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BMC Cancer

, 17:316

Translational oncology

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.

KeywordsExosome γ-glutamyltransferase 1 γ-glutamyl transpeptidase Prostate cancer Benign prostatic hyperplasia Diagnostic marker AbbreviationsBPHBenign prostatic hyperplasia

CHCChronic hepatitis C

CRPCCastration-resistant prostate cancer

EVExtracellular vesicles

gGlu-HMRGγ-glutamyl hydroxymethyl rhodamine green

GGT1Gamma-glutamyltransferase 1

GSHGlutathione

MDR1Multi-drug resistance protein 1

NAFLDNon-alcoholic fatty liver disease

PCProstate cancer

PCA3Prostate cancer antigen 3

P-gpP-glycoprotein

PSAProstate-specific antigen

PSMAProstate-specific membrane antigen

PVDFPolyvinylidene difluoride

γGluAMCgamma-glutamyl-7-amido-4-methylcoumarin

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





Autor: Kyojiro Kawakami - Yasunori Fujita - Yoko Matsuda - Tomio Arai - Kengo Horie - Koji Kameyama - Taku Kato - Koichi Masunaga

Fuente: https://link.springer.com/



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