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BMC Cancer
Published in: BMC Cancer 1/2017

Open Access 01-12-2017 | Research article

Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer

Authors: Kyojiro Kawakami, Yasunori Fujita, Yoko Matsuda, Tomio Arai, Kengo Horie, Koji Kameyama, Taku Kato, Koichi Masunaga, Yutaka Kasuya, Masashi Tanaka, Kosuke Mizutani, Takashi Deguchi, Masafumi Ito

Published in: BMC Cancer | Issue 1/2017

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Abstract

Background

Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients.

Methods

Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4–2 and bone metastatic C4–2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody.

Results

Among proteins upregulated in C4–2 and C4–2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4–2 and C4–2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues.

Conclusions

Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.
Appendix
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Metadata
Title
Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer
Authors
Kyojiro Kawakami
Yasunori Fujita
Yoko Matsuda
Tomio Arai
Kengo Horie
Koji Kameyama
Taku Kato
Koichi Masunaga
Yutaka Kasuya
Masashi Tanaka
Kosuke Mizutani
Takashi Deguchi
Masafumi Ito
Publication date
01-12-2017
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2017
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/s12885-017-3301-x

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