# Characterisation of autophagy induction by the thiopurine drugs azathioprine, mercaptopurine and thioguanine in THP-1 macrophages

## TL;DR

This study shows that thiopurine drugs like azathioprine strongly induce autophagy in macrophages without causing cell death.

## Contribution

The study reveals that thiopurines induce autophagy independently of apoptosis and ER stress, with distinct effects on mTORC1 and eIF2α signaling.

## Key findings

- Thiopurines induce strong, time- and concentration-dependent autophagy in THP-1 macrophages.
- Autophagy activation by thiopurines occurs without apoptosis or ER stress.
- AZA inhibits mTORC1 and increases eIF2α phosphorylation, while 6-MP has minimal effects on these pathways.

## Abstract

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. 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 eIF2α-S51 phosphorylation were evaluated by immunoblotting. 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 eIF2α-S51 phosphorylation. In contrast, 6-MP had a minor effect on either signalling pathway. Thiopurines are strong inducers of autophagy, and autophagy induction should be considered among the mechanisms responsible for patient response to thiopurines.

The online version contains supplementary material available at 10.1007/s00210-024-03563-0.