Institute of Bioinformatics, International Technology Park, Bangalore, India.
Cancer Biol Ther. 2010 Oct 15;10(8):796-810. doi: 10.4161/cbt.10.8.12914.
The identification of secreted proteins that are differentially expressed between non-neoplastic and esophageal squamous cell carcinoma (ESCC) cells can provide potential biomarkers of ESCC. We used a SILAC-based quantitative proteomic approach to compare the secretome of ESCC cells with that of non-neoplastic esophageal squamous epithelial cells. Proteins were resolved by SDS-PAGE, and tandem mass spectrometry analysis (LC-MS/MS) of in-gel trypsin-digested peptides was carried out on a high-accuracy qTOF mass spectrometer. In total, we identified 441 proteins in the combined secretomes, including 120 proteins with > 2-fold upregulation in the ESCC secretome vs. that of non-neoplastic esophageal squamous epithelial cells. In this study, several potential protein biomarkers previously known to be increased in ESCC including matrix metalloproteinase 1, transferrin receptor, and transforming growth factor beta-induced 68 kDa were identified as overexpressed in the ESCC-derived secretome. In addition, we identified several novel proteins that have not been previously reported to be associated with ESCC. Among the novel candidate proteins identified, protein disulfide isomerase family a member 3 (PDIA3), GDP dissociation inhibitor 2 (GDI2), and lectin galactoside binding soluble 3 binding protein (LGALS3BP) were further validated by immunoblot analysis and immunohistochemical labeling using tissue microarrays. This tissue microarray analysis showed overexpression of protein disulfide isomerase family a member 3, GDP dissociation inhibitor 2, and lectin galactoside binding soluble 3 binding protein in 93%, 93% and 87% of 137 ESCC cases, respectively. Hence, we conclude that these potential biomarkers are excellent candidates for further evaluation to test their role and efficacy in the early detection of ESCC.
我们采用基于 SILAC 的定量蛋白质组学方法比较了食管鳞癌细胞与非肿瘤性食管鳞状上皮细胞的分泌组。蛋白质经 SDS-PAGE 分离，然后在高准确度 qTOF 质谱仪上对胶内胰蛋白酶消化肽进行串联质谱分析（LC-MS/MS）。总共在合并的分泌组中鉴定到 441 种蛋白质，其中 120 种蛋白质在 ESCC 分泌组中上调超过 2 倍，而非肿瘤性食管鳞状上皮细胞中则下调。在这项研究中，一些先前已知在 ESCC 中上调的潜在蛋白质生物标志物，包括基质金属蛋白酶 1、转铁蛋白受体和转化生长因子β诱导的 68 kDa 蛋白，被鉴定为 ESCC 来源的分泌组中过表达。此外，我们还鉴定到了一些以前没有报道与 ESCC 相关的新蛋白质。在鉴定到的新候选蛋白质中，蛋白质二硫键异构酶家族 A 成员 3（PDIA3）、GDP 解离抑制剂 2（GDI2）和凝集素半乳糖结合可溶性 3 结合蛋白（LGALS3BP）通过免疫印迹分析和组织微阵列的免疫组织化学标记进一步验证。这项组织微阵列分析显示，蛋白质二硫键异构酶家族 A 成员 3、GDP 解离抑制剂 2 和凝集素半乳糖结合可溶性 3 结合蛋白在 137 例 ESCC 病例中的表达分别上调了 93%、93%和 87%。因此，我们得出结论，这些潜在的生物标志物是进一步评估的优秀候选物，以测试它们在 ESCC 早期检测中的作用和功效。
