Fig 1.
Binding affinity of AS1411 towards HCC cell lines and tissues.
(a) Nucleolin is detected on cell surface of Huh7 (left) but not on Hu767 (primary hepatocyte, right). 1 million cells of each cell line were incubated with 2ug/ml Nucleolin Antibody and 1ug/ml secondary antibody of mouse-FITC for 1h at 4°C, which was followed by flow cytometry. (b) AS1411 can stain membrane nucleolin on Huh7 and HCO2. Live cells were incubated with 200 nM of AS1411-FITC for 30 min at room temperature and examined under a confocal microscope. (c) AS1411 stains membrane nucleolin in tumor tissues, but only nuclear nucleolin of non-tumor tissue. Paraffin embedded tissue samples from patients were incubated with 200 nM of AS1411-Biotin for 1 h at room temperature. Signal was developed using HRP-conjugated streptavidin and DAB peroxidase substrate (Dako).
Fig 2.
Growth inhibition of Huh7 by treatment with AS1411.
(a) Huh7 was incubated with AS1411 at concentrations from 1 μM to 10 μM for 2 days at 37°C then subjected to MTS assay. (b) Cell cycle arrest was tested by flow cytometry of PI-stained cells. FLOW data indicates cell cycle arrest in AS1411 treated cells and cell cycle percentage (c) Analysis of the flow cytometry data showed that a much higher proportion of treated cells were arrested at S and G2/M phases, when compared to untreated cells.
Fig 3.
Preparation and affinity of the AS1411-Dox adduct.
(a) Schematic description of the preparation of AS1411-Dox adduct by incubating Dox (500 μM), AS1411 (20 μM), and formaldehyde (0.37%) at 10°C overnight, followed by purification by reverse phase HPLC. (b) AS1411-Dox adduct remained its binding affinity towards Huh7 established by the equilibrium dissociation constant (Kd). (c) UV-Vis spectrum displaying the absorbance of purified AS1411-Dox adduct, with the characteristic absorbance peak of Dox at 490 nm. (d) Flow cytometry results indicating specific recognition ability of AS1411 and AS1411-Dox adduct to Huh7 HCC cells, note the non-specific DNA library does not detect Huh7 cells (e) Random library and control DNA aptamers and the control DNA-Dox adduct in contrast were not able to specifically bind to Huh7 cells.
Fig 4.
Intracellular staining of free Dox and AS1411-Dox with Control DNA-Dox adduct.
(a) Confocal laser scanning microscopy images displaying uptake of Dox into Huh7 cells treated with 2 μM of free Dox, AS1411-Dox adduct, or control DNA-Dox adduct, respectively. After treatment cells were stained with Hoechst 33342 (scale bar: 50 μm). Free Dox passed into any cells; however only Dox attached to AS1411 and not the control aptamer was selectively delivered to Huh7 cells due to the binding affinity of AS1411. (b) Cells were treated for 1h with either; 2 μM free Dox, AS1411, AS1411-Dox adduct or control-DNA adduct., prior to MTS assay. The results indicate the specific and potent cytotoxicity in target Huh7 cells induced by AS1411-Dox adduct.
Fig 5.
In vivo evaluation of AS1411-Dox adduct in Huh7 tumor xenograft mouse model.
(a) Mice treated with free Dox or AS1411-Dox adduct had significant lower rates of tumor growth than those treated with AS1411 or control DNA-Dox adduct. (b, c) Side effects and cytotoxicity of each drug was illustrated by total body weight lost at the end of treatment (b) or activation of Caspase-3 in tumors, heart and kidney tissues (c). Mice treated with Dox lost 21.7% of their total body weight while with AS1411-Dox only 6.6% (P<0.001). Cleaved Caspase-3, which indicates the activation of Caspase-3 and induced apoptosis, was only identified in the heart and kidney of free Dox-treated mice, suggesting the specific cytotoxicity and reduced side effects of AS1411-Dox adduct compared to Dox.