Connan D Masson
Characterisation of autophagy induction by the thiopurine drugs azathioprine, mercaptopurine and thioguanine in THP-1 macrophages
Masson, Connan D; Findlay-Greene, Fern; Henderson Sousa, Filipa; Henderson, Paul; Fraser, Jennifer A; Barlow, Peter G; Stevens, Craig
Authors
Dr Fern Findlay-Greene F.Findlay-Greene@napier.ac.uk
Research Technician
Filipa Henderson Sousa
Paul Henderson
Jennifer A Fraser
Prof Peter Barlow P.Barlow@napier.ac.uk
Professor
Dr Craig Stevens C.Stevens@napier.ac.uk
Associate Professor
Abstract
Purpose: Activating autophagy may be therapeutically beneficial, and we have previously shown that azathioprine (AZA), an immunomodulatory drug, induces autophagy. Here, we evaluated the induction of autophagy by the thiopurines AZA, mercaptopurine (6-MP) and thioguanine (6-TG) in THP-1 macrophages and investigated the mechanism of action in the context of this cellular process.
Methods: The cytotoxicity of thiopurines was evaluated using an LDH assay. Induction of endogenous LC3 by thiopurines was evaluated using immunostaining. To confirm autophagy activation by thiopurines, a GFP-RFP-LC3 reporter plasmid was used to monitor the maturation of autophagosomes to autolysosomes. Induction of apoptosis by thiopurines was evaluated using Annexin V/PI staining, and ER stress was assessed via RT‒PCR analysis of XBP1 splicing. To gain insight into the mechanism of action of thiopurines, mTORC1 activity and eIF2a-S51 phosphorylation were evaluated by immunoblotting.
Results: Thiopurines were not cytotoxic to cells and induced strong time- and concentration dependent autophagy. Thiopurines activate autophagy with complete progression through the pathway. Induction of autophagy by thiopurines occurred independently of apoptosis and ER stress. Immunoblotting revealed that AZA inhibited mTORC1 activity, and AZA and 6-TG increased eIF2a-S51 phosphorylation. In contrast, 6-MP had a minor effect on either signalling pathway.
Conclusion: Thiopurines are strong inducers of autophagy, and autophagy induction should be considered among the mechanisms responsible for patient response to thiopurines.
Citation
Masson, C. D., Findlay-Greene, F., Henderson Sousa, F., Henderson, P., Fraser, J. A., Barlow, P. G., & Stevens, C. (online). Characterisation of autophagy induction by the thiopurine drugs azathioprine, mercaptopurine and thioguanine in THP-1 macrophages. Naunyn-Schmiedeberg's Archives of Pharmacology, https://doi.org/10.1007/s00210-024-03563-0
Journal Article Type | Article |
---|---|
Acceptance Date | Oct 22, 2024 |
Online Publication Date | Nov 1, 2024 |
Deposit Date | Oct 29, 2024 |
Publicly Available Date | Oct 29, 2024 |
Journal | Naunyn-Schmiedeberg's Archives of Pharmacology |
Print ISSN | 0028-1298 |
Electronic ISSN | 1432-1912 |
Publisher | Springer |
Peer Reviewed | Peer Reviewed |
DOI | https://doi.org/10.1007/s00210-024-03563-0 |
Keywords | Thiopurines, autophagy, mTORC1, EIF2α |
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Characterisation of autophagy induction by the thiopurine drugs azathioprine, mercaptopurine and thioguanine in THP-1 macrophages
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http://creativecommons.org/licenses/by/4.0/
Copyright Statement
CC BY 4.0
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