Interleukin 7 signaling prevents apoptosis by regulating bcl-2 and bax via the p53 pathway in human non-small cell lung cancer cells

Abstract

Interleukin 7/Interleukin 7 receptor (IL-7/IL-7R) signaling induces the upregulation of cyclin D1 to promote cell proliferation in lung cancer, but its role in preventing the apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. To study the role of IL-7 in lung cancer cell apoptosis, normal HBE cells as well as A549 and H1299 NSCLC cells were examined using flow cytometry. The results showed that the activation of IL-7R by its specific ligand, exogenous interleukin-7, was associated with a significant decline in apoptotic cells. Western blot and real-time PCR assays indicated that the activation of IL-7/IL-7R significantly upregulated anti-apoptotic bcl-2 and downregulated pro-apoptotic bax and p53 at both protein and mRNA levels. The knockdown of IL-7R through small interfering RNAs significantly attenuated these effects of exogenous IL-7. However, there was no significant anti-apoptotic effect in H1299 (p53-) cells. Furthermore, the inhibition of p53 significantly abolished the effects of IL-7/IL-7R on lung cancer cell apoptosis. These results strongly suggest that IL-7/IL-7R prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax, potentially via the p53 pathway in A549 and HBE cells.

Keywords: Interleukin 7; NSCLC; apoptosis; interleukin 7 receptor; p53.

Figure 1

Expression of IL-7R in NSCLC cell lines. A, B: The protein and mRNA expression level of IL-7R was analyzed by western blotting and real-time PCR in HBE, A549, and H1299 cell lines. C, D: Western blot analysis and real-time PCR analysis of IL-7R depletion efficiency in HBE, A549, and H1299 cells at the protein and transcriptional level.

Figure 2

Effect of IL-7/IL-7R on apoptosis in A549 and HBE cells. A, B: A549 and HBE cells were treated with IL-7 (20 ng/ml) for 24 h after transfection with IL-7R siRNA (siIL-7R). C: After treatment, apoptosis was estimated using Annexin V staining. Each bar represents the mean±SD of three independent experiments. *p<0.05 or **p<0.01, compared with negative control cells.

Figure 3

Effect of IL-7/IL-7R on the expression of bcl-2 and bax in A549 and HBE cells. A, B: A549 and HBE cells were treated with IL-7 (20 ng/ml) for 24 h after transfection with IL-7R siRNA (siIL-7R). After treatment, the expression of bcl-2 and bax at both protein and mRNA was estimated using western blotting and real-time PCR. Each bar represents the mean±SD of three independent experiments. *p<0.05 or **p<0.01, compared with negative control cells.

Figure 4

Effect of IL-7/IL-7R on the expression of p53 in A549 and HBE cells. A, B: A549 and HBE cells were treated with IL-7 (20 ng/ml) for 24 h after transfection with IL-7R siRNA (siIL-7R). After treatment, the expression of p53 at both protein and mRNA was estimated using western blotting and real-time PCR. Each bar represents the mean±SD of three independent experiments. *p<0.05 or **p<0.01, compared with negative control cells.

Figure 5

Effect of IL-7/IL-7R on apoptosis in A549 and HBE cells after inhibiting p53 activation. A: A549 and HBE cells were divided into five groups as described in Methods section. B: Apoptosis was estimated using the Annexin V staining. Each bar represents the percentage of Annexin positive cells of three independent experiments. *p<0.05, or **p<0.01, compared with negative control cells.

Figure 6

Effect of IL-7/IL-7R on the expression of bcl-2 and bax in A549 and HBE cells after inhibiting p53 activation. A: A549 and HBE cells were divided into five groups. The expression of IL-7R, p53, bcl-2 and bax protein level was estimated using western blotting. B: Densitometry analysis of western blot experiments. Data were normalized against GAPDH and each bar represents the mean±SD of three independent experiments. *p<0.05 or **p<0.01, compared with negative control cells.

Figure 7

Effect of IL-7/IL-7R on apoptosis in H1299 cells. A: H1299 cells were treated with IL-7 (20 ng/ml) for 24 h after transfection with IL-7R siRNA (siIL-7R). After treatment, apoptosis was estimated using Annexin V staining. B: Each bar represents the percentage of Annexin-positive cells from three independent experiments. C: The expression of bcl-2 and bax protein was estimated using western blotting. D: Densitometry analysis of western blot experiments. Data were normalized against GAPDH and each bar represents the mean±SD of three independent experiments.