ALPHA-1-6 FUCOSYLTRANSFERASE ANTISENSE-OLIGONUCLEOTIDE THERAPY FOR HEPATOCELLULAR CARCINOMA.

ALPHA-1-6 岩藻糖基转移酶反义寡核苷酸治疗肝细胞癌。

• 批准号: 09670520
• 负责人: AOYAGI Yutaka
• 金额: $ 1.98万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670520/
• 关键词: Alpha 1-6 fucosyltransferase; fucosylation; hepatocellular carcinoma; antisense oligonucleotide; Lens culinaris agglutinin; treatment

Hepatocellular carcinoma (HCC) is one of the most prevalent human cancers, and the prevention of intra and extrahepatic metastasis of HCC is still difficult even with the development of several therapeutic modalities. We have investigated serological features indicating a malignant potential of HCC. Although, in general, the serum concentration of alpha-fetoprotein (AFP) has been used for the diagnosis of HCC, we have shown that the relative amount of the Lens culinaris agglutinin (LCA)-reactive species of AFP is significantly greater in HCC than in non-neoplastic liver diseases. In addition, we have elucidated that the higher proportion of (LCA)-reactive species of AFP is significantly associated with the tumor invasiveness. The molecular basis of the LCA-reactive species of AFP is the fucosylation at the innermost N-acetylglucosamine residue of the biantennary sugar chain of AFP. Furthermore, it was reported that N-linked β1-6 branched oligosaccharides might contribute directly to th … More e malignant phenotype including metastatic potential of several types of cancers including HCC. It is supposed that these phenotypic changes must be provided by GDP-L-Fuc : N-acethyl-β-D-glucosaminide : β1-6 fucosyltransferase (αFT), which catalyzes the addition of fucose from GDP-fucose through an αl-6 linkage to the reducing end of N-acetylglucosamine residue of N-linked oligosaccharides of glycoproteins, and N-acetylglucosaminyltransferase V (GnT V), which catalyzes β1-6 branching, respectively.Therefore, in this research term, at first we determined weather the amount of message of αFT and GnT V in each liver tissue were really associated with each enzyme activity in the tissues and the serum. Consequently, quantitative reverse transcription coupled polymerase chain reaction revealed that the quantity of each mRNA in HCC was significantly associated with each enzyme activity in HCC and the corresponding serum. From the point of these observations, next we tried to investigate effects of antisense RNAs against αFT and GnT V. In order to evaluate the effects in vivo, we employed the high-level gene expression system based on tetracycline-responsive element (TRE). Although this system has a great advantage of capability for strict regulation of gene expression in vivo according to the presence or absence of tetracycline, it is relatively difficult to establish stable cell lines. Because it requires transfection of two critical components, the tetracycline-controlled transactivator (TA) and TRE fused to the gene of interest. Furthermore, none of genes for the selection is integrated into the plasmid consisting of TRE and the gene of interest. Finally, three components have to be transformed including a selectable marker. We have finished to clone αFT and GnT V genes in an appropriate construct, and create a stable cell line of TA. In a near future, we will be able to develop double-stable cell lines, and elucidate a biological significance of fucosylation and glucosaminylation at a molecular level on cancer invasiveness. Less

肝细胞癌是人类最常见的癌症之一，尽管已有多种治疗方法问世，但其肝内外转移的预防仍是一大难题。我们已经研究了表明肝细胞癌有恶性潜能的血清学特征。虽然血清甲胎蛋白(AFP)浓度一般被用于肝细胞癌的诊断，但我们发现肝细胞癌中AFP的晶状体凝集素(LCA)反应物的相对含量显著高于非肿瘤性肝病。此外，我们还阐明了较高比例的(LCA)反应性AFP与肿瘤侵袭性显著相关。AFP的LCA活性物种的分子基础是AFP的双天线糖链最内侧N-乙酰氨基葡萄糖残基上的岩藻糖化。此外，据报道，N-连接的β1-6支链寡糖可能直接与…有关更多的恶性表型包括包括肝细胞癌在内的几种癌症的转移潜能。推测这些表型变化必须由Gdp-L-Fuc：N-乙酰-β-D-氨基葡萄糖苷：β1-6岩藻糖基转移酶(αFT)和N-乙酰氨基葡萄糖转移酶V(GnT V)分别提供，前者催化Gdp-岩藻糖通过αL-6链从β岩藻糖加成到N-乙酰氨基葡萄糖残基的N-乙酰氨基葡萄糖残基还原端，后者催化GnT 1-6分支。因此，在本研究中，我们首先确定了肝组织中α-FT和GnT V的信息量是否真的与组织和血清中的每种酶的活性有关。定量逆转录耦合聚合酶链式反应结果显示，肝细胞癌组织中各酶的量与肝细胞癌及相应血清中各酶的活性显著相关。从这些观察的角度来看，接下来我们试图研究反义RNA对αFT和GnT V的影响。为了评估其体内作用，我们使用了基于四环素反应元件(TrE)的高水平基因表达系统。尽管该系统具有根据四环素的存在与否对体内基因表达进行严格调控的能力，但建立稳定的细胞系是相对困难的。因为它需要两个关键成分的转染，四环素控制的反式激活因子(TA)和tre与目的基因融合。此外，没有一个用于选择的基因整合到由tre和目的基因组成的质粒中。最后，必须转换三个组件，包括一个可选择的标记。我们已完成了αFT和GnTV基因的克隆，并建立了稳定的TA细胞系。在不久的将来，我们将能够建立双稳定的细胞系，并在分子水平上阐明岩藻糖基化和氨基葡萄糖基化对肿瘤侵袭性的生物学意义。较少

项目成果

見田 有作: "Usefulness of sensitive determination of des- γ -carboxy prothrombin in the early diagnosis of patients with hepatocellular carcinoma"Cancer. 82. 1643-1648 (1998)

Yusaku Mita：“敏感测定脱-γ-羧基凝血酶原在肝细胞癌患者早期诊断中的作用”癌症。 82. 1643-1648 (1998)

佐伯晃一: "Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells"Biochim. Biophys. Acta. 63(3). 585-587 (1999)

Koichi Saeki：“源自茶儿茶素的多酚化合物在人组织溶解性淋巴瘤 U937 细胞中的细胞凋亡诱导活性”Biochim。 585-587 (1999)。

斉藤 崇: "Elevation of TFF1 (pS2) gene expression during healing of gastric ulcer at 'non-ulcerated' sites in the stomach: Semi-quantification using single tube method of polymerase chain reaction."J Gastroenterol Hepatol, 2000. (in press). (2000)

Takashi Saito：“胃溃疡愈合过程中胃‘非溃疡’位点 TFF1 (pS2) 基因表达的升高：使用聚合酶链式反应的单管方法进行半定量。”J Gastroenterol Hepatol，2000 年。（出版中） ）（2000）。

青柳豊: "Fucosylation index of alpha-fetoprotein as a possible prognostic indicator in patients with hepatocellular carcinoma." Cancer. 83. 2076-2082 (1998)

Yutaka Aoyagi：“甲胎蛋白岩藻糖基化指数作为肝细胞癌患者的可能预后指标。”83。2076-2082 (1998)

曽我憲二: "Scrum des-γ-carboxy prothrombin level by a modified enzyme immunoassay method in hepatocel-lular carcinoma-Significance of des-γ-carboxy prothrombin in small hepatocellular carcinoma" Hepato-Gasroenterology. (in press). (1998)

Kenji Soga：“肝细胞癌中采用改良酶免疫测定法测定的 Scrum des-γ-羧基凝血酶原水平 - 小肝细胞癌中 des-γ-羧基凝血酶原的意义”《肝胃肠病学》（出版中）。