Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells
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Toxicol Res. https://doi.org/10.1007/s43188-020-00067-w
Toxicological Research
ORIGINAL ARTICLE
Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells Takamichi Ojima1 · Masashi Kawami1 · Ryoko Yumoto1 · Mikihisa Takano1 Received: 14 July 2020 / Revised: 21 August 2020 / Accepted: 23 September 2020 © Korean Society of Toxicology 2020
Abstract Epithelial-mesenchymal transition (EMT), a biological process through which epithelial cells transdifferentiate into mesenchymal cells, is involved in several pathological events, such as cancer progression and organ fibrosis. So far, we have found that methotrexate (MTX), an anticancer drug, induced EMT in the human A549 alveolar adenocarcinoma cell line. However, the relationship between EMT and the cytotoxicity induced by MTX remains unclear. In this study, we compared the processes of MTX-induced EMT and apoptosis in A549 cells. Q-VD-Oph, a caspase inhibitor, suppressed MTX-induced apoptosis, but not the increase in mRNA expression of α-smooth muscle actin (SMA), a representative EMT marker. In addition, SB431542, an EMT inhibitor, did not inhibit MTX-induced apoptosis. By using isolated clonal cells from wild-type A549 cells, the induction of EMT and apoptosis by MTX in each clone was analyzed, and no significant correlation was observed between the MTX-induced increase in α-SMA mRNA expression and the proportion of cells undergoing apoptosis. Furthermore, the increase in the mRNA expression of α-SMA was well correlated with cyclin-dependent kinase inhibitor 1A, a cell cycle arrest marker, but not with BCL-2 binding component 3 and Fas cell surface death receptor, which are both pro-apoptotic factors, indicating that the MTX-induced EMT may be related to cell cycle arrest, but not to apoptosis. These findings suggested that different mechanisms were involved in the MTX-induced EMT and apoptosis. Keywords Apoptosis · Epithelial-mesenchymal transition · Methotrexate · p21 · α-Smooth muscle actin
Introduction Epithelial-mesenchymal transition (EMT) is a cellular process that transiently places epithelial cells into a mesenchymal cell type. During EMT, epithelial cells lose polarity and cobblestone morphology, but acquire a spindle-shaped morphology with the upregulation of mesenchymal markers, including fibronectin, α-smooth muscle actin (SMA), and vimentin [1]. In general, there are three types of EMT, depending on to the biological context in which they occur [2]. In brief, type I EMT is observed during embryonic development, type II EMT occurs during wound healing and Takamichi Ojima and Masashi Kawami have contributed equally to this manuscript. * Mikihisa Takano takanom@hiroshima‑u.ac.jp 1
Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima 734‑8553, Japan
tissue regeneration, and type III EMT occurs during cancer progression, such as metastasis and invasion. As some serious pathological probl
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