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

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• 作者：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. Bohl ; er ; Richard B. Lock ; William R. Wilson
• 关键词：Aldo-keto reductase 1C3 ; Leukaemia ; PR-104 ; SN34037 ; Fluorogenic assays
• 刊名：Biochemical Pharmacology
• 出版年：1 March, 2014
• 年：2014
• 卷：88
• 期：1
• 页码：36-45
• 全文大小：1168 K

文摘

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.