P082 An investigation of azathioprine on autophagy pathway activity
Hooper K.*1, Barlow P.1, Henderson P.2,3, Stevens C.1
1Edinburgh Napier University, Edinburgh, United Kingdom 2Royal Hospital For Sick Children, Department of Pediatric Gastroenterology, Edinburgh, United Kingdom 3University Of Edinburgh, Department of Child Life and Health, Edinburgh, United Kingdom
Autophagy is an intracellular process that degrades damaged or aged proteins and organelles to maintain cellular homeostasis. Defective autophagy has been strongly linked to inflammatory bowel disease (IBD) pathogenesis, with evidence that enhancing autophagy may be therapeutically beneficial by regulating inflammation and clearing intestinal pathogens. Due to the high cost associated with the development of new drugs, a more comprehensive characterisation of commonly used IBD drugs and their mechanism of action are required. Our aim is to investigate the effect of azathioprine on autophagy pathway activity and to determine the molecular mechanisms involved.
The autophagy response to azathioprine was assessed
A significant increase in autophagy was observed in response to 120 μM of azathioprine, with optimal autophagy activity at 6 hours post-treatment. Confocal microscopy showed an increase in the percentage of cells exhibiting GFP-LC3 foci, and flow cytometry showed an increase in the fluorescent intensity of GFP-LC3 in cells treated with azathioprine compared to control cells. Western immunoblotting also showed that azathioprine treatment leads to an accumulation of LC3-II, the lipidated and active form of LC3. By monitoring cells transiently expressing the GFP-RFP-LC3 fusion protein we show that azathioprine stimulates autophagy pathway activity, and rules out the possibility that accumulation of LC3 positive autophagosomes is due to reduced fusion with lysosomes. Analysis of mTORC1 activity revealed that azathioprine treatment causes a decrease in phospho-rpS6, suggesting that azathioprine may stimulate autophagy via modulation of mTORC1 signalling.
We have used several complimentary methods to demonstrate that the immunomodulatory drug azathioprine strongly induces autophagy