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Specific inhibition of human telomerase activity by transfection reagent, FuGENE6-antisense phosphorothioate
M Tao1, N Miyano-Kurosaki, K Takai
1Department of Industrial Chemistry, Chiba Institute of Technology, Narashino, Japan.
Abstract:
Human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The ODNs and S-ODNs were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 was used to enhance the cellular uptake of oligonucleotides in cell cultures. The results showed that S-ODN-3 (19-mer) encapsulated with FuGENE6 clearly inhibited the telomerase activity in HeLa cells, and the inhibitory efficiency increased with an increase in the S-ODN-3. However, free S-ODN-3 showed no inhibitory activity. On the other hand, ODN-3 encapsulated with FuGENE6 had no detectable inhibitory activity. The encapsulated S-ODNs exhibited higher inhibitory activities than the free S-ODNs, and showed sequence specific inhibition. Thus, the activities of the S-ODNs were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, and is appropriate for use in vitro and in vivo.
Insights
Phosphorothioate oligonucleotides (S-ODNs) effectively inhibit human telomerase activity in HeLa cells when delivered with FuGENE6. This combination shows promise for targeted antitumor drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Human telomerase is implicated in malignant tumor development.
- Telomerase is a potential target for novel antitumor drug design.
- Oligonucleotides are being explored for therapeutic applications.
Purpose of the Study:
- To investigate the ability of antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides to inhibit telomerase activity.
- To evaluate the role of the transfection reagent FuGENE6 in enhancing oligonucleotide delivery and efficacy.
- To assess the potential of S-ODNs as targeted antitumor agents.
Main Methods:
- Design of ODNs and S-ODNs complementary to the telomerase RNA active site.
- Use of FuGENE6 as a transfection reagent for cellular uptake in HeLa cells.
- Assay of telomerase activity inhibition by free and encapsulated oligonucleotides.
Main Results:
- S-ODN-3 (19-mer) encapsulated with FuGENE6 significantly inhibited telomerase activity in HeLa cells.
- Inhibitory efficiency of S-ODN-3 correlated with concentration.
- Free S-ODN-3 and encapsulated ODN-3 showed no significant telomerase inhibition.
- Encapsulated S-ODNs demonstrated enhanced and sequence-specific inhibition compared to free S-ODNs.
Conclusions:
- FuGENE6 effectively enhances the cellular uptake and inhibitory activity of S-ODNs against telomerase.
- S-ODNs, when delivered via FuGENE6, show potential as targeted antitumor therapeutics.
- FuGENE6 is a promising delivery vehicle for oligonucleotide-based therapies in vitro and in vivo.
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