Edinburgh Napier University

  Background
Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis, are chronic diseases of the gastrointestinal tract. Genetic studies have identified strong association between genes involved in autophagy, ER-stress/unfolded protein response (UPR) and IBD. Stimulating autophagy may be beneficial for the treatment of IBD, and thiopurines, a class of drugs in clinical use for IBD, have been shown to induce autophagy. The aim of this study was to investigate the mechanism of action of thiopurines azathioprine (AZA), 6-MP and 6-TG in the context of autophagy, with focus on the interplay between autophagy and ER-stress/UPR signalling pathways.

Methods
Autophagy was evaluated by confocal fluorescence microscopy and live-cell imaging of autophagy marker LC3. Cell viability was assessed by Alamar Blue assay, and induction of apoptosis assessed by Annexin V/PI staining. Activation of ER stress and UPR signalling was investigated by reverse transcription polymerase chain reaction (RT-PCR), quantitative RTqPCR (RT-qPCR) and immunoblotting. Modulation of mechanistic target of rapamycin (mTORC1) was assessed by immunoblotting.

Results
AZA, 6-MP and 6-TG are strong inducers of autophagy, with AZA inducing autophagy more rapidly than 6-MP or 6-TG. AZA and 6-TG increased early apoptosis, while 6-TG reduced cell viability and inhibited cell growth. Thiopurines did not induce ER stress, however, 6-TG activated the UPR pathway. AZA induced autophagy via mechanisms involving modulation of mTORC1 signalling.

Conclusions
Thiopurines are strong inducers of autophagy. There are differences in AZA, 6-MP and 6-TG mechanism of action, therefore, a better understanding may lead to personalised application, and improve the therapeutic efficacy of thiopurines in IBD.