Polymorphism of genes encoding drug-metabolizing and inflammation-related enzymes for susceptibility to cholangiocarcinoma in Thailand

BACKGROUND Cholangiocarcinoma(CCA)is an intractable cancer,and its incidence in north eastern Thailand is the highest worldwide.Infection with the liver fluke Opisthorchis viverrini(OV)has been associated with CCA risk.However,animal experiments have suggested that OV alone does not induce CCA,but its combination with a chemical carcinogen like nitrosamine can cause experimentally induced CCA in hamsters.Therefore,in humans,other environmental and genetic factors may also be involved.AIM To examine relations between risk for CCA and genetic polymorphisms in carcinogenmetabolizing and inflammation-related genes.METHODS This hospital-based case-control study enrolled 95 case-control pairs matched by age(±5 years)and sex.We examined relations between risk for CCA and genetic polymorphisms in carcinogenmetabolizing and inflammation-related genes,serum anti-OV,alcohol consumption,and smoking.Polymorphisms of CYP2E1,IL-6(-174 and-634),IL-10(-819),and NF-κB(-94)and their cooccurrence with polymorphisms in the drug-metabolizing enzyme gene GSTT1 or GSTM1 were also analyzed.RESULTS Although CCA risk was not significantly associated with any single polymorphism,persons with the GSTT1 wild-type and CYP2E1 c1/c2+c2/c2 genotype had an increased risk(OR=3.33,95%CI:1.23-9.00)as compared with persons having the GSTT1 wild-type and CYP2E1 c1/c1 wild genotype.The presence of anti-OV in serum was associated with a 7-to 11-fold increased risk,and smoking level was related to an OR of 1.5-1.8 in multivariable analyses adjusted for each of the seven genetic polymorphisms.CONCLUSION In addition to infection with OV,gene-gene interactions may be considered as one of the risk factors for CCA development.

MUC1 and MUC5AC mucin expression in liver fluke-associated intrahepatic cholangiocarcinoma

AIM: To investigate the expressions of MUC1 and MUC5AC in intrahepatic cholangiocarcinoma (ICC). Association of expressions of mucins MUC1 and MUC5AC with clinical findings, metastasis, and survival of the liver fluke-associated ICC patients was determined.METHODS: The expressions of MUC1 and MUC5AC mucins were examined by immunohistochemical staining in 87cases of histologically-proven ICC. The expressions of mucins in relationship between clinicopathological significance and prognosis of the patients were evaluated.RESULTS: Fifty-two patients (60%) exhibited both MUC1 and MUC5AC expressions, whereas 31% expressed either MUC1or MUC5AC, and 9% expressed neither. High MUC1immunoreactivity displayed a significant correlation with tumor progression as reflected by vascular invasion (P＜0.001),whereas high expression of MUC5AC significantly correlated with neural invasion (P = 0.022) and advanced ICC stage (P = 0.008). Patients with high expression of MUC1 had a significantly shorter survival (P = 0.0002). According to multivariate analyses, MUC1 reactivity (P = 0.026),histological grading and stage of tumor represented the least probability of survival.CONCLUSION: MUC1 is overexpressed in liver flukeassociated cholangiocarcinoma and relates to vascular invasion and poor prognosis, whereas MUC5AC mucin is neoexpressed and relates to neural invasion and advanced ICC stage. High MUC1 expression in tumor may be useful for predicting the poor outcome of ICC patients.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory /secretory product

瞄准：为了调查成纤维细胞房间增长的机制，由麝后睾吸虫刺激了分泌 / 能分泌(ES ) 产品。方法：NIH-3T3，老鼠成纤维细胞房间与 O 被对待。由与成年寄生虫 co 有教养的非接触的 viverrini ES 产品。从 NIH-3T3 的全部的 RNA 对待并且与 O 未经治疗。viverrini 被提取，抄录的颠倒和有老鼠 15K 的 hybridized 互补 DNA (cDNA ) 数组。结果被 ArrayVision 版本 5 和 GeneSpring 版本 5 软件分析。在正规化以后，寄生虫的基因表示的比率对待到第 2-a 更多褶层的未经治疗的 NIH-3T3 房间在上面调整当差别表示了基因，被定义。信号转导变异基因的表达式层次被半量的基于 SYBR 的即时 RT-PCR 验证。结果：在 15,000 genes/ESTs 的一个总数之中，有确定的房间的 239 基因增长相关的功能是 2 褶层 -- 并且由 O 的 more-up-regulated。在没有到寄生产品的暴露的房间的与那些相比的 viverrini ES 产品。这些基因被分类进包括的组精力和新陈代谢，信号转导变异，蛋白质合成和翻译，矩阵和结构的蛋白质，抄写控制，房间周期和 DNA 复制。而且， serine-threonine 激酶受体，受体酷氨酸激酶和骨胶原的表情生产相关的基因由 O 是起来调整的。viverrini ES 产品。信号转导变异基因的表达式水平；pkC， pdgfr 高山哈， jak 1， eps 8，即时 RT-PCR 测量的 tgf 贝它 1i4，带和 h 地岬证实了他们的表示层次到从 cDNA 获得的那些数组。然而，仅仅 pkC 的起来调整的表示， eps 8 并且是表皮的生长因素(EGF ) 或转变生长因素贝它(TGF 贝它) 显示出的任何一个的下游的发信号分子的 tgfbeta 1i4 统计意义(P 【 0.05 ) 。结论：O。viverrini ES 产品在几个功能的范畴和这些刺激基因表示的重要变化主要包括与房间增长有关的抄本。TGF 贝它和 EGF 信号转导变异小径作为 O 的可能的小径被显示。驾驶 viverrini 的房间增长。

Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line (KKU-100)

AIM To establish and dharacterize a nev cholangiocarcinoma cell line from a patient living in the Opisthorchis viverrini (O. viverrini) endemic area of Northeast Thailand.METHODS: Fresh liver biopsy and bile specimens were obtained from a 65-year-old Thai woman with cholangiocarcinoma of the porta hepatis. After digestion, the cells were cultured in Ham's F12 media. The established cell line was then characterized for growth kinetics, cell morphology, imm unocytochemistry and cytogenetics. Tumorigenicity of the cell line was determined by heterotransplanting in nude mice. RESULTS: The primary tumor was a poorly differentiated tubular adenocarcinoma. Examination of the bile revealed malignant cells with O. viverrini eggs. The cholangiocarcinoma cell line KKU-100 was established 4 mo after the primary culture-population doubling time was 72 h. KKU-100 possesses compact and polygonal-shapedepithelial cells. Immunocytochemically, this cell line exhibited cytokeratin, EMA, CEA, and CA125, but not α-fetoprotein (AFP), CA19-9, desmin, c-met, or p53. Such protein expressions parallel those of the primary tumor. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 78 and marked chromosomal structural changes. Inoculation of KKU-100 cells into nude mice produced a transplantable, poorly differentiated aden-ocarcinoma, similar to the original tumor.CONCLUSION: KKJ-100 is the first egg-proven, Opisthorchis- associated cholangiocarcinoma cell line, which should prove useful for further investigations of the tumor biology of this cancer.

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.

Immunohistochemical expression of mismatch repair genes:A screening tool for predicting mutator phenotype in liver fluke infection-associated intrahepatic cholangiocarcinoma

瞄准：为了澄清 DNA 的可能的贡献，错配修理(MMR ) 在肝吸虫的致癌作用的系统由使用免疫的联系感染的肝内 cholangiocarcinoma (国际计算中心) 组织化学的试金。方法：29 件国际计算中心样品的一个总数，被一个基于 PCR 的方法为 genomic 不稳定性估计了，被用于学习。他们组织化学地是检验免疫表明二 MMR 基因， hMSH2 和 hMLH1 的蛋白质表示。获得的结果与他们以前估计的 mutator 显型相比。结果：hMSH2 或 hMLH1 蛋白质显然在 29 中的 28 个被表示(96.6%) 国际计算中心样品。hMSH2 或 hMLH1 蛋白质的积极原子本地化在 86.2% 被观察(25/29 ) 或 93.1%(27/29 ) 国际计算中心盒子，分别地当他们的否定原子反应仅仅在 13.8% 被检测时(4/29 ) 或 6.9%(2/29 ) 国际计算中心盒子分别地分析了。结论：我们的学习，可能第一次，通过免疫显示出那个 DNA MMR 系统不玩的 hMSH2 和 hMLH1 基因的组织化学的察觉在肝吸虫的一个突出的角色联系感染的 cholangiocarcinogenesis。这些结果在通过一个基于 PCR 的方法估计的这些基因的突变地位上证实以前的调查结果。组织化学的分析证明了是为屏蔽 MMR 缺乏不管的一条有效、敏感的途径的免疫在激活或 hMSH2 或 hMLH1 基因的倡导者 hypermethylation 体。而且，免疫组织化学以简洁，耗时的更少和为在肿瘤发生屏蔽目标 MMR 基因的可能的参与的费用有效性与 mutator phenotyping 试金相比是更有益的。