IADR Abstract Archives
Nicotinamide N-Methyltransferase (NNMT) is an Anti-Cancer Target in Cultured Human Oral Squamous Cell Carcinoma (OSCC).
Objectives: Oral squamous cell carcinoma makes up 90% of oral malignancies. With a poor five-year survival, a targeted approach may prove a useful adjunct to established cancer therapeutics. The enzyme, N-Nicotinamide Methyltransferase (NNMT) is overexpressed in a variety of cancers and has been investigated as an anti-cancer target. We recently identified two small molecule inhibitors of NNMT (AG-670 and AO-022) based on a pharmacophore of the in-silico nicotinamide binding site. The effect of these modulators of NNMT on OSCC cell viability and metabolism was evaluated.
Methods: All cells were cultured as per ATCC recommendations. Detection of NNMT was assessed by immunoblot (Novus Biologicals NBP2-00537) from cell lysates. Cell viability was determined by alamar blue assay using a range of inhibitor concentrations from 1uM to 200uM over 72h. Cellular respiration and extracellular acidification rate were determined by Seahorse XF analyser. Mitochondria were isolated from rat liver and NADH-dependent oxygen consumption in the presence/absence of NNMT inhibitors was determined using a Rank Oxygen Electrode. An NNMT Inhibitor Screening Assay kit (Sigma-Aldrich) was used to determine the potency of inhibitors on the isolated enzyme.
Results: This investigation confirmed the presence of NNMT in SCC-4 and DOK cells. Both inhibitors resulted in cell death of the SCC-4 cells over the aforementioned concentration range. The IC50 values for each compound were subsequently calculated. Significantly, our data has shown that both NNMT inhibitors reduce oxidative phosphorylation and increase glycolysis in DOK and SCC-4 cells. Neither inhibitor showed a significant effect on NADH-dependant oxygen consumption in the coupled and uncoupled rat liver mitochondria.
Conclusions: Our data suggests that NNMT is expressed in both SCC-4 and DOK cells. Both inhibitors had a cytotoxic effect on the SCC-4 cells. These compounds inhibit oxygen consumption in intact SCC4 and DOK cells, resulting in increased glycolytic flux. The NNMT inhibitors do not, however, directly inhibit mitochondrial electron transport chain function. It is hypothesized that these small molecule inhibitors cause cell death of SCC-4 cells indirectly, via NNMT, through a mechanism that is yet to be determined.
Methods: All cells were cultured as per ATCC recommendations. Detection of NNMT was assessed by immunoblot (Novus Biologicals NBP2-00537) from cell lysates. Cell viability was determined by alamar blue assay using a range of inhibitor concentrations from 1uM to 200uM over 72h. Cellular respiration and extracellular acidification rate were determined by Seahorse XF analyser. Mitochondria were isolated from rat liver and NADH-dependent oxygen consumption in the presence/absence of NNMT inhibitors was determined using a Rank Oxygen Electrode. An NNMT Inhibitor Screening Assay kit (Sigma-Aldrich) was used to determine the potency of inhibitors on the isolated enzyme.
Results: This investigation confirmed the presence of NNMT in SCC-4 and DOK cells. Both inhibitors resulted in cell death of the SCC-4 cells over the aforementioned concentration range. The IC50 values for each compound were subsequently calculated. Significantly, our data has shown that both NNMT inhibitors reduce oxidative phosphorylation and increase glycolysis in DOK and SCC-4 cells. Neither inhibitor showed a significant effect on NADH-dependant oxygen consumption in the coupled and uncoupled rat liver mitochondria.
Conclusions: Our data suggests that NNMT is expressed in both SCC-4 and DOK cells. Both inhibitors had a cytotoxic effect on the SCC-4 cells. These compounds inhibit oxygen consumption in intact SCC4 and DOK cells, resulting in increased glycolytic flux. The NNMT inhibitors do not, however, directly inhibit mitochondrial electron transport chain function. It is hypothesized that these small molecule inhibitors cause cell death of SCC-4 cells indirectly, via NNMT, through a mechanism that is yet to be determined.