Identification and validation of autophagy modulating small molecules

Human health is strongly dependent of the accurate coordination and regulation of diverse cellular and metabolic processes, including autophagy. The latter is an evolutionarily conserved process which is active in all human cells. Accordingly, dysregulation of autophagic activity is involved in many pathologies. The main aim of this PhD thesis was the pharmacological modulation (inhibition or induction) of autophagy. The experimental work existed of two major parts. In part 1, we aimed to investigate the possible beneficial effects of UAMC-2526, a ATG4B inhibitor in a Panc02 mouse model of PDAC and in a HT-29 colorectal cancer model. In chapter 3 we used female c57/BL6 mice that were inoculated with Panc02 cancer cells. These mice were treated for 28 days with gemcitabine, autophagy inhibitor UAMC-2526, gemcitabine combined with UAMC-2526, or vehicle. We investigated the mechanism of the tumor growth inhibition to determine whether this inhibition is due to the autophagy inhibition or are other mechanisms involved such as inhibition of proliferation or cell death. The combination of oxaliplatin and bevacizumab is the backbone of therapy for advanced colorectal cancer. Based on the previous results of UAMC-2526 in chapter 4 we determined the influence of UAMC-2526 in combination with these two cancer therapies. We used CD1-/- xenograft mouse model inoculated with HT-29 colorectal cancer cells. These mice were treated for 28 days with oxaliplatin, bevacizumab, oxaliplatin combined with bevacizumab, oxaliplatin combined with bevacizumab and UAMC-2526, bevacizumab and vehicle. In part 2 of the thesis we focused on autophagy inducing molecules. In chapter 5, we used an image-based high-throughput method to screen 10,240 compounds. GFP-LC3 transfected L929 fibroblasts were used to identify novel autophagy inducing small molecules. Thirty compounds, for which their effects on autophagosome accumulation were confirmed in triplicate, were selected for further validation. These hits were re-evaluated in chapter 6, using HeLa cells transfected with mRFP-GFP-LC3 construct. The latter approach allowed us to distinguish compounds that can truly induce autophagic flux from those that induce autophagosome accumulation. mTOR independency of the compounds were investigated via western blot. Additionally, TEM was used as gold standard method to validate the inducers. In summary, we can conclude that modulation of autophagy is complicated and extremely condition dependent. In addition, it is crucial to note that monitoring autophagic flux is not straightforward and requires critical assessment. Combining different methods and comparing their results has to be a golden rule in monitoring autophagic activity.

Jaar van publicatie:2022

Trefwoorden:Doctoral thesis

Toegankelijkheid:Closed