Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597.
J Proteome Res. 2013 Feb 1;12(2):980-90. doi: 10.1021/pr3010259. Epub 2013 Jan 9.
In this study, we aim to identify biomarkers for gastric cancer metastasis using a quantitative proteomics approach. The proteins extracted from a panel of 4 gastric cancer cell lines, two derived from primary cancer (AGS, FU97) and two from lymph node metastasis (AZ521, MKN7), were labeled with iTRAQ (8-plex) reagents and analyzed by 2D-LC-MALDI-TOF/TOF MS. In total, 641 proteins were identified with at least a 95% confidence. Using cutoff values of >1.5 and <0.67, 19 proteins were found to be up-regulated and 34 were down-regulated in the metastatic versus primary gastric cancer cell lines respectively. Several of these dysregulated proteins, including caldesmon, were verified using Western blotting. It was found that caldesmon expression was decreased in the two metastasis-derived cell lines, and this was confirmed by further analysis of 7 gastric cancer cell lines. Furthermore, immunohistochemical staining of 9 pairs of primary gastric cancer and the matched lymph node metastasis tissue also corroborated this observation. Finally, knockdown of caldesmon using siRNA in AGS and FU97 gastric cancer cells resulted in an increase in cell migration and invasion, while the overexpression of caldesmon in AZ521 cells led to a decrease in cell migration and invasion. This study has thus established the potential role of caldesmon in gastric cancer metastasis, and further functional studies are underway to delineate the underlying mechanism of action of this protein.
在这项研究中，我们旨在使用定量蛋白质组学方法鉴定胃癌转移的生物标志物。从四株胃癌细胞系（两株源自原发性肿瘤[AGS、FU97]，两株源自淋巴结转移[AZ521、MKN7]）中提取的蛋白质用 iTRAQ（8 重）试剂标记，并通过 2D-LC-MALDI-TOF/TOF MS 进行分析。总共鉴定到了 641 种具有至少 95%置信度的蛋白质。使用 >1.5 和 <0.67 的截止值，发现 19 种蛋白质在转移性与原发性胃癌细胞系中上调，34 种蛋白质下调。这些失调蛋白中的几种，包括钙调蛋白，使用 Western blot 进行了验证。发现钙调蛋白在两个转移衍生细胞系中的表达降低，这在对 7 株胃癌细胞系的进一步分析中得到了证实。此外，对 9 对原发性胃癌和相应淋巴结转移组织的免疫组织化学染色也证实了这一观察结果。最后，使用 siRNA 在 AGS 和 FU97 胃癌细胞中敲低钙调蛋白导致细胞迁移和侵袭增加，而在 AZ521 细胞中过表达钙调蛋白导致细胞迁移和侵袭减少。因此，这项研究确立了钙调蛋白在胃癌转移中的潜在作用，并且正在进行进一步的功能研究以阐明该蛋白的作用机制。
