| Research Abstract |
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
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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
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