: state-of-the-art care

    Ca-A Cancer J Clin

    (1998)
  • The challange we face: the latest cancer statistics

  • S. Holmang et al.

    The relationship among multiple recurrences, progression and prognosis of patients with stages Ta and T1 transitional cell cancer of the bladder followed for at least 20 years

    J Urol

    (1995)
  • C. Booth et al.

    Stromal and vascular invasion in an human in vitro bladder cancer model

    Lab Invest

    (1997)
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