title: Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells - Effects on gene expression levels and thiopurine metabolism.
abstract: Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP's effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress.
authors.affiliation: Laboratory Medicine, Region Jönköping County, Sweden.|Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sofie
authors.initials: S
authors.lastname: Haglund
authors.affiliation: Division of Drug Research, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden.
authors.forename: Svante
authors.initials: S
authors.lastname: Vikingsson
authors.affiliation: Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden.|Center for Digestive Diseases, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Sven
authors.initials: S
authors.lastname: Almer
authors.affiliation: Laboratory Medicine, Region Jönköping County, Sweden.|Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Söderman
mesh_terms: D000493:Allopurinol / Q000494:pharmacology*; D000963:Antimetabolites / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D004359:Drug Therapy, Combination; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D053263:Gene Regulatory Networks; D057809:HEK293 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D015122:Mercaptopurine / Q000494:pharmacology*; D008780:Methyltransferases / Q000378:metabolism; D011687:Purines / Q000378:metabolism*
chemical_list: D000963:Antimetabolites; D011687:Purines; D000493:Allopurinol; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase