Abstract
BackgroundExosomes 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.MethodsExosomes 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.ResultsAmong 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.ConclusionsOur results suggest that serum exosomal GGT activity could be a useful biomarker for PC.
Highlights
Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer
We found that serum exosomal GGT activity was significantly higher in prostate cancer (PC) patients than in benign prostatic hyperplasia (BPH) patients, which was supported by the finding that gamma-glutamyltransferase 1 (GGT1) expression was increased in PC tissues compared with BPH tissues
Identification of GGT1 as a potential exosomal marker for PC based on proteomic analysis of exosomes isolated from PC cells by differential centrifugation We analyzed androgen-dependent LNCaP cell line and its sublines of C4, C4–2 and C4–2B cell lines [17, 18]
Summary
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. Exosomes exist in the body fluids such as blood and urine and are expected to be a new marker for various diseases including cancer. Melo et al recently reported that exosomes expressing glypican-1 in blood can differentiate patients with pancreatic cancer from healthy subjects and those with benign pancreatic disease [3]. PC well responds to androgen deprivation therapy, but 10 to 20% of patients develop castration-resistant prostate cancer (CRPC) [5]. The recent introduction of cabazitaxel, enzalutamide and abiraterone has expanded treatment options for metastatic CRPC patients [8]
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