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

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3301-x) contains supplementary material, which is available to authorized users.

Promise and Implementation of Proteomic Prostate Cancer Biomarkers

“…Significantly higher activity was detected in PC vs BPH. This finding was further supported through the analysis of tissue samples (n = 50 PC, n = 50 BPH) [ 77 ]. Based on the measurement of GGT1 activity, ROC analysis was performed, resulting in an AUC of 0.71 [ 77 ].…”

“…Exosomes were isolated from the conditioned media of androgen-dependent (LNCaP) and androgen-independent (C4, C4–2, and C4–2B; sublines of LNCaP) cell lines [ 77 ]. The exosomal proteome was analyzed using iTRAQ and MALDI-TOF/TOF, resulting in identification of a total of 153 proteins.…”

Serum Exosomal Gamma-Glutamyltransferase Activity Increased in Patients with Renal Cell Carcinoma with Advanced Clinicopathological Features

“…Cells were seeded on 150-mm dishes and grown for 72 h in serum-free advanced DMEM or advanced RPMI medium (Thermo Fisher Scientific, Waltham, MA, USA). Exosomes were harvested from culture medium by differential centrifugation according to the method we described previously [7]. In brief, culture medium was sequentially centrifuged at 2,000 and 12,000 g. After filtration of the supernatant through a 0.22-μm PVDF (polyvinylidene difluoride) membrane, the filtrate was centrifuged at 110,000 g. Western Blot Analysis Western blot analyses of the cell lysates and exosomes were performed according to the method we described previously [7].…”

“…Exosomes were harvested from culture medium by differential centrifugation according to the method we described previously [7]. In brief, culture medium was sequentially centrifuged at 2,000 and 12,000 g. After filtration of the supernatant through a 0.22-μm PVDF (polyvinylidene difluoride) membrane, the filtrate was centrifuged at 110,000 g. Western Blot Analysis Western blot analyses of the cell lysates and exosomes were performed according to the method we described previously [7]. The antibodies used were as follows: anti-CD9 antibody and antiβ-actin antibody (Cell Signaling Technology, Danvers, MA, USA); and anti-GGT1 antibody and anti-CD63 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).…”

Male Accessory Glands and Sperm Function

“…The prostate produces a variety of substances such as zinc (Zn), citric acid (citrate), acid phosphatase and gamma-glutamyltransferase into others. These four have been considered reliable markers of the prostate gland [15,16]. Zinc has a tendency to bind with other elements of semen; it can sometimes be bound to the surface of the sperm cells [17].…”