Bcl-XL and STAT3 mediate malignant actions of gamma-irradiation in lung cancer cells

Abstract

Previous reports suggest that, in addition to its therapeutic effects, ionizing radiation (IR) increases the invasiveness of surviving cancer cells. Here, we demonstrate that this activity of IR in lung cancer cells is mediated by a signaling pathway involving p38 kinase, phosphoinositide 3-kinase, Akt, and matrix metalloproteinase (MMP-2). The invasion-promoting doses of IR also increased and reduced the levels of vimentin and E-cadherin, respectively, both of which are markers for the epithelial-mesenchymal transition (EMT). Interestingly, all of these malignant actions of IR were mimicked by the overexpression of Bcl-X(L), a pro-survival member of the Bcl-2 family, in lung cancer cells. Moreover, both RNA and protein levels of Bcl-X(L) were elevated upon irradiation of the cells, and the prevention of this event using small-interfering RNAs of Bcl-X(L) reduced the ability of IR to promote invasion signals and EMT-associated events. This suggests that Bcl-X(L) functions as a signaling mediator of the malignant effects of IR. It was also demonstrated that IR enhances signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the reduction of STAT3 levels via RNA interference prevented IR-induced Bcl-X(L) accumulation, and thus all the tested Bcl-X(L)-dependent events. Overall, the data suggest that IR induces Bcl-X(L) accumulation via STAT3, which then promotes cancer cell invasion and EMT-associated markers. Our findings demonstrate a novel function of Bcl-X(L) in cancer, and also advance our understanding of the malignant actions of IR significantly.

Figure 1

Ionizing radiation (IR) promotes malignant behaviors in A549 cells. (a) A549 cells were irradiated with 10 Gy of γ‐rays. After 24 h of incubation, cell lysates and conditioned media were prepared. Cell lysates were analyzed for p‐p38, p38, p‐Akt, Akt, vimentin, and E‐cadherin levels by western blotting using β‐actin as a loading control. The conditioned media were analyzed to compare the levels of secreted matrix metalloproteinase (MMP‐2). (b) A549 cells were incubated with or without inhibitors for phosphoinositide 3‐kinase (PI3K) (LY294002, 5 μM), Akt (Akt inhibitor I, 5 μM), p38 kinase (SB202190, 5 μM), or MMP‐2 (OA‐Hy, 5 μM) for 30 min, and then irradiated. After 24 h of incubation, cellular invasiveness was compared on Matrigel‐coated polycarbonate filters. The experiment was repeated three times, and the means and SDs were determined. (c) The control and irradiated cells were immunostained to analyze vimentin and E‐cadherin. DAPI staining to detect nuclei was also carried out. Stained cells were visualized with a confocal microscope.

Figure 2

Bcl‐X_L is a common target of ionizing radiation (IR) in both A549 and H460 cells. A549 and H460 cells were exposed to sublethal doses of IR, 10 and 1 Gy, respectively. At the indicated times after irradiation, the levels of Bcl‐X_L, Bcl‐2, and Bak were compared via western blotting.

Figure 3

Bcl‐X_L overexpression mimics the effects of ionizing radiation (IR). (a) A549 cells were transiently transfected with empty pcDNA3 vectors or vectors containing Bcl‐X_L cDNA. The levels of the indicated components were compared via western blotting. (b) The control and Bcl‐X_L transfectants were incubated with or without indicated inhibitors for 30 min, then analyzed for their invasiveness. (c) The transfectants were immuno‐stained and analyzed for vimentin and E‐cadherin.

Figure 4

Bcl‐X_L mediates the malignant actions of ionizing radiation (IR). (a) The transfectants were irradiated (10 Gy), and incubated for 24 h. Cellular levels of Bcl‐X_L and cellular invasiveness were compared. (b) Control and Bcl‐X_L siRNAs were introduced into A549 cells. After 24 h of incubation, the cells were irradiated, then incubated for an additional 24 h. Levels of the indicated components were compared via western blotting. (c) Cellular invasiveness was analyzed.

Figure 5

Signal transducer and activator of trans‐cription 3 (STAT3) mediates ionizing radiation (IR)‐induced Bcl‐X_L accumulation. (a) A549 cells were irradiated. At the indicated times, the levels of Bcl‐X_L mRNA were compared via RT‐PCR, using GAPDH as an internal control. (b) The irradiated cells were analyzed for levels of STAT3 protein and its phosphorylation via western blotting. (c) Control and STAT3 siRNAs were introduced into A549 cells. After 24 h of incubation, the cells were irradiated and incubated for an additional 24 h. Levels of the indicated components were compare via western blotting. (d) Cellular invasiveness was compared.

Figure 6

Ionizing radiation (IR) promotes invasion in a transient mode. (a) Levels of Bcl‐X_L, matrix metalloproteinase‐2 (MMP‐2), vimentin, and E‐cadherin were analyzed by western blotting at the indicated times after irradiation. (b) Cellular invasiveness was compared at the times specified.

Figure 7

Role of Bcl‐X_L and signal transducer and activator of transcription 3 (STAT3) in H460 cells. (a) H460 cells were irradiated (1 Gy), and incubated for 24 h. Levels of the indicated components were analyzed by western blotting. (b) H460 cells were transiently transfected with the control and Bcl‐X_L expression vectors. Levels of the indicated components were compared. (c) Control and Bcl‐X_L siRNAs were introduced into H460 cells. After 24 h of incubation, the cells were irradiated, incubated for an additional 24 h, and analyzed for invasiveness. (d) Control and STAT3 siRNAs were introduced into H460 cells. After 24 h of incubation, the cells were irradiated, incubated for an additional 24 h, and subsequently analyzed for the relevant components by western blotting. (e) Cellular invasiveness was compared.