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3-hydroxykynurenine is a ROS-inducing cytotoxic tryptophan metabolite that disrupts the TCA cycle
Preprint   Open access

3-hydroxykynurenine is a ROS-inducing cytotoxic tryptophan metabolite that disrupts the TCA cycle

Jane L. Buchanan, Adam J. Rauckhorst and Eric B. Taylor
bioRxiv
Cold Spring Harbor Laboratory
07/10/2023
DOI: 10.1101/2023.07.10.548411
PMCID: PMC10369892
PMID: 37502990
url
https://doi.org/10.1101/2023.07.10.548411View
Preprint (Author's original)This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

Tryptophan is an essential amino acid that is extensively characterized as a regulator of cellular function through its metabolism by indoleamine 2,3-deoxygenase (IDO) into the kynurenine pathway. However, despite decades of research on tryptophan metabolism, the metabolic regulatory roles of it and its metabolites are not well understood. To address this, we performed an activity metabolomics screen of tryptophan and most of its known metabolites in cell culture. We discovered that treatment of human colon cancer cells (HCT116) with 3-hydroxykynurenine (3-HK), a metabolite of kynurenine, potently disrupted TCA cycle function. Citrate and aconitate levels were increased, while isocitrate and all downstream TCA metabolites were decreased, suggesting decreased aconitase function. We hypothesized that 3HK or one of its metabolites increased reactive oxygen species (ROS) and inhibited aconitase activity. Accordingly, we observed almost complete depletion of reduced glutathione and a decrease in total glutathione levels. We observed a dose-dependent decrease in cell viability after 48 hours of 3HK treatment. These data suggest that raising the intracellular levels of 3HK could be sufficient to induce ROS-mediated apoptosis. We modulated the intracellular levels of 3HK by combined induction of IDO and knockdown of kynureninase (KYNU) in HCT116 cells. Cell viability decreased significantly after 48 hours of KYNU knockdown compared to controls, which was accompanied by increased ROS production and Annexin V staining revealing apoptosis. Finally, we identify xanthommatin production from 3-HK as a candidate radical-producing, cytotoxic mechanism. Our work indicates that KYNU may be a target for disrupting tryptophan metabolism. Interestingly, many cancers exhibit overexpression of IDO, providing a cancer-specific metabolic vulnerability that could be exploited by KYNU inhibition.

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