Fig 1.
Nucleolin expression in HCC cells.
(A) Nucleolin (green fluorescence) was present on the surface of SNU-761 cells based on fluorescence microscopy. (B−D) Flow cytometry analysis of cells immunostained with Cy-3 (1:400, 100 nM) showed that AS1411- and modified AS1411-aptamers had relatively higher affinity to SNU-761 (B), Huh-7 (C) and SNU3058 cells (D) compared to control.
Fig 2.
Anti-tumor effects of modified AS1411-aptamers on SNU-761 cells.
(A) Significant cytotoxic effects on SNU-761 cells were noted with modified AS1411-aptamer treatment under both normoxic and hypoxic conditions. (B) Cell cycle analysis revealed that modified AS1411-aptamer significantly decreased the proportion of S-phase cells as compared to control AS1411-aptamer, especially under hypoxic conditions (P = 0.009). The experiment was repeated three times. (C) In an in vivo xenograft model, modified AS1411-aptamers significantly suppressed HCC tumor volume by 53.2% compared to control aptamers at day 10 (*P<0.05).
Fig 3.
The mechanism of AS1411-or modified AS1411-aptamersregarding anti-tumor effects in SNU-761 cells.
(A) No significant induction of SNU-761 cell apoptosis was noted with AS1411- or modified AS1411-aptamers. Immunoblot analysis was performed using anti-caspase 9, anti-caspase 8, anti-caspase 7 and anti-actin antibodies. (B) No significant changes in phosphorylated Akt (P’-Akt) and phosphorylated ERK1/2 (P’-ERK1/2) expression were noted following AS1411- or modified AS1411-aptamer treatment. (C) The percentage of nuclei stained with NK-κB (p65) was not significantly decreased followingAS1411- or modified AS1411-aptamer treatment (all P>0.05).
Fig 4.
AS1411-aptamer suppresses hepatocellular carcinoma by up-regulating galectin-14.
(A) DNA microarray results (volume plot of expression level, hierarchical clustering analysis) (B) Induction of LGALS-14 by the AS1411-aptamer was confirmed by RT-PCR under normoxic (20% O2 and 5% CO2 at 37°C) and hypoxic conditions (1% O2, 5% CO2, and 94% N2 at 37°C). Galectin-14 protein expression was also enhanced by both AS1411- and modified AS1411-aptamers based on immunoblot analysis. (C) Decreased galectin-14 expression by siRNA enhanced HCC proliferation compared to control siRNA, particularly with treatment of the modified AS1411-aptamer.