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

Biochem Pharmacol. 2014 Mar 1;88(1):36-45. doi: 10.1016/j.bcp.2013.12.019. Epub 2014 Jan 13.

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.

Keywords: Aldo-keto reductase 1C3; Fluorogenic assays; Leukaemia; PR-104; SN34037.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • 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