Fig 1.
Characterization of SLN and SLN-STAT3 decoy ODN complexes.
(A) TEM image of bare SLN (scale bar, 100 nm). (B) TEM image of SLN–STAT3 decoy ODN complexes (scale bar, 100 nm). (C) Gel retardation assay of SLN-decoy ODN complexes. A total of 0.5 μg of decoy ODN per hole was mixed with SLN. The weight ratios of SLN to ODN from left to right are as follows, lane 1: naked ODN control; lane 2: SLN:ODN = 5:1; lane 3: SLN:ODN = 10:1; lane 4: SLN:OND = 20:1; lane 5: SLN:ODN = 50:1; lane 6: SLN:ODN = 100:1.
Fig 2.
Cell viability of ODN formulations by MTT assay.
SKOV3 (A) and A2780 (B) cells were treated with negative agents (negative control, NC), SLN-decoy ODN complexes, SLN-scrambled ODN complexes, naked ODN, Lipofectamine 2000-decoy ODN complexes (Lipo-decoy ODN), SLN and Lipofectamine 2000 at different concentrations (12.5, 25 and 50 nmol/L). At 48 hours after treatment, an MTT assay was performed. SKOV3 (C) and A2780 (D) cells were transfected with serial concentrations of SLN-decoy ODN complexes (12.5, 25 and 50 nmol/L), and cell viability was assessed at 24, 48 and 72 hours. The data are expressed as the means ± SD of three independent experiments, * P < 0.05, ** P < 0.01 compared with NC.
Fig 3.
Uptake of SLN-decoy ODN complexes in ovarian cancer cells.
(A) Fluorescent micrographs of SKOV3 and A2780 cells transfected with naked decoy ODN, SLN-decoy ODN and Lipofectamine 2000-decoy ODN at a concentration of 25 nmol/L at 24 and 48 hours post transfection. (B) Flow cytometry analysis of cellular uptake of SKOV3 and A2780 cells transfected with SLN-decoy ODN complexes at concentrations of 0, 12.5, 25 and 50 nmol/L at 48 hours post transfection. (C) Flow cytometry analysis of cellular uptake of SKOV3 and A2780 cells transfected with naked decoy ODN, SLN-decoy ODN and Lipofectamine 2000-decoy ODN complexes at a concentration of 25 nmol/L at 48 hours post transfection. (D) and (E) Western blot analysis of STAT3 and p-STAT3 (Ser727, Tyr705) protein expression. The relative protein levels were expressed as the ratio of protein of interest/β-actin. The data are expressed as the means ± SD of three independent experiments, ** P < 0.01, # P > 0.05.
Fig 4.
SLN-STAT3 decoy ODN complexes induce apoptosis of ovarian cancer cells.
(A) SLN-decoy ODN induced a significant increase in apoptosis in SKOV3 and A2780 cells. After 48 hours of transfection, cell apoptosis was detected by annexin V-FITC/PI double-staining assay with flow cytometry. (B) Western blot assays showed that SLN-decoy ODN led to distinct up-regulation of Bax and cleaved caspase 3 and down-regulation of Bcl-2, pro-caspase 3 and Survivin. The data are expressed as the means ± SD of three independent experiments, ** P < 0.01 compared with NC.
Fig 5.
SLN-STAT3 decoy ODN complexes induce autophagy of ovarian cancer cells.
(A) Transmission electron micrographs showing autophagic vesicles (AVs; white arrows) after transfection with the SLN-STAT3 decoy ODN in A2780 (a) and SKOV3 (b) cells. (c) Double-membrane AVs. (d) AVs containing cytosolic components. (B) Acridine orange staining at 48 hours after treatment in each group. (D) Western blot analysis showed that SLN-STAT3 decoy ODN reduced the expression of p-Akt, p-mTOR and increased the expression of LC3A-II, LC3B-II and Beclin-1.
Fig 6.
SLN-STAT3 decoy ODN complexes inhibit migration and invasion of ovarian cancer cells.
(A) SLN-decoy ODN exhibited slower wound recovery at 24 and 48 hours after wounding compared with the NC and scrambled ODN groups. (B) A transwell invasion assay showed that SLN-decoy ODN inhibited cell invasion. At 48 hours after transfection, cells were reseeded to the upper transwell chamber for 24 hours and stained with crystal violet. (C) Western blot analysis showed that SLN-decoy ODN increased E-cadherin expression and reduced Snail and MMP9 expression. The data are expressed as the means ± SD of three independent experiments, * P < 0.05, ** P < 0.01 compared with NC.