Regulation of Epithelial-Mesenchymal Transition of A549 Cells by Prostaglandin D2

Abstract

Background/aims: Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D₂ (PGD₂) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD₂ during the process of epithelial-mesenchymal transition (EMT) in A549 cells.

Methods: A549 cells were stimulated with PGD₂ and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-β1 and TGFβ type I receptor (TGFβRI), and protein expression was assessed by immunoblotting and immunofluorescence.

Results: Here, we found that stimulation of A549 cells with PGD₂ resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD₂ resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD₂. Slug expression was markedly upregulated by stimulating A549 cells with PGD₂, and stimulation of A549 cells with PGD₂ significantly enhanced TGF-β1 expression, and silencing of TGF-β1 significantly blocked PGD₂-induced EMT and Smad2 phosphorylation. In addition, PGD₂-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFβRI. PGD₂-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type.

Conclusion: Given these results, we suggest that tumor microenvironmental factors such as PGD₂, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

Keywords: PGD2; EMT; Lung cancer; MET; TGF-β1.