Trefoil factors:Tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma

AIM:To investigate trefoil factor(TFF) gene copy number,mRNA and protein expression as potential biomarkers in cholangiocarcinoma(CCA).METHODS:TFF mRNA levels,gene copy number and protein expression were determined respectively by quantitative reverse transcription polymerase chain reaction(PCR),quantitative PCR and immunohistochemistry in bile duct epithelium biopsies collected from individuals with CCA,precancerous bile duct dysplasia and from disease-free controls.The functional impact of recombinant human(rh) TFF2 peptide treatment on proliferation and epidermal growth factor receptor(EGFR) /mitogenactivated protein kinase(MAPK) signaling was assessed in the CCA cell line,KMBC,by viable cell counting and immunoblotting,respectively.RESULTS:TFF1,TFF2 and TFF3 mRNA expression was significantly increased in CCA tissue compared to disease-free controls,and was unrelated to gene copy number.TFF1 immunoreactivity was strongly increased in both dysplasia and CCA,whereas TFF2 immunoreactivity was increased only in CCA compared to diseasefree controls.By contrast,TFF3 immunoreactivity was moderately decreased in dysplasia and further decreased in CCA.Kaplan-Meier analysis found no association of TFF mRNA,protein and copy number with age,gender,histological subtype,and patient survival time.Treatment of KMBC cells with rhTFF2 stimulated proliferation,triggered phosphorylation of EGFR and downstream extracellular signal related kinase(ERK),whereas co-incubation with the EGFR tyrosine kinase inhibitor,PD153035,blocked rhTFF2-dependent proliferation and EGFR/ERK responses.CONCLUSION:TFF mRNA/protein expression is indicative of CCA tumor progression,but not predictive for histological sub-type or survival time.TFF2 is mitogenic in CCA via EGFR/MAPK activation.

Prognostic significance of microsatellite alterations at 1p36 in cholangiocarcinoma

瞄准：在 cholangiocarcinoma (CCA ) 在染色体区域 1p36 磅上调查杂合现象(LOH ) 和微卫星不稳定性(MSI ) 的损失病人并且决定在微卫星改变和 clinicopathological 参数之间的协会。方法：十个多态的微卫星标记用 GS-3000 胶化扫描碎片自动分析器为 LOH 和 MSI 被决定。结果：68 在至少一部位从 90 个案例(75.6%) 显示出 LOH。LOH 最经常被发现在 D1S199 (40.0%) ， D1S507 (34.6%) ， D1S2845 (30.5%) ，和 D1S2734 (30.1%) 。MSI 在至少一部位在 90 个案例(37.8%) 中的 34 个中被发现。在 1p36 的好印射显示出普通损失的二个特殊区域，它是在 D1S507 和 D1S2734 之间的 D1S2845 和 25.5 厘米的区域，显示通常认为的肿瘤的存在压制或对可能的基因在 CCA 的发展起重要作用。有在 D1S234 的 LOH 的病人显示出不太淋巴的侵略(P = 0.017 ) ，而没有，有在 D1S2676 的 LOH 的病人比那些展出了更淋巴的侵略(P = 0.031 ) 。在 D1S2845 的 LOH 与神经侵略显示出重要关联(P = 0.029 ) 。而且，在 D1S228 表明了 MSI 的病人显示出差的预后(P = 0.0026 ) 。结论：突变而产生之遗传的损失在染色体 1p36 在微卫星改变起一个主要作用，它可以贡献肝吸虫的致癌作用和致病相关 CCA 和这些改变能为 CCA 病人被用作分子的预示的指示物。

Amplification of chromosome 21q22.3 harboring trefoil factor family genes in liver fluke related cholangiocarcinoma is associated with poor prognosis

瞄准：在 cholangiocarcinoma (CCA ) 包括翘摇因素家庭基因(TFF ) 在染色体区域 21q22-qter 上决定突变而产生之遗传的不平衡病人和分析在突变而产生之遗传的不平衡和 clinicopathological 参数之间的关联。方法：量的 PCR 扩大用一条标准曲线和格林·西布尔在四个微卫星标记和翘摇因素家庭基因(TFF1， TFF2，和 TFF3 ) 上被执行我染料方法。相对拷贝数字被测试地点的 DNA 拷贝数字决定引用地点。当由有正常参考的比较的删除或扩大变化，相对拷贝数字被解释。在突变而产生之遗传的不平衡和 CCA 病人的 clinicopathological 参数之间的协会被 chi (2 ) 评估测试。Kaplan-Meier 方法被用来分析幸存。结果：在 D21S1890， D21S1893，和 TFF3 的扩大的频率分别地是 32.5% ， 30.0% ，和 28.7% 。在盖住 D21S1893， D21S1890，和 TFF 的区域有扩大的病人显示出差的预后，而有删除的病人显示出有利预后(平均数：51.7 wk 对 124.82 wk， P = 0.012 ) 。穆尔蒂瓦里伊特·考克斯回归分析表明 D21S1893， D21S1890 和 TFF，血管侵略，和阶段的那扩大与差的预后被联系。结论：D21S1893-D21S1890 区域可以特别怀有候选人基因 TFF 和丝氨酸朊酶家庭，它可能涉及肿瘤侵略和转移贡献穷人幸存。在这个区域的扩大可以在 CCA 病人的治疗被用作一个预示的标记。

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.

Amplification of D22S283 as a favorable prognostic indicator in liver fluke related cholangiocarcinoma

瞄准：为了分析目标基因 NF2， TIMP3， ST13， TOB2， BIK，和 TP 和参考的 DNA 拷贝数字，在肝吸虫在 22q12-qter 上印射的微卫星标记 D22S283， D22S423，和 D22S274 联系了 cholangiocarcinoma (CCA ) 并且与临床的参数定义它的关联。方法：量的实时 PCR (qPCR ) 被用于决定在 65 肝吸虫的突变而产生之遗传的不平衡联系了 CCA 纸巾。在突变而产生之遗传的不平衡和 clinicopathological 参数之间的统计关联，即年龄，性别，肿瘤阶段，组织学的类型，血管侵略，神经侵略和淋巴的侵略借助于 chi2 测试被评估。考克斯回归分析被用于决定病人的幸存。结果： TP ( 22q13.33 )的扩大， TOB2 ( 22q13.2-13.31 )， D22S283 ( 22q12.3 )， TIMP3 ( 22q12.3 )和 NF2 ( 22q12.2 )在 35 被发现(53.8%)， 28 (43.1%)， 27 (41.5%)， 24 (36.9%)，并且 24 (36.9%)，分别地。在 D22S423 (22q13.1-13.2 ) 和 BIK (22q13.31 ) 的损失在 26 被检测(40%) 并且 23 (35.4%) 分别地。重要关联在淋巴的侵略和 BIK 的突变而产生之遗传的损失之间被观察(P = 0.025 ) 并且 D22S283 (P = 0.041 ) 。 Univariate 并且多，变量考克斯回归分析作为好预后的一个独立预言者揭示了 D22S283 扩大( P = 0.006 ，死亡危险比率= 0.411 ，95% CI = 0.217-0.779 )并且是的血管侵略一个独立人士差的预示的因素( P = 0.042 ，死亡危险比率= 1.911 ，95% CI = 1.022-3.571 )在 CCA 病人。结论：这研究提供证据因为在肝吸虫的染色体 22q 上的基因扩大和删除的参与联系了 CCA。当在肝吸虫的有利预后的独立指示物联系了 CCA，这是 D22S283 扩大的第一份报告。

Genetic and environmental determinants of risk for cholangiocarcinoma in Thailand

Cholangiocarcinoma(CCA) is a difficult cancer to diagnose in the early stage and to treat by curative resec-tion. The incidence of CCA in the northeast of Thailand is the highest in the world. To make progress in detecting a high risk group and in the prevention and detection of CCA, we have been analyzing the risk factors for CCA. Although liver fluke infection is known to be a risk factor, there are patients who are not infected with the liver fluke and not all people infected with the liver fluke will suffer from the disease. Therefore, it is of the utmost importance to analyze the risk factors and the mechanism to prevent the disease and also to detect the disease in its early stage to save patients' lives. Through collaboration among Thai and Japanese researchers, we analyzed the genetic and environmental determinants of risks for CCA. Also, we have been trying to develop methods to detect the disease in a non-invasive way. Without repeating findings reported in various reviews on CCA, we will first discuss the environmental and genetic determinants of the risks for CCA. Second, we will discuss the properties of CCA, including the etiological agents and the mechanism of cholangiocarcinogenesis, and finally, we will discuss future approaches to prevent and cure CCA from the standpoint of evidence-based medicine. We will discuss these points by including the data from our laboratories. We would like to emphasize the importance of the genetic data, especially whole genome approaches, to understand the properties of CCA, to find a high risk population for CCA and to develop effective preventative methods to stop the carcinogenic steps toward CCA in the near future. In addition, it is of the upmost importance to develop a non-invasive, specific and sensitive method to detect CCA in its early stage for the application of modern medical approaches to help patients with CCA.