TREATMENT OF HEPATOCELLULAR CARCINOMA BY THE REGULATION OF GLYCOSYLTRANSFERASE

通过糖基转移酶的调节治疗肝细胞癌

• 批准号: 13670500
• 负责人: AOYAGI Yutaka
• 金额: $ 2.3万
• 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670500/
• 关键词: Alpha 1-6 fucosyltransferase; hepatocellular carcinoma; lipofection; antisense oligonucleotide; Lens culinaris agglutinin; treatment; アルファフェトプロテイン

In Japan, hepatocellular carcinoma (HCC) is one of the most prevalent human cancers. The diagnosis and treatment for HCC are still difficult although several therapeutic modalities and serological tumor markers, such as alpha-fetoprotein (AFP), have been developed. We have already 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 and 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. Thus, it is supposed that these phenotypic changes must be provided by GDP-L-Fuc: N-acethyl-β-D-glucosaminide: α1-6 fucosyltransferase (αFT), which catalyzes the addition of fucose from GDP-fucose through an α1-6 linkage to the reducing end of N-acetylglucosamine residue of … More N-linked oligosaccharides of glycoproteins. Accordingly, we attempt to perform the treatment of HCC by the regulation of αFT in the present project.For this issue, we evaluated whether the proportion of LCA-reactive species of AFP could be diminished by the transfer of antisense oligonucleotide, which was deduced from the sequence reported as αFT : GenBank D89289. Because the conformation of αFT mRNA estimated by RNA structure software (David H Mathews, et al) indicated that the vicinity at the nucleotide position 861 would be available for antisense access, 2'-methoxyethyl phosphorothioate RNA oligonucleotide, 5'-CACTCATCTTGGA-3', was transfected to HepG2 cells by lipofection. The AFP concentrations at 24 hours after lipofection were 3.2 ± 0.4 ng/ml, 3.3 ± 0.3 ng/ml, 3.8 ± 0.2 ng/ml, and 2.4 ± 0.1 ng/ml in the medium of the cells without lipofection, with RNA(-)-lipofection, sense-lipofection, and antisense-lipofection, respectively. The concentration in antisense-lipofection was significantly lower than that in sense-lipofection (Dunn's test P<0.05). The proportions of LCA-reactive species of AFP at 24 hours after lipofection were 76.2 ± 2.8 %, 82.8 ± 0.8 %, 58.7 ± 2.9 %, and 48.0 ± 9.6 % in the medium of the cells without lipofection, with RNA(-)-lipofection, sense-lipofection, and antisense-lipofection, respectively. The proportion in antisense lipofection was significantly lower than that in RNA(-)-lipofection (P<0.01). These results suggested that αFT (D89289) is responsible for the fucosylation of AFP and that the depletion of αFT activity may reduce AFP production affecting the cell viability.In the next step, we are engaged in an establishment of αFT knock-down cells by inducing small interfering RNA against αFT. The 19 nucleotides from the nucleotide position 1002 were cloned into pSUPER.retro vector (OligoEngine, USA), which expresses a target RNA connected with the complementary sequence forming double-stranded RNA in an intrastranded fashion. Although the clones carrying empty and mutated sequences have been established, none of carrying the target sequence has been established yet. There are some possibilities that αFT activity is critical for cell viability. Less

在日本，肝细胞癌（HCC）是最常见的人类癌症之一。肝癌的诊断和治疗仍然是困难的，虽然一些治疗方式和血清学肿瘤标志物，如甲胎蛋白（AFP），已开发。我们已经发现，肝癌中与透镜凝集素（LCA）反应的AFP种类的相对量显著高于非肿瘤性肝病，并且与（LCA）反应的AFP种类的较高比例与肿瘤侵袭性显著相关。AFP的LCA反应性物质的分子基础是AFP的双触角糖链的最内侧N-乙酰葡糖胺残基的岩藻糖基化。因此，推测这些表型变化必须由GDP-L-Fuc：N-乙酰乙基-β-D-氨基葡萄糖苷：α1-6岩藻糖基转移酶（αFT）提供，其催化从GDP-岩藻糖通过α1-6键加成到N-乙酰氨基葡萄糖苷的还原末端。 ...更多信息 糖蛋白的N-连接寡糖。因此，本研究试图通过αFT的调控来治疗肝癌，为此，我们评估了是否可以通过转移αFT：GenBank D89289序列推导的反义寡核苷酸来减少AFP的LCA反应性物种的比例。由于通过RNA结构软件（大卫H马修斯等）估计的αFT mRNA的构象表明核苷酸位置861附近可用于反义接近，因此通过脂质体转染将2 '-甲氧基乙基硫代磷酸酯RNA寡核苷酸，5'-CACTCATCTTGGA-3 '转染至HepG 2细胞。脂质体转染后24小时，在无脂质体转染、有RNA（-）-脂质体转染、正义-脂质体转染和反义-脂质体转染的细胞培养基中，AFP浓度分别为3.2 ± 0.4 ng/ml、3.3 ± 0.3 ng/ml、3.8 ± 0.2 ng/ml和2.4 ± 0.1 ng/ml。反义脂质体转染组的细胞内脂质体浓度显著低于正义脂质体转染组（Dunn's检验P<0.05）。在脂质转染后24小时，在无脂质转染、有RNA（-）-脂质转染、正义-脂质转染和反义-脂质转染的细胞的培养基中，AFP的LCA反应性种类的比例分别为76.2 ± 2.8%、82.8 ± 0.8%、58.7 ± 2.9%和48.0 ± 9.6%。反义脂质体转染组细胞比例显著低于RNA（-）脂质体转染组（P<0.01）。这些结果表明，αFT（D89289）参与了AFP的岩藻糖基化，αFT活性的缺失可能会降低AFP的产生，从而影响细胞的存活率。将来自核苷酸位置1002的19个核苷酸克隆到pSUPER.retro载体（OligoEngine，USA）中，该载体表达与互补序列连接的靶RNA，从而以链内方式形成双链RNA。虽然已经建立了携带空序列和突变序列的克隆，但尚未建立携带靶序列的克隆。有一些可能性，αFT活性对细胞活力至关重要。少

项目成果

Waguri N, Suda T, Kamura K, Aoyagi Y.: "Heterogeneous hepatic enhancement on CT angiography in idiopathic portal hypertension"Liver. 22. 276-280 (2002)

Waguri N、Suda T、Kamura K、Aoyagi Y.：“特发性门静脉高压 CT 血管造影的异质肝脏增强”肝脏。

Shoji Y, Mita T, Isemura M, Mega T, Hase S, Isemura S, Aoyagi Y: "A fibronectin-binding protein from rice bran with cell adhesion activity for animal tumor cells"Biosci. Biotechnol. Biochem.. 65. 1181-1186 (2001)

Shoji Y、Mita T、Isemura M、Mega T、Hase S、Isemura S、Aoyagi Y：“来自米糠的纤连蛋白结合蛋白，对动物肿瘤细胞具有细胞粘附活性”Biosci。

Suda T, Fujiyama A, Takimoto M, Igarashi M, Kuroiwa T, Waguri N, Kawai H, Mita Y, Aoyagi Y: "Interchromosomal telomere length variation"Biochem Bioph Res Comm. 291. 210-214 (2002)

Suda T、Fujiyama A、Takimoto M、Igarashi M、Kuroiwa T、Waguri N、Kawai H、Mita Y、Aoyagi Y：“染色体间端粒长度变异”Biochem Bioph Res Comm。

Furukawa K, Hrada T, Aoaygi Y: "Successful endoscopic sphincoterotomy using a papillotome tipped with a balloon for cases of choledocholithiasis complicated by duodenal diverticula"Am J Gastroenterol. 96. 3215-3216 (2001)

Furukawa K、Hrada T、Aoaygi Y：“使用顶端带有气球的乳头刀成功进行内镜下乳头括约肌切开术，治疗胆总管结石并发十二指肠憩室的病例”Am J Gastroenterol。

Aoyagi Y, Mita Y, Suda T, et al.: "The fucosylation index of serum α-fetoprotein as useful prognostic factor in patients with hepatocellular carcinoma in special reference to chronological changes"Hepatolgy Res.. 23. 287-295 (2002)

Aoyagi Y、Mita Y、Suda T 等人：“血清甲胎蛋白的岩藻糖基化指数作为肝细胞癌患者的有用预后因素，特别参考时间顺序变化”Hepatolgy Res.. 23. 287-295 (2002)