RESUMEN
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.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Antineoplásicos/metabolismo , Fluorometría/métodos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Compuestos de Mostaza Nitrogenada/metabolismo , Profármacos/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/genética , Aerobiosis , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Médula Ósea/enzimología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Células HCT116 , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/genética , Leucocitos/enzimología , Morfolinas/química , Morfolinas/metabolismo , Compuestos de Mostaza Nitrogenada/farmacocinética , Compuestos de Mostaza Nitrogenada/farmacología , Oxidación-Reducción , Profármacos/farmacocinética , Profármacos/farmacología , Especificidad por Sustrato , Factores de Tiempo , Urea/análogos & derivados , Urea/química , Urea/metabolismoRESUMEN
Activation of prodrugs in tumors (e.g., by bioreduction in hypoxic zones) has the potential to generate active metabolites that can diffuse within the tumor microenvironment. Such "bystander effects" may offset spatial heterogeneity in prodrug activation but the relative importance of this effect is not understood. Here, we quantify the contribution of bystander effects to antitumor activity for the first time, by developing a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model for PR-104, a phosphate ester pre-prodrug that is converted systemically to the hypoxia-activated prodrug PR-104A. Using Green's function methods we calculated concentrations of oxygen, PR-104A and its active metabolites, and resultant cell killing, at each point of a mapped three-dimensional tumor microregion. Model parameters were determined in vitro, using single cell suspensions to determine relationships between PR-104A metabolism and clonogenic cell killing, and multicellular layer (MCL) cultures to measure tissue diffusion coefficients. LC-MS/MS detection of active metabolites in the extracellular medium following exposure of anoxic single cell suspensions and MCLs to PR-104A confirmed that metabolites can diffuse out of cells and through a tissue-like environment. The SR-PK/PD model estimated that bystander effects contribute 30 and 50% of PR-104 activity in SiHa and HCT116 tumors, respectively. Testing the model by modulating PR-104A-activating reductases and hypoxia in tumor xenografts showed overall clonogenic killing broadly consistent with model predictions. Overall, our data suggest that bystander effects are important in PR-104 antitumor activity, although their reach may be limited by macroregional heterogeneity in hypoxia and reductase expression in tumors. The reported computational and experimental techniques are broadly applicable to all targeted anticancer prodrugs and could be used to identify strategies for rational prodrug optimization.
RESUMEN
Hypoxia contributes to resistance of tumors to some cytotoxic drugs and to radiotherapy, but can in principle be exploited with hypoxia-activated prodrugs (HAP). HAP in clinical development fall into two broad groups. Class I HAP (like the benzotriazine N-oxides tirapazamine and SN30000), are activated under relatively mild hypoxia. In contrast, Class II HAP (such as the nitro compounds PR-104A or TH-302) are maximally activated only under extreme hypoxia, but their active metabolites (effectors) diffuse to cells at intermediate O2 and thus also eliminate moderately hypoxic cells. Here, we use a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model to compare these two strategies and to identify the features required in an optimal Class II HAP. The model uses a Green's function approach to calculate spatial and longitudinal gradients of O2, prodrug, and effector concentrations, and resulting killing in a digitized 3D tumor microregion to estimate activity as monotherapy and in combination with radiotherapy. An analogous model for a normal tissue with mild hypoxia and short intervessel distances (based on a cremaster muscle microvessel network) was used to estimate tumor selectivity of cell killing. This showed that Class II HAP offer advantages over Class I including higher tumor selectivity and greater freedom to vary prodrug diffusibility and rate of metabolic activation. The model suggests that the largest gains in class II HAP antitumor activity could be realized by optimizing effector stability and prodrug activation rates. We also use the model to show that diffusion of effector into blood vessels is unlikely to materially increase systemic exposure for realistic tumor burdens and effector clearances. However, we show that the tumor selectivity achievable by hypoxia-dependent prodrug activation alone is limited if dose-limiting normal tissues are even mildly hypoxic.
RESUMEN
PURPOSE: Benzotriazine-N-oxide bioreductive prodrugs such as tirapazamine and its improved analogue CEN-209 (SN30000) have potential for exploiting hypoxia in tumors. Here, we test the hypothesis that the 2-nitroimidazole EF5, in clinical development for both immunohistochemical and positron emission tomography imaging of hypoxia, can detect not only hypoxia but also the one-electron reductases required for activation of these hypoxia-targeted prodrugs. EXPERIMENTAL DESIGN: Aerobic and hypoxic covalent binding of [(14)C]-EF5 was determined in human tumor cell lines, including lines with overexpression of NADPH:cytochrome P450 oxidoreductase (CYPOR), and reductive metabolism of tirapazamine and CEN-209 by mass spectrometry. DNA damage response was measured by γH2AX formation. Bioreductive metabolism was modulated in HCT116 tumor xenografts by overexpression of CYPOR and breathing of hyperbaric oxygen or 10% oxygen. RESULTS: Overexpression of CYPOR induced similar 2- to 4-fold increases in EF5 binding and metabolic reduction of tirapazamine and CEN-209 in SiHa and HCT116 cell lines, and similar enhancement of γH2AX formation. EF5 binding and metabolic reduction of the prodrugs were highly correlated in a panel of 14 hypoxic tumor cell lines. In HCT116 xenografts, CYPOR overexpression also significantly increased EF5 binding and CEN-209 reduction, and modification of tumor hypoxia caused similar changes to the bioreductive activation of both agents, resulting in a strong correlation between EF5 binding and CEN209-induced DNA damage (R(2) = 0.68, P < 0.0001) at the individual tumor level. CONCLUSIONS: EF5 binding is a promising stratification biomarker for benzotriazine-N-oxide bioreductive prodrugs because of its potential for interrogating reductase activity as well as hypoxia in individual tumors.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Etanidazol/análogos & derivados , Hidrocarburos Fluorados/análisis , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Óxidos N-Cíclicos/metabolismo , Etanidazol/análisis , Etanidazol/metabolismo , Femenino , Células HCT116 , Histonas/metabolismo , Humanos , Hidrocarburos Fluorados/metabolismo , Indicadores y Reactivos , Ratones , Oxidorreductasas/metabolismo , Profármacos/farmacología , Tirapazamina , Trasplante Heterólogo , Triazinas/metabolismoRESUMEN
BACKGROUND & AIMS: TNF was the first cytokine employed for cancer therapy, but its use was limited due to its insufficient selectivity towards malignant cells. Fructose induces transient hepatic ATP depletion in humans and rodents due to the liver-specific fructose metabolism via fructokinase, while other cells e.g. Muscle cells metabolize fructose via hexokinase. Under ATP depleted conditions hepatocytes are protected against TNF-induced apoptosis. Our aim was to identify metabolic differences between normal and malignant liver cells that can be exploited for selective immunotherapy. METHODS: We analyzed the expression and activities of enzymes involved in fructose metabolism in primary hepatocytes and hepatoma cell lines. Furthermore, we studied the influence of hexokinase II (HKII) on fructose-mediated ATP depletion and cytoprotection in murine hepatocytes. RESULTS: Primary mouse, rat and human hepatocytes depleted of ATP by fructose were fully protected against TNF-induced cytotoxicity. By contrast, hepatic tumor cell lines showed increased HKII expression, lack of fructose-mediated ATP depletion and, therefore, remained susceptible to TNF/ActD-induced apoptosis. Inhibition of hexokinases restored fructose-induced ATP depletion in hepg2 cells. Finally, hypoxia-inducible factor1 (HIF1)-mediated up-regulation of HKII prevented fructose-induced ATP depletion and overexpression of HKII inhibited fructose-mediated cytoprotection against TNF-induced apoptosis in primary murine hepatocytes. CONCLUSION: Increased expression of HKII in malignant cells of hepatic origin shifts the fructose metabolism from liver- to muscle-type, thereby preventing ATP depletion and subsequent cytoprotection of the target cells. Therefore, healthy liver cells are transiently protected from TNF-mediated cell death by fructose-induced ATP depletion, while malignant cells can be selectively eliminated through TNF-induced apoptosis.