OBJECTIVE: The nicotinamide phosphoribosyltransferase (Nampt) inhibitor APO866 depletes intracellular nicotinamide adenine dinucleotide (NAD(+)) and shows promising anticancer activity in preclinical studies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and induces apoptosis via caspase-8 and -10. Here we have explored the interaction between APO866 and TRAIL in leukemia cell lines and in primary B-cell chronic lymphocytic leukemia cells. MATERIALS AND METHODS: Cells were treated with APO866, TRAIL, or their combination. Viability and mitochondrial transmembrane potential (ΔΨ(m)) were determined by cell staining with propidium iodide and tetramethylrhodamine ethyl ester, respectively, and flow cytometry. Nampt and γ-tubulin levels, as well as caspase-3 cleavage were detected by immunoblotting. DR4 and DR5 expression were assessed by immunostaining and flow cytometry. Caspases were inhibited with zVAD-FMK and zDEVD-FMK; autophagy with 3-methyladenine, LY294002, and wortmannin. Intracellular NAD(+) and adenosine triphosphate (ATP) were measured by cycling assays and high-performance liquid chromatography (HPLC), respectively. RESULTS: APO866 induced NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage in leukemia cells, thereby leading to autophagic cell death. TRAIL induced caspase-dependent apoptosis. TRAIL addition to APO866 synergistically increased its activity in leukemia cells by enhancing NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage. No DR5 upregulation at the cell surface in response to APO866 was observed. Remarkably, in healthy leukocytes APO866 and TRAIL were poorly active and failed to show any cooperation. CONCLUSIONS: Activation of the extrinsic apoptotic cascade with TRAIL selectively amplifies the sequelae of Nampt inhibition in leukemia cells, and appears as a promising strategy to enhance APO866 activity in hematological malignancies.

Potent synergistic interaction between the Nampt inhibitor APO866 and the apoptosis activator TRAIL in human leukemia cells.

ZOPPOLI, GABRIELE;CEA, MICHELE;SONCINI, DEBORA;FRUSCIONE, FLORIANA;MORAN, EVA;CAFFA, IRENE;MOTTA, GIOVANNI;GHIO, RICCARDO;FERRANDO, FABIO;BALLESTRERO, ALBERTO;PARODI, SILVIO;PATRONE, FRANCO;BRUZZONE, SANTINA;NENCIONI, ALESSIO
2010

Abstract

OBJECTIVE: The nicotinamide phosphoribosyltransferase (Nampt) inhibitor APO866 depletes intracellular nicotinamide adenine dinucleotide (NAD(+)) and shows promising anticancer activity in preclinical studies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and induces apoptosis via caspase-8 and -10. Here we have explored the interaction between APO866 and TRAIL in leukemia cell lines and in primary B-cell chronic lymphocytic leukemia cells. MATERIALS AND METHODS: Cells were treated with APO866, TRAIL, or their combination. Viability and mitochondrial transmembrane potential (ΔΨ(m)) were determined by cell staining with propidium iodide and tetramethylrhodamine ethyl ester, respectively, and flow cytometry. Nampt and γ-tubulin levels, as well as caspase-3 cleavage were detected by immunoblotting. DR4 and DR5 expression were assessed by immunostaining and flow cytometry. Caspases were inhibited with zVAD-FMK and zDEVD-FMK; autophagy with 3-methyladenine, LY294002, and wortmannin. Intracellular NAD(+) and adenosine triphosphate (ATP) were measured by cycling assays and high-performance liquid chromatography (HPLC), respectively. RESULTS: APO866 induced NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage in leukemia cells, thereby leading to autophagic cell death. TRAIL induced caspase-dependent apoptosis. TRAIL addition to APO866 synergistically increased its activity in leukemia cells by enhancing NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage. No DR5 upregulation at the cell surface in response to APO866 was observed. Remarkably, in healthy leukocytes APO866 and TRAIL were poorly active and failed to show any cooperation. CONCLUSIONS: Activation of the extrinsic apoptotic cascade with TRAIL selectively amplifies the sequelae of Nampt inhibition in leukemia cells, and appears as a promising strategy to enhance APO866 activity in hematological malignancies.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/263002
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