DAP3 Is Involved in Modulation of Cellular Radiation Response by RIG-I-Like Receptor Agonist in Human Lung Adenocarcinoma Cells

Abstract

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) mediate anti-viral response through mitochondria. In addition, RLR activation induces anti-tumor effects on various cancers. We previously reported that the RLR agonist Poly(I:C)-HMW/LyoVec™ (Poly(I:C)) enhanced radiosensitivity and that cotreatment with Poly(I:C) and ionizing radiation (IR) more than additively increased cell death in lung adenocarcinoma cells, indicating that Poly(I:C) modulates the cellular radiation response. However, it remains unclear how mitochondria are involved in the modulation of this response. Here, we investigated the involvement of mitochondrial dynamics and mitochondrial ribosome protein death-associated protein 3 (DAP3) in the modulation of cellular radiation response by Poly(I:C) in A549 and H1299 human lung adenocarcinoma cell lines. Western blotting revealed that Poly(I:C) decreased the expression of mitochondrial dynamics-related proteins and DAP3. In addition, siRNA experiments showed that DAP3, and not mitochondrial dynamics, is involved in the resistance of lung adenocarcinoma cells to IR-induced cell death. Finally, we revealed that a more-than-additive effect of cotreatment with Poly(I:C) and IR on increasing cell death was diluted by DAP3-knockdown because of an increase in cell death induced by IR alone. Together, our findings suggest that RLR agonist Poly(I:C) modulates the cellular radiation response of lung adenocarcinoma cells by downregulating DAP3 expression.

Keywords: death-associated protein 3; ionizing radiation; lung adenocarcinoma; mitochondria; radiosensitivity; retinoic acid-inducible gene-I-like receptor.

Figure 1

Effect of Poly(I:C)-HMW/LyoVec™ (Poly(I:C)) and/or ionizing radiation (IR) on mitochondrial dynamics in A549 cells. (A,B) A549 cells were incubated with Poly(I:C). After incubation for 1 h, the cells were irradiated with 4 Gy. After culturing for 48 or 72 h, the cells were harvested for western blotting. (A) Representative images of immunoblots are shown. Actin was used as a loading control. (B) The relative values of Mfn1/actin, L-OPA1/actin and Drp1/actin ratio are presented as mean ± SD of three independent experiments. For the Drp1 proteins, both bands were quantified together. One sample t-test was performed using the GraphPad QuickCalcs. * p < 0.05, ** p < 0.01 versus control. (C) A549 cells cultured for 72 h in the presence of Poly(I:C) were harvested for mitochondrial morphology analysis using the MitoTracker^TM Green FM. (D) A549 cells transfected with control, Drp1, or Mfn1 siRNA were cultured for 72 h and harvested for mitochondrial morphology analysis. Scale bar = 20 μm.

Figure 2

Effects of Drp1-knockdown on IR-induced cell death in A549 cells. (A) A549 cells transfected with control or Drp1 siRNA were harvested, and the Drp1 protein expression was analyzed by western blotting. Representative images of immunoblots are shown. Actin was used as a loading control. The relative values of Drp1/actin ratio are presented. For the Drp1 proteins, both bands were quantified together. (B) Drp1-knockdown A549 cells were treated with 4 Gy. After culturing for 72 h, the cells were harvested for cell death analysis using annexin V-FITC/propidium iodide (PI) staining. Representative cytograms of annexin V/PI staining are shown. The inset numbers indicate the fractions of annexin V+/PI− or annexin V+/PI+ cells.

Figure 3

Death-associated protein 3 (DAP3) expression in human lung adenocarcinoma cells treated with Poly(I:C) and/or IR. (A,B) A549 and H1299 cells were incubated with Poly(I:C). After incubation for 1 h, the cells were irradiated with 4 Gy. After culturing for 72 h, the cells were harvested for western blotting. (A) Representative images of immunoblots are shown. Actin was used as a loading control. (B) The relative values of DAP3/actin ratio are presented as mean ± SD of three independent experiments. One sample t-test was performed using the GraphPad QuickCalcs. * p < 0.05, ** p < 0.01 versus control.

Figure 4

Effects of DAP3-knockdown on IR-induced cell death and radiosensitivity of human lung adenocarcinoma cells. (A) A549 and H1299 cells transfected with control or DAP3 siRNA were harvested, and DAP3 protein expression was analyzed by western blotting. Representative images of immunoblots are shown. Actin was used as a loading control. The relative values of DAP3/actin ratio are presented. (B) DAP3-knockdown A549 and H1299 cells were irradiated with 4 Gy. After culturing for 72 h, the cells were harvested for cell death analysis using annexin V-FITC/PI staining. Representative cytograms of annexin V/PI staining are shown. The inset numbers indicate the fractions of annexin V+/PI− or annexin V+/PI+ cells. (C) DAP3-knockdown A549 and H1299 cells were irradiated with X-rays. After a 20-h incubation, the cells were harvested and seeded in fresh media and further cultured until noticeable growth. The surviving fraction of A549 and H1299 cells is shown. Data are presented as mean ± SD of three independent experiments. ** p < 0.01 versus control siRNA.

Figure 5

DAP3 is involved in the more-than-additive effect of cotreatment with Poly(I:C) and IR on induction of cell death. DAP3-knockdown A549 (A) and H1299 cells (B) were incubated with Poly(I:C). After incubation for 1 h, the cells were irradiated with 4 Gy. After culturing for 72 h, the cells were harvested for cell death assay using annexin V/PI staining. (left) Representative cytograms of annexin V/PI staining are shown. The inset numbers indicate the fractions of annexin V+/PI− or annexin V+/PI+ cells. (right) The results are presented as the net increase in the fraction of annexin V+ cells (the sum of annexin V+/PI− cells and annexin V+/PI+ cells). Data are presented as mean ± SD of three independent experiments. * p < 0.05 versus control siRNA.

Figure 6

Effects of Poly(I:C) on the DAP3 mRNA expression and p-eIF-2α protein expression in A549 cells. (A) A549 cells were cultured with Poly(I:C) for 24–72 h and harvested for qRT-PCR. Data are presented as mean ± SD of three independent experiments. (B,C) A549 cells treated with Poly(I:C) were cultured for 24–72 h and harvested for western blotting. (B) Representative images of immunoblots are shown. Actin was used as the loading control. (C) The relative values of phosphorylated eukaryotic initiation factor-2α (p-eIF-2α)/eIF-2α and DAP3/actin ratio are presented as mean ± SD of three independent experiments. One sample t-test was performed using the GraphPad QuickCalcs. * p < 0.05, ** p < 0.01 versus control.