Denatonium inhibits growth and induces apoptosis of airway epithelial cells through mitochondrial signaling pathways

Abstract

Background: Denatonium, a widely used bitter agonist, activates bitter taste receptors on many cell types and plays important roles in chemical release, ciliary beating and smooth muscle relaxation through intracellular Ca(2+)-dependent pathways. However, the effects of denatonium on the proliferation of airway epithelial cells and on the integrity of cellular components such as mitochondria have not been studied. In this study, we hypothesize that denatonium might induce airway epithelial cell injury by damaging mitochondria.

Methods: Bright-field microscopy, cell counting kit-8 (CCK-8) assay and flow cytometry analysis were used to examine cellular morphology, proliferation and cell cycle, respectively. Transmission electron microscopy (TEM) was used to examine mitochondrial integrity. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and protein expression, respectively.

Results: For airway epithelial cells, we observed that denatonium significantly effects cellular morphology, decreases cell proliferation and reduces the number of cells in S phase in a dose-dependent manner. TEM analysis demonstrated that denatonium causes large amplitude swelling of mitochondria, which was confirmed by the loss of mitochondrial membrane potential, the down-regulation of Bcl-2 protein and the subsequent enhancement of the mitochondrial release of cytochrome c and Smac/DIABLO after denatonium treatment.

Conclusions: In this study, we demonstrated for the first time that denatonium damages mitochondria and thus induces apoptosis in airway epithelial cells.

Figure 1

Functional expression of bitter taste receptors and their downstream signaling effectors. A) Immunohistochemistry images showed that bitter taste receptor TAS2R10 and its downstream signaling effectors GNAT3 (B) and TRPM5 (C) were highly expressed on airway epithelial cells in mice. Scale bar: 100 μm. D) Western blot showed that bitter taste receptor TAS2R4 and GNAT3 were expressed on A549 cells. E) In vitro experiments showed that 1 mM denatonium triggered Ca²⁺ oscillations in A549 human epithelial cells. A549 cells were stained with Fluo-4 to visualize intracellular free Ca²⁺. Red arrow points to the region of interest (ROI) in an A549 cell. F) Ca²⁺ oscillations started immediately after denatonium application and lasted for a few cell cycles.

Figure 2

Denatonium inhibits A549 and 16HBE cell proliferation and induces cell morphological changes. A) Bright-field images of cultured A549 cells showed that treatment with denatonium for 72 h induced cell morphological changes. B) Bright-field images of cultured 16HBE cells showed that treatment with denatonium for 24 h induced cellular morphology changes. C) Denatonium markedly inhibited the growth of A549 cells in a dose-dependent manner. D) Denatonium markedly inhibited the growth of 16HBE cells in a dose-dependent manner. One representative experiment with n = 3 is shown. The error bars represent mean values ± SEM. ***indicates significant difference at p < 0.001 versus control.

Figure 3

Flow cytometric analysis of apoptosis induction and cell cycle distribution in A549 and 16HBE cells. A) A549 cells were treated with denatonium (1 mM or 2 mM) for 72 h, stained with FITC-annexin V/PI and then analyzed by flow cytometry. The right panel shows the apoptosis rates of the cells of the various groups. Flow cytometry was also used to analyze DNA at the G1, S, and G2 phases of the cell cycle. B) 16HBE cells were treated with denatonium (0.5 mM, 1 mM or 2 mM) for 24 h. Then the number of apoptotic cells and the number of cells in different stages of the cell cycle were detected by flow cytometry. Data are representative of three similar experiments. **indicates significant difference at p < 0.01 versus control and ***indicates significant difference at p < 0.001 versus control.

Figure 4

Denatonium reduces mitochondrial membrane potential. A) The TEM microphotographs show the ultrastructure of control cells (a1,a2) and denatonium (2 mM, 72 h) treated A549 cells (b1, b2, b3). Swollen mitochondria (***) were observed in treated cells (b2, b3). B) A549 cells were stained with the cationic lipophilic dye, JC-1 and analyzed by FACS after treatment with denatonium for 72 h. C) 16HBE cells were stained with the cationic lipophilic dye JC-1 and analyzed by FACS after treatment with denatonium for 24 h. The figures are representative profiles of at least three experiments. The histogram shows the quantification of mitochondrial membrane potential. (**p < 0.01, ***p < 0.001).

Figure 5

Denatonium down-regulates the expression of the anti-apoptotic protein Bcl-2 and enhances the release of cytochrome c and Smac/DIABLO from mitochondria in 16HBE cells. A) The expression of the anti-apoptotic protein Bcl-2 was significantly reduced in 16HBE cells after treatment with denatonium for 24 h. B) In 16HBE cells treated with denatonium for 24 h, the cytochrome c and Smac/DIABLO levels in mitochondria were significantly reduced, whereas, the cytochrome c and Smac/DIABLO levels in cytosol were significantly increased (C). The figures are representative profiles of at least three experiments. (***p < 0.001).