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。尽管已经开发了多种治疗方式和血清学肿瘤标志物，例如甲胎蛋白（AFP），但 HCC 的诊断和治疗仍然很困难。我们已经表明，HCC 中 AFP 的 AFP 晶状体凝集素 (LCA) 反应性种类的相对量显着高于非肿瘤性肝病，并且 AFP (LCA) 反应性种类的较高比例与肿瘤侵袭性显着相关。 AFP LCA 反应物质的分子基础是 AFP 双触角糖链最里面的 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-乙酰基-β-D-氨基葡萄糖胺：α1-6 岩藻糖基转移酶 (αFT) 提供，该酶催化 GDP-岩藻糖通过 α1-6 连接与 N-连接寡糖的 N-乙酰氨基葡萄糖残基的还原端添加岩藻糖。 糖蛋白。因此，我们在本项目中尝试通过调节αFT来治疗HCC。对于这个问题，我们评估了是否可以通过反义寡核苷酸的转移来减少AFP的LCA反应性种类的比例，反义寡核苷酸是从报告为αFT的序列推导出来的：GenBank D89289。由于RNA结构软件（David H Mathews等人）估计的αFT mRNA构象表明核苷酸位置861附近可用于反义访问，因此通过脂转染将2'-甲氧基乙基硫代磷酸酯RNA寡核苷酸5'-CACTCATCTTGGA-3'转染至HepG2细胞。脂转染后24小时，在未经脂转染、RNA(-)-脂转染、有义-脂转染和反义-脂转染的细胞培养基中，AFP浓度分别为3.2±0.4ng/ml、3.3±0.3ng/ml、3.8±0.2ng/ml和2.4±0.1ng/ml。反义脂转染中的浓度显着低于正义脂转染中的浓度（Dunn's检验P<0.05）。脂转染后24小时，在未进行脂转染、进行了RNA(-)-脂转染、正义-脂转染和反义-脂转染的细胞培养基中，AFP的LCA反应种类的比例分别为76.2±2.8%、82.8±0.8%、58.7±2.9%和48.0±9.6%。反义脂转染中的比例显着低于RNA(-)-脂转染中的比例(P<0.01)。这些结果表明，αFT (D89289) 负责 AFP 的岩藻糖基化，并且 αFT 活性的消耗可能会减少影响细胞活力的 AFP 产生。下一步，我们将通过诱导针对 αFT 的小干扰 RNA 来建立 αFT 敲低细胞。将来自核苷酸位置1002的19个核苷酸克隆到pSUPER.retro载体(OligoEngine，美国)中，该载体表达与互补序列以链内方式连接形成双链RNA的靶RNA。尽管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。 αFT 活性有可能对细胞活力至关重要。较少的

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)