Abstract
Mercaptopurine (MP) is a cytotoxic thiopurine important for the treatment of cancer and autoimmune diseases. MP and other thiopurine drugs undergo extensive intracellular metabolism, but the mechanisms of action are poorly characterized. In particular, it is unknown how different metabolites contribute to cytotoxicity and incorporation of thiopurine bases into DNA. The aim of this study was to ask whether cytotoxicity results from the incorporation of thioguanosine nucleotides into DNA, an alternative thiopurine metabolite, or a combination of factors. Therefore, we measured the cytotoxicity, metabolism, and incorporation of thioguanosine into DNA in response to MP or MP metabolites. Thiopurine metabolites varied in cytotoxicity, with methyl-thioinosine-mono-phosphate and thioguanosine-tri-phosphate the most toxic, and the methyl-thioguanosine nucleotides the least. We show, using liquid chromatography-tandem mass spectrometry, how different metabolites may perturb biochemical pathways, particularly disrupting guanosine nucleotide homeostasis, that may contribute to the mechanism of action of thiopurines. Although there was no correlation between metabolite cytotoxicity and the levels of 6-methylthioinosine-mono-phosphate or thioguanosine incorporation into DNA as individual factors, a combined analysis suggested that these factors together had a major influence on cytotoxicity. This study emphasizes the importance of enzymes of nucleotide homeostasis, methylation, and demethylation in thiopurine effects. These results will facilitate the development of dynamic biochemical models of thiopurine biochemistry that will improve our understanding of mechanisms of action in relevant target tissues.
Highlights
The thiopurine drugs, azathioprine, mercaptopurine (MP), and 6-thioguanine have been used as immunosuppressants since the early 1950s for treatment of inflammatory bowel disease (IBD), childhood acute lymphoblastic leukemia, autoimmune hepatitis, rheumatic disease, and as a drug to reduce organ transplant rejection (Coulthard and Hogarth, 2005; Fotoohi et al, 2010)
MP is used to treat childhood acute lymphoblastic leukemia as part of maintenance therapy; we employed MOLT-4 cells, a T-cell acute lymphoblastic leukemia cell line, as an experimental model. For this proof-of-principle study we developed a new liquid chromatography–mass spectrometry (LS-MS/MS) method to investigate the metabolism of MP and its metabolites, and to test the link between cytotoxicity, thiopurine metabolites, and incorporation of fraudulent bases into DNA
Cells were exposed to each thiopurine metabolite individually on three separate occasions for 72 hours; this is the time required for incorporation of thioguanosine nucleotides into the DNA of replicating cells and the minimum exposure time to elicit cell death by thiopurine drugs at the EC50 concentrations determined by the MTS assay
Summary
The thiopurine drugs, azathioprine, mercaptopurine (MP), and 6-thioguanine have been used as immunosuppressants since the early 1950s for treatment of inflammatory bowel disease (IBD), childhood acute lymphoblastic leukemia, autoimmune hepatitis, rheumatic disease, and as a drug to reduce organ transplant rejection (Coulthard and Hogarth, 2005; Fotoohi et al, 2010). Thiopurines are prodrugs and have to be extensively metabolized to exert their cytotoxic effect. MP and 6-thioguanine undergo metabolism before exerting cytotoxicity: both drugs generate thioguanosine nucleotides via hypoxanthine guanine phosphoribosyltransferase (HPRT), but the metabolism of MP is more complex, with a different range of metabolites compared with 6-thioguanine. MP or its metabolites can be diverted from pathways of nucleotide incorporation by xanthine oxidase-mediated inactivation to thiouric acid, or by thiopurine methyltransferase (TPMT)-mediated methylation of thiopurine nucleotides or thioinosine nucleotide precursors (Coulthard and Hogarth, 2005; Fotoohi et al, 2010; Coulthard, 2012) (Fig. 1)
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