Reversal effect of adenovirus-mediated human interleukin 24 transfection on the cisplatin resistance of A549/DDP lung cancer cells

Abstract

Interleukin-24 (IL-24) is a tumor-suppressor gene that has been documented in human melanoma cells. IL-24 has marked antitumor activities on various types of human cancer, but its underlying mechanism remains unclear. In the present, we investigated the effects of human IL-24 (hIL-24) on the chemotherapy resistance of lung cancer cells. The cisplatin (DDP)-resistant lung carcinoma cell line A549/DDP was subjected to adenovirus-mediated transfection with the human IL-24 gene (Ad-hIL-24). The growth-inhibitory and apoptotic effects of Ad-hIL-24 on A549/DDP cells were observed, and the expression levels of AKT, phosphorylated-AKT (p-AKT) and P-glycoprotein (P-gp) were detected. Ad-hIL-24 significantly decreased the levels of p-AKT and P-gp, and effectively inhibited A549/DDP cell growth. Furthermore, A549/DDP cells exhibited a significantly increased rate of apoptosis, as well as G2/M-phase arrest, following transfection with Ad-hIL-24, and these effects were increased in cells treated with Ad-IL-24 combined with DDP when compared with those treated with Ad-hIL-24 or DDP alone. These results suggest that hIL-24 can reverse the DDP resistance of lung cancer cells, and that the associated mechanism involves the induction of apoptosis and G2/M-phase arrest through the phosphoinositide3-kinase (PI3K)/AKT signaling pathway, as well as a decrease in drug resistance through P-gp expression.

Figure 1.

Adenovirus-mediated human interleukin 24 gene (Ad-hIL-24) infects A549/DDP cells. A549/DDP cells were infected with Ad-hIL-24 and Ad-GFP, and incubated for 24 or 48 h. Ad-GFP served as an internal control. The treated cells were fixed with paraformaldehyde and stained with a fluorescent antibody. Saline (DDP solvent) served as the blank controls. (A) Green fluorescent protein (GFP) expression was observed under fluorescence microscopy. Scale bar, 25 µm. Magnification, ×100. Upper panel, fluorescence microscopy images: a, saline; b, cells infected with Ad-hIL-24 for 24 h; c, cells infected with Ad-hIL-24 for 48 h; Lower panel, light-field images: d, saline; e, cells infected with Ad-hIL-24 for 24 h; f, cells infected with Ad-hIL-24 for 48 h. (B) GFP cells were counted under fluorescence microscopy, and the infected rates were assessed.

Figure 2.

hIL-24 expression and the growth curve of A549/DDP cells infected with Ad-hIL-24. A549/DDP cells were treated with DDP, Ad-hIL-24, and Ad-hIL-24 plus DDP, and then incubated for 48 h. Saline (DDP solvent) served as blank controls and Ad-GFP served as the vector control. (A) hIL-24 expression was detected in the infected cells using western-blotting. (B) The media of the infected cells were harvested, and hIL-24 was detected using ELISA. (C) The cell viability of the infected cells was detected using CCK-8 assay. (D) The inhibitory rates were assessed. Data are presented as the means ± SD from three independent experiments statistically using the Students t test (*P<0.05).

Figure 3.

P-pg, AKT and p-AKT expression in A549/DDP cells transfected with Ad-hIL-24. A549/DDP cells were treated with DDP, Ad-hIL-24, and Ad-hIL-24 plus DDP, and incubated for 48 h. Ad-GFP served as the vector control. Total proteins in the treated cells were extracted. The protein samples were subjected to western-blotting. (A) P-pg, AKT and p-AKT expression was detected in the treated cells with DDP, Ad-hIL-24 or Ad-hIL-24 plus DDP. (B) The relative photographic density was quantitated. GAPDH was used as an internal control to ascertain basal level expression and equal protein loading. The abundance ratio relative to GAPDH was counted. Data are presented as the mean ± SD from three independent experiments statistically using the Students t test (*P<0.05).

Figure 4.

Morphological analysis of Ad-hIL-24-mediated A549/DDP cell apoptosis. A549/DDP cells were treated with DDP, Ad-hIL-24, and Ad-hIL-24 plus DDP, and incubated for 48 h. The treated cells were fixed with paraformaldehyde and then stained with Hoechst 33342. Saline served as the blank control, and Ad-GFP as the vector control. (A) The apoptotic cells were observed under fluorescence microscopy: a, saline; b, Ad-vector; c, Ad-hIL-24; d, DDP; e, Ad-hIL-24 plus DDP. Scale bar, 50 µm. Magnification, ×400. (B) The rates of apoptotic cells were counted. Data are presented as the means ± SD from three independent experiments statistically using the Students t test (*P<0.05).

Figure 5.

Flow cytometric analysis of Ad-hIL-24-mediated A549/DDP cell apoptosis. A549/DDP cells were treated with DDP, Ad-hIL-24, and Ad-hIL-24 plus DDP, and incubated for 48 h. Then the treated cells were fixed with ethanol and stained with Annexin V-FITC. Saline served as the blank control, and Ad-GFP as the vector control. (A) The apoptotic cells were analyzed with flow cytometry: a, saline; b, Ad-vector; c, Ad-hIL-24; d, DDP; e, Ad-hIL-24 plus DDP. Scale bar, 50 µm. Magnification ×400. (B) The rates of apoptotic cells were counted. Data are presented as the means ± SD from three independent experiments statistically using the Students t test (*P<0.05).

Figure 6.

Cell cycle analysis of A549/DDP cells treated with Ad-hIL-24. A549/DDP cells were treated with DDP, Ad-hIL-24 and Ad-hIL-24 plus DDP, and then incubated for 48 h. The cells were harvested for flow cytometry. Ad-GFP was used as the vector control. (A) The cell cycle of treated cells was analyzed using flow cytometry. (B) Cells at the sub/G1, /G1, S and G2/M stages were assessed. Data are presented as the means ± SD from three independent experiments statistically using the Students t test (*P<0.05).