Kurimoto Ryota, Ebata Takahiro, Iwasawa Shunichiro, Ishiwata Tsukasa, Tada Yuji, Tatsumi Koichiro, Takiguchi Yuichi
Department of Medical Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.
Department of Respirology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.
Oncol Lett. 2017 Jul;14(1):944-950. doi: 10.3892/ol.2017.6188. Epub 2017 May 17.
The epithelial-to-mesenchymal transition (EMT) in cancer is associated with invasion, metastasis and chemoresistance. Recent studies have revealed the increased expression of programmed death-ligand 1 (PD-L1) in cells undergoing EMT. The underlying mechanism of EMT involves transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2). Pirfenidone and the known EMT-suppressor nintedanib suppress pulmonary fibrosis partially through suppression of TGF-β. The present study aimed to determine whether pirfenidone has the potential to induce EMT-reversion, using nintedanib as a reference. The human lung adenocarcinoma cell lines A-549, HCC-827, and PC-9 were treated with TGF-β and FGF-2 to induce EMT. The EMT-induced cells were further treated with pirfenidone or nintedanib. Phenotypic alterations associated with EMT were assessed by examining the following: i) The expression levels of E-cadherin, vimentin, fibronectin and slug, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescent immunohistochemistry; ii) cell motility via wound-healing assays; and iii) the expression of PD-L1 using RT-qPCR. The combination of TGF-β and FGF-2 successfully induced EMT in all three cell lines, characterized by a significant reduction in E-cadherin expression in the A-549 and HCC-827 cells, increased expression levels of vimentin, fibronectin, slug and PD-L1, and increased cell motility in all three cell lines. Pirfenidone and nintedanib reverted all of these phenotypes, with the exception of unaltered E-cadherin expression in all three cell lines, and inconsistent expression of vimentin in the HCC-827 and PC-9 cells. Thus, pirfenidone and nintedanib have the ability to induce EMT-reversion in human lung adenocarcinoma.
癌症中的上皮-间质转化（EMT）与侵袭、转移和化疗耐药相关。最近的研究表明，经历EMT的细胞中程序性死亡配体1（PD-L1）的表达增加。EMT的潜在机制涉及转化生长因子-β（TGF-β）和成纤维细胞生长因子-2（FGF-2）。吡非尼酮和已知的EMT抑制剂尼达尼布通过抑制TGF-β部分抑制肺纤维化。本研究旨在以尼达尼布为参照，确定吡非尼酮是否具有诱导EMT逆转的潜力。用人肺腺癌细胞系A-549、HCC-827和PC-9用TGF-β和FGF-2处理以诱导EMT。对诱导EMT的细胞进一步用吡非尼酮或尼达尼布处理。通过检查以下方面评估与EMT相关的表型改变：i）使用逆转录定量聚合酶链反应（RT-qPCR）和荧光免疫组织化学检测E-钙黏蛋白、波形蛋白、纤连蛋白和蛞蝓蛋白的表达水平；ii）通过伤口愈合试验检测细胞运动性；iii）使用RT-qPCR检测PD-L1的表达。TGF-β和FGF-2的组合成功在所有三种细胞系中诱导了EMT，其特征是A-549和HCC-827细胞中E-钙黏蛋白表达显著降低，波形蛋白、纤连蛋白、蛞蝓蛋白和PD-L1表达水平增加，并且所有三种细胞系中的细胞运动性增加。吡非尼酮和尼达尼布逆转了所有这些表型，但所有三种细胞系中E-钙黏蛋白表达未改变，以及HCC-827和PC-9细胞中波形蛋白表达不一致除外。因此，吡非尼酮和尼达尼布具有诱导人肺腺癌EMT逆转的能力。
