Gene and Cell Therapy Center for Vessel-Associated Disease, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine, Gyungnam, Republic of Korea.
Department of Pharmacy, Yeonsei University, Incheon, Republic of Korea.
Cell Physiol Biochem. 2022 Mar 25;56(2):89-104. doi: 10.33594/000000506.
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 D (PGD) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD during the process of epithelial-mesenchymal transition (EMT) in A549 cells.
A549 cells were stimulated with PGD 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.
Here, we found that stimulation of A549 cells with PGD resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD 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 PGD. Slug expression was markedly upregulated by stimulating A549 cells with PGD, and stimulation of A549 cells with PGD significantly enhanced TGF-β1 expression, and silencing of TGF-β1 significantly blocked PGD-induced EMT and Smad2 phosphorylation. In addition, PGD-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFβRI. PGD-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.
Given these results, we suggest that tumor microenvironmental factors such as PGD, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.
背景/目的：尽管在诊断和手术技术方面取得了重大进展，但肺癌仍然是全球最致命的恶性肿瘤之一。由于前列腺素如前列腺素 D（PGD）参与了多种病理生理过程，包括炎症和肿瘤发生，因此本研究旨在研究 PGD 在 A549 细胞上皮-间充质转化（EMT）过程中的作用。
用 PGD 刺激 A549 细胞，并用免疫印迹和免疫荧光法分析 EMT 标志物的表达。用定量实时聚合酶链反应（qPCR）评估 EMT 相关基因 Slug 的表达。分别在趋化性和 Matrigel 侵袭测定中测定 A549 细胞的迁移和侵袭能力。我们还使用受体抑制剂或 TGF-β1 和 TGFβ 型 I 受体（TGFβRI）的沉默来抑制 TGF/Smad 信号通路，并通过免疫印迹和免疫荧光法评估蛋白表达。
在这里，我们发现 PGD 刺激 A549 细胞在低血清条件下向间充质样表型发生形态变化。PGD 刺激 A549 细胞导致增殖显著减少，而侵袭和迁移增强。E-钙粘蛋白的表达明显下调，而 Vimentin 表达上调。PGD 刺激 A549 细胞后 Slug 表达明显上调，PGD 刺激 A549 细胞显著增强 TGF-β1 表达，TGF-β1 沉默显著阻断 PGD 诱导的 EMT 和 Smad2 磷酸化。此外，PGD 诱导的 Smad2 磷酸化和 EMT 均被 TGFβRI 的药理学抑制或沉默显著阻断。PGD 诱导的 Slug 表达和 EMT 在低营养和低血清条件下显著增强。最后，在正常培养条件下，间充质型 A549 细胞的后续培养将细胞表型逆转回上皮型。
鉴于这些结果，我们认为肿瘤微环境因素如 PGD、营养和生长因子可能是治疗转移性癌症的潜在治疗靶点。
