Thanan Raynoo, Pairojkul Chawalit, Pinlaor Somchai, Khuntikeo Narong, Wongkham Chaisiri, Sripa Banchob, Ma Ning, Vaeteewoottacharn Kulthida, Furukawa Ayako, Kobayashi Hatasu, Hiraku Yusuke, Oikawa Shinji, Kawanishi Shosuke, Yongvanit Puangrat, Murata Mariko
Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan; Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie, 513-8670, Japan.
Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Free Radic Biol Med. 2013 Dec;65:1464-1472. doi: 10.1016/j.freeradbiomed.2013.07.034. Epub 2013 Jul 31.
Nitrative and oxidative DNA damage plays an important role in inflammation-related carcinogenesis. Chronic inflammation such as parasite infection and primary sclerosing cholangitis can be an etiological factor of cholangiocarcinoma. Using a proteomic approach and double-fluorescent staining, we identified high expression and colocalization of albumin and cytokeratin-19 in liver fluke-associated cholangiocarcinoma tissues, compared with normal livers from cholangiocarcinoma patients and cadaveric donors, respectively. Albumin was detected not only in cells of hyperplastic bile ducts and cholangiocarcinoma, but also in liver stem/progenitor cell origin, such as canal of Hering, ductules, and ductular reactions, suggesting the involvement of stem/progenitor cells in cholangiocarcinoma development. To clarify the involvement of liver stem/progenitor cells in cholangiocarcinoma, we examined several stem/progenitor cell markers (CD133, CD44, OV6, and Oct3/4) in cholangiocarcinoma tissues analyzed by immunohistochemical staining, and measured 8-oxodG levels by using HPLC-ECD as an inflammation-related DNA lesion. In addition, a stem/progenitor cell factor Bmi1, 8-nitroguanine (formed during nitrative DNA damage), DNA damage response (DDR) proteins (phosphorylated ATM and γ-H2AX), and manganese-SOD (Mn-SOD) were analyzed by immunohistochemistry. Stem/progenitor cell markers (CD133, OV6, CD44, and Oct3/4) were positively stained in 56, 38, 47, and 56% of 34 cholangiocarcinoma cases, respectively. Quantitative analysis of 8-oxodG revealed significantly increased levels in CD133- and/or Oct3/4-positive tumor tissues compared to negative tumor tissues, as well as 8-nitroguanine formation detected by immunohistochemistry. In the cases of CD44- and/or OV6-positive tissue, no significant difference was observed. Cholangiocarcinoma patients with CD133- and/or Oct3/4-positive tumor tissues showed significantly lower expression of Mn-SOD and higher DDR protein, γ-H2AX. Moreover, CD133- and/or Oct3/4-positive cholangiocarcinoma patients had significant associations with tumor histology types, tumor stage, and poor prognoses. Our results suggest that CD133 and Oct3/4 in cholangiocarcinoma are associated with increased formation of DNA lesions and the DDR protein, which may be involved in genetic instability and lead to cholangiocarcinoma development with aggressive clinical features.
硝化和氧化DNA损伤在炎症相关的致癌过程中起重要作用。慢性炎症如寄生虫感染和原发性硬化性胆管炎可能是胆管癌的病因。通过蛋白质组学方法和双荧光染色，我们分别与胆管癌患者的正常肝脏和尸体供体的肝脏相比，发现肝吸虫相关胆管癌组织中白蛋白和细胞角蛋白-19高表达且共定位。白蛋白不仅在增生性胆管和胆管癌细胞中被检测到，还在肝干细胞/祖细胞起源处被检测到，如赫林管、胆小管和小胆管反应，提示干细胞/祖细胞参与胆管癌的发生发展。为了阐明肝干细胞/祖细胞在胆管癌中的作用，我们通过免疫组织化学染色检测了胆管癌组织中的几种干细胞/祖细胞标志物（CD133、CD44、OV6和Oct3/4），并使用高效液相色谱-电化学检测法测量8-氧代鸟嘌呤水平作为炎症相关的DNA损伤。此外，通过免疫组织化学分析了干细胞/祖细胞因子Bmi1、8-硝基鸟嘌呤（在硝化DNA损伤过程中形成）、DNA损伤反应（DDR）蛋白（磷酸化的ATM和γ-H2AX）以及锰超氧化物歧化酶（Mn-SOD）。在34例胆管癌病例中，干细胞/祖细胞标志物（CD133、OV6、CD44和Oct3/4）分别在56%、38%、47%和56%的病例中呈阳性染色。8-氧代鸟嘌呤的定量分析显示，与阴性肿瘤组织相比，CD133和/或Oct3/4阳性肿瘤组织中的水平显著升高，免疫组织化学检测也发现了8-硝基鸟嘌呤的形成。在CD44和/或OV6阳性组织的病例中，未观察到显著差异。CD133和/或Oct3/4阳性肿瘤组织的胆管癌患者显示出Mn-SOD的表达显著降低，DDR蛋白γ-H2AX的表达升高。此外，CD133和/或Oct3/4阳性的胆管癌患者与肿瘤组织学类型、肿瘤分期和不良预后显著相关。我们的结果表明，胆管癌中的CD133和Oct3/4与DNA损伤和DDR蛋白的形成增加有关，这可能参与遗传不稳定并导致具有侵袭性临床特征的胆管癌发生发展。
