Alessia Norbedo1*, Marianna Lucafò1, Carlotta Bidoli1, Marco Gerdol1, Metka Lenassi2, Giuliana Decorti1,3, Gabriele Stocco1
1University of Trieste, Department of Life Science Trieste, Italy
2University of Ljubljana, Faculty of Medicine, Institute of Biochemistry, VrazovTrg 2, 1000 Ljubljana, Slovenia
3Institute for Maternal and Child Health I.R.C.C.S. Burlo Garofolo, Trieste, Italy
alessia.norbedo [at] phd.units.it
Abstract
Thiopurines, such as mercaptopurine, are antimetabolites, used in the treatment of acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD). PACSIN2 rs2413739 is associated with gastrointestinal toxicity in children with ALL and with drug-efficacy in IBD pediatric patients. PACSIN2 is involved in vesicular trafficking and may affect the release and content of extracellular vesicles (EVs), which mediate cell communication and whose cargo modifies phenotypes of target cells. This study evaluates mechanisms associating PACSIN2 polymorphism with interindividual variability in efficacy of thiopurines, by considering the role of PACSIN2 in sorting specific miRNA in EVs.
Effects of stable PACSIN2 knock-down (KD) were evaluated in intestinal LS180 cells. MTT cytotoxicity assay was used to verify mercaptopurine-sensitivity. EVs, released by LS180 KD and MOCK control cells were isolated by ultracentrifuge and characterized by nanoparticle tracking analysis (NTA). EVs miRNA-sequencing was performed by Illumina Hi-seq 2000. EVs may alter drug cytotoxicity, therefore LS180 MOCK and KD cells were co-treated with mercaptopurine and EVs. Statistical analysis was performed using t-test and ANOVA.
Mercaptopurine was more cytotoxicity in LS180 KD cells (IC50 MOCK 3.23; IC50 KD 2.18 µM). No differences were observed by NTA in release of EVs between MOCK and KD cells (t-test, p = 0.13). PACSIN2 KD altered intracellular and EVs expression of 6 and 24 miRNAs respectively. EVs released by reduced mercaptopurine cytotoxicity (about 10%) and Rac1 protein expression in KD cells (ANOVA, p < 0.001), probably because they transport different miRNAs.
In conclusion, PACSIN2 KD increase mercaptopurine cytotoxicity, probably, by deregulation of miRNA expression in cells and EVs. These results will be further investigated to better explain the link between PACSIN2 and EVs, whose miRNAs could provide a new scenario in personalizing thiopurine treatment.
Keywords: PACSIN2, extracellular vesicles, miRNA-sequencing, mercaptopurine
Acknowledgement: This work was supported by the Italian Ministry of Health, through the contribution given to the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy, grant RC 23-23.
