doi: 10.3967/bes2022.066
Exosomes from PM2.5-treated Human Bronchial Epithelial Cells Increase Lung Cancer Metastatic Potential
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Abstract:
Objective Fine particulate matter (PM2.5) is an air pollutant that has become of great concern in recent years. Numerous studies have found that PM2.5 may contribute to lung cancer, but the pathogenesis has not yet been fully elucidated. In this study, we explored the roles of exosomes from bronchial epithelial cells in PM2.5-promoted lung cancer metastasis. Methods Exosomes were isolated from cell supernatants. An animal model of lung metastasis (established by tail vein injection of A549-luc) and in vitro studies with lung cancer cell lines were used to investigate the effects of exosomes derived from PM2.5-treated human bronchial epithelial cells (PHBE-exo). Results The animal experiments revealed that PHBE-exo-treated mice showed stronger luciferase activity and a larger relative metastatic region in the lungs, thus indicating that PHBE-exo promoted the metastatic potential of lung cancer. Additionally, PHBE-exo promoted the migration, invasion and epithelial-to-mesenchymal transition of lung cancer cells, in a manner mediated by activation of c-Jun N-terminal kinase. Conclusion These results implied that PM2.5 may promote the development of lung cancer through exosomes derived from bronchial epithelial cells, thus providing a potential interventional target for lung cancer. These findings broadened our understanding of cancer-promoting mechanisms of environmental pollutants. -
Key words:
- Fine particulate matter /
- Exosome /
- Lung cancer /
- C-Jun N-terminal kinase /
- Bronchial epithelial cell
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Figure 1. Identification of exosomes derived from HBE cells (HBE-exo) and exosomes derived from PM2.5-treated HBE cells (PHBE-exo). (A) Transmission electron microscopy (TEM) pictures of HBE-exo and PHBE-exo. Scale bar: 100 μm. (B) Nanoparticle tracking analysis (NTA) tested the size distribution of HBE-exo and PHBE-exo. (C) Western blotting results of exosomal markers CD9 and CD81 of control cells, HBE-exo and PHBE-exo.
Figure 2. Exosomes derived from PM2.5-treated HBE cells promoted metastasis of lung cancer. (A) The procedure of animal experiment. (B&C) The representative images of the mice in each group (B) and total luciferase counts of each mouse (C) were assessed by biofluorescence image system. (D–F) Hematoxylin and Eosin (H&E) staining analysis of lung tissues. The representative histopathological pictures of each mice group (D): left panel, magnification ×10; right panel, magnification ×100. Red arrows: metastatic nodules. Scale bars: 200 μm. Metastatic nodule numbers (E) and relative metastatic regions (tumor region / tissue region) (F) of each mouse were calculated and shown as scatter graph with median, n ≥ 7.
S4. The effects of exosomes derived from PM2.5-treated HBE cells on the viability of lung cancer cells. Lung cancer cells were treated with PBS, exosomes derived from human bronchial epithelial cell (HBE-exo) and exosomes derived from human bronchial epithelial cells treated with PM2.5 (PHBE-exo). After 24 h, cell viabilities of lung cancer cells were assessed by cck8 kit (mean ± SD, n = 3).
Figure 3. Exosomes derived from PM2.5-treated HBE cells promoted the migration and invasion capacity of lung cancer cells. Lung cancer cells were treated with PBS, exosomes derived from HBE cells (HBE-exo) or exosomes derived from PM2.5-treated HBE cells (PHBE-exo). Representative figures of A549 (A) and H1975 (C) cells in transwell assays. Scale bar: 100 μm. A549 (B) and H1975 (D) cell numbers were quantified by image J (mean ± SD, n = 3). *P < 0.05, different from HBE-exo-treated group.
Figure 4. Exosomes derived from PM2.5-treated HBE cells changed the protein expression of epithelial-to-mesenchymal transition (EMT) markers of lung cancer cells. Lung cancer cells were treated with PBS, exosomes derived from HBE cells (HBE-exo) or exosomes derived from PM2.5-treated HBE cells (PHBE-exo). Protein expression of EMT markers in A549 (A&C) and H1975 (B&D) cells were quantified by western blotting (mean ± SD, n = 3). The intensities of protein bands were analyzed and normalized to GAPDH. *P < 0.05, different from HBE-exo-treated group.
Figure 5. Exosomes derived from PM2.5-treated HBE cells affected the activation of MAPKs. A549 cells were treated with PBS, exosomes derived from HBE cells (HBE-exo) or exosomes derived from PM2.5-treated HBE cells (PHBE-exo). Representative figures of western blotting (A) and the protein expression of JNK, P-JNK, ERK, P-ERK, P-38, and P-P38 were quantified by image J (B) (mean ± SD, n = 3). The intensities of protein bands were analyzed and normalized to GAPDH. *P < 0.05, different from HBE-exo treated group.
S5. Exosomes derived from PM2.5-treated HBE cells affected JNK signaling pathway in H1975 cells. Lung cancer cells H1975 were treated with PBS, exosomes derived from human bronchial epithelial cell (HBE-exo) and exosomes derived from human bronchial epithelial cell treated with PM2.5 (PHBE-exo). Protein expression of JNK and phosphorylated JNK in H1975 cells were quantified by western blotting (mean ± SD, n = 3). *P < 0.05, different from HBE-exo treated group.
S6. The JNK pathway activated by exosomes was effectively inhibited by SP600125. Lung cancer cells were treated with DMSO, exosomes derived from human bronchial epithelial cell treated with PM2.5 (PHBE-exo), PHBE-exo+SP600125. Protein expression of JNK and phosphorylated JNK in A549 (A&C) and H1975 (B&D) were quantified by western blotting (mean ± SD, n = 3). *P < 0.05, different from PHBE-exo treated group.
Figure 6. Inhibition of the JNK signaling pathway suppressed the migration and invasion capacity enhanced by exosomes derived from PM2.5-treated HBE cells. Lung cancer cells were treated with DMSO, exosomes derived from PM2.5-treated HBE cells (PHBE-exo), or SP600125+PHBE-exo. Representative figures of A549 (A) and H1975 (C) cells in transwell assays. Scale bar: 100 μm. Cell numbers of A549 (B) and H1975 (D) were quantified by image J (mean ± SD, n = 3). *P < 0.05, different from PHBE-exo-treated group.
Figure 7. Inhibition of the JNK signaling pathway suppressed EMT induced by exosomes derived from PM2.5-treated HBE. Lung cancer cells were treated with DMSO, exosomes derived from PM2.5-treated HBE cells (PHBE-exo), PHBE-exo+SP600125. A549 (A&C) and H1975 (B&D) cell protein expression of EMT markers were quantified by western blotting (mean ± SD, n = 3). The intensities of protein bands were analyzed and normalized to GAPDH. *P < 0.05, different from PHBE-exo-treated group.
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22070Supplementary Materials.pdf