Protein pattern difference in the colon cancer cell lines examined by two-dimensional differential in-gel electrophoresis and mass spectrometry
Protein pattern difference in the colon cancer cell lines examined by two-dimensional differential in-gel electrophoresis and mass spectrometry
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DOI：
10.1007/s00595-006-3301-y
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发表时间：
2006-12-01
期刊：
影响因子：
2.5
通讯作者：
Otsubo, Takehito
中科院分区：
文献类型：
--
作者：
Katayama, Masafumi;Nakano, Hiroshi;Otsubo, Takehito
目的.结直肠癌（CRC）的关键转移过程尚未通过全面的蛋白质分析进行充分研究。我们采用双向凝胶电泳（2D-DIGE）和液相色谱-串联质谱（LC/MS/MS）技术研究结直肠癌转移过程中蛋白质谱的变化。研究两种结直肠癌细胞系：来源于原发灶的SW 480和来源于同一患者淋巴结转移灶的SW 620。使用2D-DIGE与马来酰亚胺CyDye荧光蛋白标记技术比较两种细胞系，该技术在低浓度下对许多蛋白质具有增强的灵敏度。采用Cy 3和Cy 5双标记法和LC/MS/MS技术对细胞进行蛋白质组学分析，并将两种细胞株分别注入裸鼠脾脏进行体内转移实验。在大约1500个蛋白质中，我们检测到9个蛋白质点在两个细胞系之间具有明确的显著变化。通过Western印迹分析验证了9种蛋白质中的3种。与SW 480相比，SW 620中α-烯醇化酶和磷酸丙糖异构酶显著上调。与SW 480相比，SW 620中膜联蛋白A2（膜联蛋白II）显著下调。在使用SW 480的转移性实验中既没有建立肝转移也没有建立腹膜播散，但是在使用SW 620的实验中发生了一些肝和腹膜转移。使用SW 620的体内转移实验显示，与脾植入病变中的α-烯醇化酶和磷酸丙糖异构酶的表达相比，肝转移中的α-烯醇化酶和磷酸丙糖异构酶的表达增加。与脾脏移植性病变相比，腹膜移植性病变中磷酸丙糖异构酶的表达增加。2D-DIGE和LC/MS/MS技术鉴定了9种蛋白质，其在SW 620中的增加显著高于在SW 480中的增加。我们的体内转移实验的发现表明，α-烯醇化酶和磷酸丙糖异构酶，至少部分，可能与这两个细胞系的转移过程。
Purpose. The pivotal metastatic processes of colorectal cancer (CRC) have yet to be fully investigated by a comprehensive all-inclusive protein analysis. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) to investigate the protein pattern changes during the metastasis of CRC. Two CRC cell lines were investigated: SW480 derived from the primary lesion and SW620 derived from lymph node metastasis in the same patient.Methods. The two cell lines were compared using 2D-DIGE with a maleimide CyDye fluorescent protein labeling technique, which has an enhanced sensitivity for many proteins at a low concentration. A comprehensive proteomics analysis was performed by the dual-labeling method using Cy3 and Cy5 and by LC/MS/MS. In addition, an in vivo experiment of metastasis using nude mice was performed by the injection of the two cell lines into the spleen.Results. Among approximately 1500 proteins, we detected 9 protein spots with definitively significant changes between the two cell lines. Three out of the nine proteins were validated by a Western blot analysis. Alpha-enolase and triosephosphate isomerase were significantly upregulated in SW620 in comparison to SW480. Annexin A2 (annexin II) was significantly downregulated in SW620 compared to SW480. Neither liver metastasis nor peritoneal dissemination was established in the metastatic experiment using SW480 but some liver and peritoneal metastases occurred in the experiment using SW620. An in vivo metastatic experiment using SW620 showed the expressions of alpha-enolase and triosephosphate isomerase to increase in the liver metastases in comparison to those in the splenic implanted lesion. The expressions of triosephosphate isomerase increased in the peritoneal lesions in comparison to those in the splenic implanted lesion.Conclusions. 2D-DIGE and LC/MS/MS techniques identified nine proteins that increased significantly more in SW620 than in SW480. The finding of our in vivo metastatic experiment suggests that alpha-enolase and triosephosphate isomerase, at least in part, may be associated with the metastatic process of these two cell lines.