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Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells

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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.

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Acknowledgements

This work was supported in part by the Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JP18H02586, JP18K06749, and JP19K16447).

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Author notes

  1. Takamichi Ojima and Masashi Kawami have contributed equally to this manuscript.

Authors and Affiliations

  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

    Takamichi Ojima, Masashi Kawami, Ryoko Yumoto & Mikihisa Takano

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  1. Takamichi Ojima
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  2. Masashi Kawami
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  4. Mikihisa Takano
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Correspondence to Mikihisa Takano.

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Ojima, T., Kawami, M., Yumoto, R. et al. Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells. Toxicol Res. 37, 293–300 (2021). https://doi.org/10.1007/s43188-020-00067-w

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  • DOI: https://doi.org/10.1007/s43188-020-00067-w

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