A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells.

Abstract

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.

• Authors: Stephen M F Jamieson, Yongchuan Gu, Donya Moradi Manesh, Jad El-Hoss, Duohui Jing, Karen L Mackenzie, Christopher P Guise, Annika Foehrenbacher, Susan M Pullen, Juliana Benito, Jeffrey B Smaill, Adam V Patterson, Medhanie A Mulaw, Marina Konopleva, Stefan K Bohlander, Richard B Lock, William R Wilson
• Journal: Biochemical pharmacology (Biochem Pharmacol) Vol. 88 Issue 1 Pg. 36-45 (Mar 01 2014) ISSN: 1873-2968 [Electronic] England
• PMID: 24434189 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• Antineoplastic Agents
• Enzyme Inhibitors
• Heterocyclic Compounds, 4 or More Rings
• Morpholines
• Nitrogen Mustard Compounds
• PR-104A
• Prodrugs
• SN 34037
• coumberone
• Urea
• 3-Hydroxysteroid Dehydrogenases
• Hydroxyprostaglandin Dehydrogenases
• AKR1C3 protein, human
• Aldo-Keto Reductase Family 1 Member C3

Topics

• 3-Hydroxysteroid Dehydrogenases (antagonists & inhibitors, genetics, metabolism)
• Aerobiosis
• Aldo-Keto Reductase Family 1 Member C3
• Antineoplastic Agents (metabolism, pharmacokinetics, pharmacology)
• Bone Marrow (enzymology)
• Cell Line, Tumor
• Cell Survival (drug effects)
• Dose-Response Relationship, Drug
• Drug Resistance, Neoplasm (drug effects)
• Enzyme Inhibitors (chemistry, metabolism)
• Fluorometry (methods)
• HCT116 Cells
• Heterocyclic Compounds, 4 or More Rings (chemistry, metabolism)
• Humans
• Hydroxyprostaglandin Dehydrogenases (antagonists & inhibitors, genetics, metabolism)
• Leukocytes (enzymology)
• Morpholines (chemistry, metabolism)
• Nitrogen Mustard Compounds (metabolism, pharmacokinetics, pharmacology)
• Oxidation-Reduction
• Prodrugs (metabolism, pharmacokinetics, pharmacology)
• Substrate Specificity
• Time Factors
• Urea (analogs & derivatives, chemistry, metabolism)