Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer.
Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer.
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DOI：
10.1186/s12885-017-3301-x
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发表时间：
2017-05-05
期刊：
影响因子：
3.8
通讯作者：
Ito M
中科院分区：
文献类型：
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作者：
Kawakami K;Fujita Y;Matsuda Y;Arai T;Horie K;Kameyama K;Kato T;Masunaga K;Kasuya Y;Tanaka M;Mizutani K;Deguchi T;Ito M
外泌体或细胞外囊泡具有作为包括癌症在内的各种疾病的诊断标志物的潜力。为了鉴定前列腺癌（PC）的新型外泌体标记物，我们对从PC细胞系分离的外泌体进行蛋白质组学分析，并检查标记物在患者中的有用性。对通过差速离心从雄激素依赖性LNCaP前列腺癌细胞系及其部分雄激素非依赖性C4、雄激素非依赖性C4-2和骨转移性C4-2B的亚系的培养基分离的外泌体进行基于iTRAQ的蛋白质组学分析。还通过免疫捕获分离外泌体，并通过尺寸排阻色谱和密度梯度离心分离。通过Western印迹分析测定蛋白质表达。使用荧光探针γ-谷氨酰羟甲基罗丹明绿色（gGlu-HMRG）测量GGT活性。使用抗GGT 1抗体进行组织的免疫组织化学分析。在C4-2和C4-2B细胞中比LNCaP细胞中上调的蛋白质中，我们专注于γ-谷氨酰转移酶1（GGT 1），这是一种调节细胞外谷胱甘肽催化的细胞表面酶。通过差速离心和用抗CD 9或前列腺特异性膜抗原（PSMA）抗体免疫捕获从C4-2和C4-2B细胞分离的外泌体中，GGT 1大小亚基的水平升高。在细胞裂解物和外来体中，GGT 1表达与GGT活性相关。人血清的尺寸排阻色谱证明了CD 9阳性组分中存在GGT活性和GGT 1亚基。密度梯度离心揭示了GGT 1亚基与CD 9在通过差速离心从人血清分离的外来体中的共存。由于GGT活性与通过差速离心分离的血清外泌体中的GGT 1表达相关，我们测量了患者的血清外泌体GGT活性。出乎意料的是，我们发现PC患者的血清外泌体GGT活性显著高于良性前列腺增生（BPH）患者。为了支持这一发现，免疫组化分析显示，与BPH组织相比，PC组织中GGT 1表达增加。我们的研究结果表明，血清外泌体GGT活性可能是一个有用的生物标志物PC。本文的在线版本（doi：10.1186/s12885-017-3301-x）包含补充材料，可供授权用户使用。
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 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. 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. Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC. The online version of this article (doi:10.1186/s12885-017-3301-x) contains supplementary material, which is available to authorized users.