Kawakami Kyojiro, Fujita Yasunori, Matsuda Yoko, Arai Tomio, Horie Kengo, Kameyama Koji, Kato Taku, Masunaga Koichi, Kasuya Yutaka, Tanaka Masashi, Mizutani Kosuke, Deguchi Takashi, Ito Masafumi
Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, 173-0015, Japan.
Department of Pathology, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakae-cho, Itabashi-ku, Tokyo, 173-0015, Japan.
BMC Cancer. 2017 May 5;17(1):316. doi: 10.1186/s12885-017-3301-x.
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.
外泌体或细胞外囊泡有潜力作为包括癌症在内的多种疾病的诊断标志物。为了鉴定前列腺癌（PC）的新型外泌体标志物，我们对从PC细胞系中分离出的外泌体进行了蛋白质组学分析，并检测了该标志物在患者中的实用性。
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亚系的培养基中分离外泌体，进行基于iTRAQ的蛋白质组学分析。外泌体也通过免疫捕获分离，并用尺寸排阻色谱法和密度梯度离心法分离。通过蛋白质印迹分析确定蛋白质表达。使用荧光探针γ-谷氨酰羟甲基罗丹明绿（gGlu-HMRG）测量γ-谷氨酰转移酶（GGT）活性。使用抗GGT1抗体对组织进行免疫组织化学分析。
在C4-2和C4-2B细胞中上调而在LNCaP细胞中未上调的蛋白质中，我们重点关注γ-谷氨酰转移酶1（GGT1），一种调节细胞外谷胱甘肽分解代谢的细胞表面酶。通过差速离心以及用抗CD9或前列腺特异性膜抗原（PSMA）抗体进行免疫捕获，从C4-2和C4-2B细胞中分离出的外泌体中GGT1大亚基和小亚基的水平均升高。在细胞裂解物和外泌体中，GGT1表达与GGT活性相关。人血清的尺寸排阻色谱显示在CD9阳性组分中存在GGT活性和GGT1亚基。密度梯度离心显示在通过差速离心从人血清中分离出的外泌体中GGT1亚基与CD9共存。由于通过差速离心分离的血清外泌体中GGT活性与GGT1表达相关，我们测量了患者血清外泌体GGT活性。出乎意料的是，我们发现PC患者血清外泌体GGT活性显著高于良性前列腺增生（BPH）患者。支持这一发现的是，免疫组织化学分析显示与BPH组织相比，PC组织中GGT1表达增加。
我们的结果表明血清外泌体GGT活性可能是PC的一种有用生物标志物。
