Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides

Abstract

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA 'gapmer' oligonucleotide comprising a phosphorothioate central sequence flanked by 5' and 3' HNA sequences to conventional phosphorothioate oligonucleotides and to a 2'-O-methoxyethyl (2'-O-ME) phosphorothioate 'gapmer'. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2'-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate 'gap') was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.

Figure 1

Chemical structures of oligonucleotides employed in this study as compared to phosphodiester oligonucleotides.

Figure 2

Cellular uptake and distribution of HNA derivatives. NIH 3T3 MDR cells were treated with fluorescein-labeled HNA derivatives (10 nM) complexed with Lipofectamine 2000. (A) HNA (GS1954); (B) HNA gapmer (GS1956). (a) fluor-labeled oligonucleotide; (b) phase contrast image; (c) overlay image. Note that some of the cells lack fluorophore (arrow).

Figure 3

Cytotoxicity of oligonucleotides. The cytotoxicities of antisense oligonucleotides were determined after treatment of NIH 3T3 MDR cells with various concentrations of antisense/Lipofectamine 2000 complexes and further incubating them for 64 h as described. Closed circles, control cells treated with Lipofectamine 2000 only; closed squares, HNA (GS1954); closed triangles, HNA gapmer (GS1956); open squares, phosphorothioate (5995); open triangles, 2′-O-ME gapmer (13 158). Values represent cell population in percentage with 100% taken for untreated NIH 3T3 MDR cells. Means and standard errors of 3–6 determinations.

Figure 4

Oligonucleotide uptake by cells. NIH 3T3 MDR cells were treated with Lipofectamine 2000 complexes of fluorescein labeled HNA gapmer as described. The fluorescence of the cells was then measured by flow cytometry. (A) x-Axes represent fluorescence intensities and y-axes represent the cell number. (a) Untreated negative control cells; (b) 1 nM HNA gapmer; (c) 3 nM HNA gapmer; (d) 9 nM HNA gapmer; (e) 27 nM HNA gapmer. (B) x-Axis represents HNA gapmer concentrations, y-axis at left and open bars represent the percentage of the cell population that is transfected; y-axis at right and closed circles represent relative fluorescence strengths. Means and standard errors of three determinations.

Figure 5

Antisense effects on P-glycoprotein expression in NIH 3T3 MDR cells. NIH 3T3 MDR cells were treated with Lipofectamine 2000 complexes of various oligonucleotides, and cell surface P-glycoprotein expression in the viable cells was evaluated as described in Materials and Methods. x-Axes represent fluorescence intensity and y-axes represent cell number. Panels (A, D and G), control (Lipofectamine 2000 only); (B) 9 nM HNA gapmer mismatched (GS1957); (C) 9 nM HNA gapmer (GS1956); (E) 9 nM phosphorothioate scrambled control (10 221); (F) 9 nM phosphorothioate (5995); (H) 9 nM 2′-O-ME gapmer scrambled (13 753) (I) 9 nM 2′-O-ME gapmer (13 758). The demarcations between the left and right boxes in the figures are set at one standard deviation below the mean of the untreated control.

Figure 6

Antisense effects on P-glycoprotein expression in NCI/ADR-RES cells. NCI/ADR-RES cells were treated with Lipofectamine 2000 complexes of various oligonucleotides, and cell surface P-glycoprotein expression in the viable cells was evaluated as described in Materials and Methods. x-Axes represent fluorescence intensity and y-axes represent cell population. Panels (A, D and G), controls (Lipofectamine 2000 only); (B) 9 nM HNA gapmer mismatched (GS1957); (C) 9 nM HNA gapmer (GS1956); (E) 9 nM phosphorothioate scrambled control (10 221); (F) 9 nM phosphorothioate (5995); (H) 9 nM 2′-O-ME gapmer scrambled (13 753) (I) 9 nM 2′-O-ME gapmer (13 758). The demarcations between the left and right boxes in the figures are set at one standard deviation below the mean of the untreated control.

Figure 7

Antisense concentration dependence of P-glycoprotein expression. Cell surface P-glycoprotein expression in NIH 3T3 MDR cells was measured by immunostaining and flow cytometry as described in Materials and Methods. Closed circles, control cells treated with Lipofectamine 2000 only; closed squares, HNA (GS1954); closed triangles, HNA gapmer (GS1956); open squares, phosphorothioate (5995); open triangles, 2′-O-ME gapmer (13 158). Values are percentage of P-glycoprotein expression with 100% taken for NIH 3T3 MDR cells treated with corresponding mismatched or scrambled sequence. The percentage was calculated on the basis of the fraction of the cell population shifted to greater than one standard deviation below the mean of the untreated controls in terms of P-glycoprotein expression. Means and standard errors of 3–6 determinations.

Figure 8

Western blots for P-glycoprotein expression. After treating NIH 3T3 MDR cells with various antisense/Lipofectamine 2000 complexes as described, cells were harvested for western analysis. Lane 1, Lipofectamine 2000 only control; lane 2, HNA gapmer (GS1956); lane 3, phosphorothioate (5995); lane 4, 2′-O-ME gapmer (13 758); lane 5, HNA gapmer mismatch (GS1957); lane 6, phosphorothioate scrambled (10 221); lane 7, 2′-O-ME gapmer scrambled (13 753). All oligonucleotides were used at a 10 nM concentration.

Figure 9

Effects of antisense oligonucleotides on Rhodamine 123 accumulation. NIH 3T3 MDR cells were treated with 9 nM of antisense oligonucleotides complexed with Lipofectamine 2000 as described. Closed bars, control oligonucleotides; open bars, oligonucleotides targeted to MDR1 gene. Values are Rhodamine 123 uptake increase, with the 100% level taken as that for untreated NIH3T3 MDR cells. Mean and standard errors of 3–6 determinations.