Cancer Res Treat.  2004 Oct;36(5):308-314.

Effective Inhibition of Cancer Cell Growth by a Novel Tripartite Transfection Complex Containing Ribbon Antisense Molecules to hTR

• Park JG

Abstract

PURPOSE
In the present study, ribbon antisense to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. MATERIALS AND METHODS: Ribbon antisense molecules to the human hTR gene (hTR-RiAS) were constructed and complexed with a short modified peptide and cationic liposomes to improve the cellular uptake of the antisense molecules. The DPL complexes containing hTR-RiAS were transfected into target cancer cells. Various assays were performed to confirm the effects of the hTR-RiAS on the gene expression and cell proliferation. RESULTS: When cancer cells were treated with hTR-RiAS, the cellular level of hTR mRNA was reduced by more than 95%, as shown by RT-PCR. Further, the telomerase activity was also affected by the antisense treatment. In contrast, both mismatched and scrambled oligonucleotides failed to reduce the levels of hTR mRNA and telomerase activity. When checked for cancer cell viability, hTR- RiAS inhibited cell growth by more than 70%, in a very rapid manner. The reduced cell viability was found to be due to apoptosis of cancer cells. CONCLUSION: These results show that hTR-RiAS is a powerful anticancer reagent, with the potential for broad efficacy to diverse malignant tumors.

Keyword

Telomerase; hTR; Ribbon antisense; Tripartite DPL transfection

MeSH Terms

Apoptosis
Cell Proliferation
Cell Survival
Gene Expression
Humans
Liposomes
Oligonucleotides
RNA
RNA, Messenger
Telomerase
Transfection*
Liposomes
Oligonucleotides
RNA
RNA, Messenger
Telomerase

Figure

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  Fig. 1 Schematic diagram for the construction of ribbon antisense to the hTR gene. hTR13701 was phosphorylated at the 5' end, and harbors complementary sequences at both 5' and 3' ends, with a single stranded sequence of GATC at the 5' extreme end. Ribbon antisense was constructed by covalently ligating two identical hTR13701 molecules. The RiAS oligos, therefore, contains two loops and a stem.

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  Fig. 2 hTR expression and specific antisense activity of ribbon antisense to hTR RNA. (A) Expression of hTR in various cancer cell lines. Expression of hTR was examined in 6 cancer cell lines by RT-PCR. Lane M, DNA size marker of 100 bp ladder; lane 1, A549; lane 2, NCI H1299; lane 3, Hep3B; lane 4, SW480; lane 5, HeLa and lane 6, A375SM. (B) Specific reduction of hTR RNA in HeLa cells by ribbon antisense to hTR RNA. Cells were transfected with a tripartite DPL complex (hTR-RiAS/Tatpeptide/Lipofectamine). Five different RiAS oligos were used, and RT-PCR assays performed. Lane M, 100 bp ladder; lane 1, sham; lane 2, liposomes alone; lane 3, hTR13701; lane 4, hTR13702; lane 5, hTR13703; lane 6, hTR13704 and lane 7, hTR13705.

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  Fig. 3 Specific reduction of hTR RNA by hTR-RiAS in various cancer cell lines. After transfecting a tripartite DPL complex (hTR-RiAS/Tat-peptide/Lipofectamine) into each target cell, RT-PCR was performed. (A) Dose dependent specific reduction of hTR RNA by hTR-RiAS in HeLa cells. Lane M, 100 bp DNA ladder; lane 1, sham; lane 2, liposome alone; lane 3, hTR-RiAS (0.1 µg); lane 4, hTR-RiAS (0.5 µg) and lane 5, hTR-RiAS (1.0 µg). (B) Specific reduction of hTR RNA by hTR-RiAS in SW480 cells: Lane M, 100 bp DNA ladder; lane 1, sham; lane 2, liposome alone; lane 3, scrambled control (1.0 µg); lane 4, mismatched control (1.0 µg) and lane 5, hTR-RiAS (1.0 µg). (C) Specific reduction of hTR RNA by hTR-RiAS in NCIH1299 cells. Lane M, 100 bp DNA ladder; lane 1, sham; lane 2, liposome alone; lane 3, scrambled control (1.0 µg); lane 4, mismatched control (1.0 µg) and lane 5, hTR-RiAS (1.0 µg).

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  Fig. 4 Reduction of telomerase activity in hTR RiAS-treated HeLa cells. After transfecting a tripartite DPL complex (hTR-RiAS/Tat-peptide/Lipofectamine) into HeLa cells, the telomerase activity was detected using the TRAP-ELISA method. Cells that were sham-treated, treated with liposomes alone, with mismatched control. A TS8 template set and heat-treated HeLa cells were used as positive and negative controls for the detection of telomerase expression, respectively. Each bar value represents the mean±S.D. of triplicate experiments. *Abbreviation: Lipo., liposomes alone

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  Fig. 5 Effect of hTR-RiAS on the proliferation of HeLa cells. The cells were treated with tripartite DPL complexes containing 0.05, 0.1 and 0.2 µg of hTR-RiAS, as indicated. The transfectants were examined for growth inhibition by an MTT assay 72 h post-transfection. Cells were sham-treated and treated with liposomes alone and simultaneously assayed. Each bar value represents the mean±S.D. of triplicate experiments. *Abbreviation: Lipo., liposomes alone

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  Fig. 6 Induction of apoptotic DNA ladder formation by hTR RiAS in HeLa cells. Genomic DNA was extracted in 48 h treatment of tripartite DPL complexes and run on a 1.8% agarose gel. Lane M, 100 bp DNA ladder; lane 1, sham; lane 2, liposomes alone; lane 3, scrambled control; lane 4, mismatched control and lane 5, hTR-RiAS.

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