Anti-insulin-like growth factor-IIP3 DNAzymes inhibit cell proliferation and induce caspase-dependent apoptosis in human hepatocarcinoma cell lines

Figures & data

Figure 1 Effect of DRz concentration on the expression of IGF-IIP3 in SMMC-7721 cells.

Notes: (A) mRNA expression of IGF-IIP3 was significantly inhibited by different concentrations of DRzs (1 μg/mL = 0.098 pmol/μL). The cells were transfected with the indicated concentrations of DRz1, DRz2, or DRz1^inv for 24 hours. Data shown are means ± SD of normalized relative IGF-IIP3 mRNA levels (n ≥3). *Significant difference to control (P < 0.05); (−) dimensionless units. (B) IGF-IIP3 protein expression was quantified via densitometry and compared with the “no treatment” control. The maximal gray value was set to 1 and all other results are expressed relative to this value (black) (n ≥3).
Abbreviations: DRz, deoxyribozyme (DNAzyme); DRz1^inv, inactive DRz; IGF-IIP3, insulin-like growth factor II promoter 3; mRNA, messenger RNA; SD, standard deviation.

Figure 2 Effect of DRzs on the expression of IGF-IIP3 in SMMC-7721 cells.

Notes: (A) mRNA expression of IGF-IIP3 was significantly inhibited by DRzs. The cells received either no treatment or were transfected with 4 μg/mL (0.39 pmol/μL) DRz1, DRz2, or DRz1^inv. Data shown are means ± SD of relative IGF-IIP3 mrna expressed compared to control (no treatment) (n ≥3). *Significant difference to control (P < 0.05). (B) Protein expression of IGF-IIP3 was equally inhibited by both DRzs. IGF-IIP3 protein expression was quantified via densitometry and compared with the “no treatment” control. The maximal gray value was set to 1 and all other results were expressed relative to this value (black). Note that the 24-hour transfection with DRz1 exhibited the strongest reduction in IGF-IIP3 protein expression levels (n ≥3).
Abbreviations: DRz, deoxyribozyme (DNAzyme); DRz1^inv, inactive DRz; IGF-IIP3, insulin-like growth factor II promoter 3; mRNA, messenger RNA; SD, standard deviation.

Figure 3 Fluorescence microscopic evaluation of DRz cellular uptake and intracellular distribution.

Note: Cells were transfected with 4 μg/mL (0.39 pmol/μL) DRz1, DRz2, or DRz1^inv (control) and cultured for 24 hours to a final cell density of 2 × 10⁴. Bar: 200 μm.
Abbreviations: Cells were transfected with 4 μg/mL (0.39 pmol/μL) DRz1, DRz2, or DRz1^inv (control) and cultured for 24 hours to a final cell density of 2 × 10⁴. Bar: 200 μm.

Figure 4 Effect of 4 μg/mL (0.39 pmol/μL) DRzs on the expression of IGF-IIP3 in various HCC cell lines.

Notes: Protein expression of IGF-IIP3 was significantly inhibited by DRzs in SMMC-7721 cells, HepG2 cells, and Huh7 cells. IGF-IIP3 protein expression was quantified by densitometry, with the maximal gray value set to 1 (n ≥3).
Abbreviations: DRz, deoxyribozyme (DNAzyme); HCC, hepatocellular carcinoma; IGF-IIP3, insulin-like growth factor II promoter 3.

Figure 5 Assessment of cell proliferation via MTT assay.

Notes: (A) Cell proliferation and survival of SMMC-7721 cells (initial density 5 × 10⁴ cells/well) in the presence of 4 μg/mL (0.39 pmol/μL) of the IGF-IIP3 DRzs for a maximum of 72 hours. (B) comparison of cell proliferation and survival of SMMC-7721, HepG2, and Huh7 cells (initial density 5 × 10⁴ cells/well) in response to 4 μg/mL (0.39 pmol/μL) of the IGF-IIP3 DRzs after 24 hours. All data are means ± SD from triplicates (n ≥3). *P < 0.05; **P < 0.01; ***P < 0.001.
Abbreviations: DRz, deoxyribozyme (DNAzyme); IGF-IIP3, insulin-like growth factor II promoter 3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation.

Figure 6 Fluorescence microscopic evaluation of nuclear morphological changes (DAPI stained) in response to IGF-IIP3 DRzs.

Notes: Cells were transfected with 4 μg/mL (0.39 pmol/μL) DRz1, DRz2, or DRz1^inv (control), cultured for 24 hours to a final cell density of 2 × 10⁴, and subsequently stained with 100 μL/mL DAPI. Arrows indicate fragmented (red), hyper-condensed (white), and nuclei entering condensation/fragmentation (orange), which are further highlighted in the magnified pseudo-color insert. CLUT = relative intensity profile indicating the amount of nuclear condensation. Bar: 20 μm.
Abbreviations: DAPI, diamidino-phenylindole; DRz, deoxyribozyme (DNAzyme); DRz1^inv, inactive DRz; IGF-IIP3, insulin-like growth factor II promoter 3.

Figure 7 Representative flow cytometric assessment of apoptosis via annexin V and PI staining.

Notes: (A) SMMC-7721 cells were treated with 4 μg/mL (0.39 pmol/μL) DRz1 and DRz2, and kept in culture for 72 hours. Every 24 hours, ~10⁵ cells were analyzed with flow cytometry following annexin V-FITC/PI staining to evaluate changes in the apoptotic population. (B) Graphical representation of the flow cytometry cell death data in (A). Notice that DRz1 induces nearly twice as much cell death as DRz2 and that the late apoptotic and necrotic cell death induction remains stable for 48 hours, whereas the number of cells in the early apoptotic stages decreases.
Abbreviations: DRz, deoxyribozyme (DNAzyme); FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 8 Representative flow cytometric assessment of apoptosis via annexin V and PI staining.

Note: HepG2 and Huh7 cells were treated with 4 μg/mL (0.39 pmol/μL) DRz1 and DRz2 and kept in culture for 24 hours (~10⁵ cells were analyzed with flow cytometry).
Abbreviations: HepG2 and Huh7 cells were treated with 4 μg/mL (0.39 pmol/μL) DRz1 and DRz2 and kept in culture for 24 hours (~10⁵ cells were analyzed with flow cytometry).

Figure 9 Evaluation of pro-caspase expression. (A) Western blot analysis. SMMC-7721 cells were transfected with 4 μg/mL (0.39 pmol/μL) DRz1 or DRz2. Procapsase-3 and -9 levels were assessed after 24 hours’ incubation via western blotting. A representative blot of three independent experiments is shown (n = 3). Densitometry analysis showed that the protein signal densities in DRz1-treated cells were lower than in control cells (no treatment). (B) Effect of IGF-IIP3 DRzs on caspase-3 or -9 activity in SMMC-7721 cells. The cells were kept in culture, treated with IGF-IIP3 DRzs for 24 hours, and subsequently analyzed for caspase-3 or -9 activity (n ≥3). *P < 0.05; **P < 0.01; ***P < 0.001.

Abbreviations: DRz, deoxyribozyme (DNAzyme); IGF-IIP3, insulin-like growth factor II promoter 3.