Characterisation of Pharmacological Mechanisms and Potential Therapeutic uses of FK866
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The finding that NAD+ plays a role in a variety of signalling pathways, including gene expression, Ca2+ signalling and DNA repair mechanisms, has sparked interest in the proteins involved in these pathways as potential pharmacological targets for drug development. Recently, FK866, a potent inhibitor of nicotinamide phosphoribosyltransferase (Nampt) an important enzyme in the NAD+ rescue pathway, has been evaluated in clinical trials against cancer. The aim of this study is to further investigate the mechanisms and therapeutic characteristics of FK866 in different cancer cell lines and to determine if decreasing intracellular NAD+ levels can be used as a co-therapy strategy to improve the efficacy of current and new chemotherapy treatments. Experiments measuring cell vitality showed that FK866 dose-dependently decreased cell vitality. To investigate NAD+ consumption during Nampt inhibition, NAD+ levels were measured in cells treated with FK866 and inhibition of each of the main NAD+ consuming enzymes (PARP, sirtuins or CD38). This revealed differential NAD+ consumption rates by the different NAD+ consuming enzymes in MDA-MB-231 cells, with sirtuins being the major NAD+ consuming enzyme. The glycolytic effects of Nampt inhibition was measured using SEAHORSE assays; which measured the oxygen consumption and extracellular acidification rates as well as measuring NAD+/ NADH ratios. In the MCF-7 and MDA-MB-231 cell lines, FK866 had no effect on the oxygen consumption rates; however there was a general decrease in extracellular acidification rates indicating an effect on glycolytic activity. When measuring the NAD+/NADH ratio however, there was only a decrease in the MDA -MB-231 cells but no change in the MCF-7 cell line. Cell vitality and NAD+ levels were measured after treatment with FK866 in addition to NAD+ consuming enzyme inhibitors or the alkylating agent, Temozolomide to see if combination therapy would have more cytotoxic potential. This co-treatment indicated that there was no real positive effect on either the MCF-7 or MDA-MB-231 cells in either the cell vitality or NAD+ levels. Finally the effects of FK866 and the oral PARP inhibitor, Olaparib, were investigated using 3D cell culture (spheroids) and compared with 2D monolayer cultures. The effects of FK866 showed little difference in spheroid or monolayer culture. However, when treating with Olaparib there was higher level of cell viability and NAD+ levels with the cells grown in spheroid culture in comparison to cells grown in monolayer. In conclusion, this study has shown that FK866, as a single treatment decreases cell vitality, NAD+ levels and glycolytic activity. However as a co -therapy with PARP or Sirtuin inhibitors there is an increase in the cell vitality and NAD+ levels. Although similarities have been seen between spheroid culture and monolayers as a single treatment, FK866 does not seem to have the beneficial effects as a therapeutic.
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