Kawase Hiroshi, Fujii Kiyonaga, Miyamoto Masaki, Kubota Kanako C, Hirano Satoshi, Kondo Satoshi, Inagaki Fuyuhiko
Department of Surgical Oncology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, Japan.
J Proteome Res. 2009 Aug;8(8):4092-103. doi: 10.1021/pr900468k.
Cholangiocarcinoma is an intractable cancer for which there is no effective therapy other than surgical resection, and many patients are not candidates for this treatment. Even for patients who undergo surgical resection, the 5-year survival rate is low. One reason for this is that the disease is often detected in late stages. Thus, there is a clear need for better biomarkers to facilitate early diagnosis and prognostication. During the biomarker discovery phase of our study, we used LC-MS-based proteomics with spectral counting, a semiquantitative approach to differential expression profiling, in paired cancerous and normal bile duct tissue samples from two cases. In total, 38 proteins up-regulated in the cancer samples were identified. These were verified using a SILAC method for MS-based validation. The results led to the identification of well-characterized proteins and proteins of unknown function that are up-regulated in cholangiocarcinoma. We used immunoblot analysis to validate four candidate biomarkers, actinin-1, actinin-4, protein DJ-1 and cathepsin B, with the test case samples and four additional cholangiocarcinoma case samples. Each of the four candidate proteins was overexpressed in a subset of five of the six cases tested. By immunohistochemistry, we further confirmed that expression of these proteins was elevated in cancer cells as compared with normal bile duct cells. Thus, we successfully identified several proteins up-regulated in cholangiocarcinoma. These proteins are candidate biomarkers and may also help to provide new insights into our understanding of the disease.
胆管癌是一种难治性癌症，除手术切除外没有有效的治疗方法，而且许多患者不适合这种治疗。即使是接受手术切除的患者，5年生存率也很低。原因之一是该疾病通常在晚期才被发现。因此，迫切需要更好的生物标志物来促进早期诊断和预后评估。在我们研究的生物标志物发现阶段，我们使用基于液相色谱-质谱联用（LC-MS）的蛋白质组学和光谱计数法（一种用于差异表达谱分析的半定量方法），对来自两例患者的配对癌组织和正常胆管组织样本进行分析。总共鉴定出38种在癌组织样本中上调的蛋白质。使用基于质谱的稳定同位素标记氨基酸细胞培养法（SILAC）对这些结果进行验证。结果鉴定出了在胆管癌中上调的特征明确的蛋白质和功能未知的蛋白质。我们使用免疫印迹分析，用测试病例样本和另外四个胆管癌病例样本验证了四种候选生物标志物，即辅肌动蛋白-1、辅肌动蛋白-4、DJ-1蛋白和组织蛋白酶B。在测试的六个病例中的五个病例的亚组中，这四种候选蛋白均有过表达。通过免疫组织化学，我们进一步证实，与正常胆管细胞相比，这些蛋白在癌细胞中的表达升高。因此，我们成功鉴定出了几种在胆管癌中上调的蛋白质。这些蛋白质是候选生物标志物，也可能有助于为我们对该疾病的理解提供新的见解。
