Background: Cancer cells have a high NAD turnover rate due to their increased cell proliferation and DNA repair. Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway, a regulator of the intracellular NAD pool and of the activity of Sirtuins (SIRTs), a class of NAD-dependent deacetylases.
Objective and hypotheses: Cancer cells are highly dependent on NAD and are expected to be more susceptible to an inhibition of NAD synthesis than non-transformed cells. Does an inhibition of NAMPT by FK866 make leukemia cells more sensitive to chemotherapeutic agents?
Method: Jurkat and Molt-4 cell lines were used as leukemia cell models. Viability was measured using WST-1 assay. Cell death was analysed by propidium iodide staining. NAD levels were measured using HPLC. NAMPT enzymatic activity was measured using 14C-nicotinamide.
Results: NAMPT expression and enzymatic activity was significantly higher in leukemia cell lines compared to normal PBMCs. Incubation with FK866 [10 nM] for 24 h reduced NAMPT activity by 91.1±3.6% in Jurkat cells and by 97.8±1.2% in Molt-4 cells. NAD levels were reduced by FK866 by 83.9±1.0% (Jurkat) or 79.2±2.8% (Molt-4). The combination of etoposide and FK866 caused increased cell death compared to each substance alone. Apoptosis induction as measured by caspase-3 and -7 activation was not further increased by the addition of FK866 to etoposide. Interestingly, etoposide decreased the expression of the NAD-dependent deacetylases SIRT1 and SIRT2. The acetylation and activation of the SIRT1 target p53 was enhanced after combining etoposide with FK866, which was associated with an increased expression of its downstream target p21.
Conclusion: The combination of etoposide and FK866 caused increased cell death which was not caspase-mediated, but led to reduced SIRT1 activity as indicated by enhanced acetylation and transcriptional activity of p53. Combining FK866 and etoposide could be a novel therapeutic strategy to enhance the efficacy of etoposide against leukemia.
10 - 12 Sep 2016
European Society for Paediatric Endocrinology