Abuetabh Yasser, Persad Sujata, Nagamori Seishi, Huggins Joanne, Al-Bahrani Redha, Sergi Consolato
Department of Lab. Medicine and Pathology, University of Alberta Hospital,NW, Edmonton, Alberta T6G 2B7, Canada.
Ann Clin Lab Sci. 2011 Summer;41(3):217-23.
Cholangiocarcinoma (CC) is the most frequent malignant epithelial tumor of the biliary system. CC has received increasing interest due to its different etiologic factors, invasiveness, and the difficulty of diagnosis at an early stage. The pathogenesis of CC has not been clearly defined, but cohesiveness of tumor cells seems to be a critical factor. Calcium-dependent adherence proteins or cadherins are a family of proteins essential for connecting the plasma membrane of adjacent cells. Linkage of cadherins with the cytoskeleton occurs through another class of proteins, called catenins. E-cadherin forms a mutually exclusive complex or unit with β-catenin. Loss of E-cadherin -β-catenin adhesion represents an important step in the progression of many epithelial malignancies. Cell lines arising from CC are not often investigated and may show a differential expression of cell adhesion molecules, particularly E-cadherin - β-catenin. We hypothesized that a moderately invasive cell line of CC may co-localize both molecules in cytoplasm and cytoplasmic membrane, indicating a greater "tightness" of the tumor cells, while a metastasizing cell line may show isolated cytoplasmic membrane localization, indicating tumor cells probably more keen to reach the blood stream and give metastases. Thus, our aim was to investigate the expression and localization of E-cadherin and β-catenin in two CC cell lines, including a rapidly metastasizing cell line and a moderately invasive cell line, correlating to a different degree of invasiveness of the primary tumor.
OZ and HuCCT1 cells represent homogeneous, functional human biliary epithelial tumor cell lines that were originally isolated in Japan. Following cell line growth we extracted total proteins. Western blot analysis, immunofluorescence and confocal laser microscopy were used to identify the protein expression and their cyto-localization and co-localization.
Both CC cell lines expressed E-cadherin and β-catenin, but they showed remarkably different localization patterns. In HuCCT1, both E-cadherin and β-catenin were localized in the cytoplasm, while in OZ these proteins were localized in the cytoplasmic membrane only. This was attributed to a different degree of invasiveness of the primitive CC from which the cell lines were characterized, OZ being a metastasizing cell line, HuCCT1 being a moderately invasive cell line.
To the best of our knowledge, this is the first time that E-cadherin and β-catenin have been studied in detail in these two cell lines. These data seem to be very promising in terms of adding insight into the cell biology of CC and initiating investigations that aim to identify cytoskeletal dynamics and ultimately provide guidelines for developing new therapeutic strategies.
胆管癌（CC）是胆道系统最常见的恶性上皮性肿瘤。由于其不同的病因、侵袭性以及早期诊断的困难，胆管癌越来越受到关注。胆管癌的发病机制尚未明确，但肿瘤细胞的黏附性似乎是一个关键因素。钙依赖性黏附蛋白或钙黏蛋白是连接相邻细胞质膜所必需的一类蛋白质。钙黏蛋白与细胞骨架的连接通过另一类称为连环蛋白的蛋白质实现。E-钙黏蛋白与β-连环蛋白形成互斥复合物或单元。E-钙黏蛋白-β-连环蛋白黏附的丧失是许多上皮性恶性肿瘤进展中的重要一步。源自胆管癌的细胞系较少被研究，可能会表现出细胞黏附分子的差异表达，尤其是E-钙黏蛋白-β-连环蛋白。我们推测，一种中度侵袭性的胆管癌细胞系可能会使这两种分子在细胞质和细胞质膜中共定位，表明肿瘤细胞具有更高的“紧密性”，而转移性细胞系可能会表现出孤立的细胞质膜定位，表明肿瘤细胞可能更倾向于进入血流并发生转移。因此，我们的目的是研究E-钙黏蛋白和β-连环蛋白在两种胆管癌细胞系中的表达和定位，这两种细胞系包括一个快速转移的细胞系和一个中度侵袭性的细胞系，它们与原发性肿瘤不同程度的侵袭性相关。
OZ和HuCCT1细胞代表最初在日本分离的同质功能性人胆管上皮肿瘤细胞系。细胞系生长后，我们提取了总蛋白。采用蛋白质印迹分析、免疫荧光和共聚焦激光显微镜来鉴定蛋白质表达及其细胞定位和共定位。
两种胆管癌细胞系均表达E-钙黏蛋白和β-连环蛋白，但它们表现出明显不同的定位模式。在HuCCT1中，E-钙黏蛋白和β-连环蛋白均定位于细胞质，而在OZ中，这些蛋白质仅定位于细胞质膜。这归因于细胞系所源自的原发性胆管癌不同程度的侵袭性，OZ是转移性细胞系，HuCCT1是中度侵袭性细胞系。
据我们所知，这是首次在这两种细胞系中对E-钙黏蛋白和β-连环蛋白进行详细研究。这些数据似乎非常有前景，有助于深入了解胆管癌的细胞生物学，并启动旨在确定细胞骨架动力学并最终为制定新的治疗策略提供指导的研究。
