treatment was a rise in PC content, which was time dependent in HT29 cells. When detectable , GPC levels decreased post belinostat treatment. The changes in PC and GPC were confirmed by 1H MRS which indicated that the tCho signal was also significantly higher following Gamma Secretase belinostat treatment in both cell lines. The rise in PC is in line with our previous findings with LAQ824 and SAHA in HT29 cells indicating that it is not inhibitor chemotype specific or cell line/tissue typedependent. Furthermore, belinostat treatment resulted in increased BCAAs, alanine, and threonine levels in both HT29 and PC3 cells as shown by 1H MRS. These changes were also timedependent in HT29 cells.Taken together, these observations suggest that belinostat treatment led to differential metabolic routing that favors the conversion of pyruvate to alanine at the expense of lactate synthesis.
A previous report showed BMS-354825 inhibition of glucose transport concomitant with reduced hexokinase activity following HDAC inhibition . Here, we did not observe a reduction in 13C glucose accumulating in the cells, and the combined level of downstream 13C intermediates formed from 13C glucose seemed to be comparable between control and treated cells . These observations suggest that glucose uptake was probably unaltered under our experimental conditions although more work is required to test this hypothesis. Furthermore, and although neither of the changes in individual content of intracellular 13C lactate or 13C glutamate following belinostat treatment were statistically significant, the glutamate/ lactate ratio increased by approximately 3 fold.
This effect could suggest an altered balance between glycolysis and Krebs cycle metabolism as previously reported . Next, surgery we investigated the basis for the PC rise following belinostat treatment. PC is formed via 2 main routes: de novo synthesis through ChoK catalyzed phosphorylation of its precursor choline and release from membrane PtdCho via PtdCho specific phospholipase C or release of choline from PtdCho via phospholipase D followed by ChoK mediated phosphorylation . To distinguish these 2 processes, we assessed levels of 13CPC in cells incubated in choline for 3 hours. This short duration ensured that the 13C PC observed was formed de novo from exogenous 13C choline, thus excluding contributions from membrane PtdCho derived PC .
13C MRS indicated that the levels of 13C PC formed were approximately 1.5 fold higher in belinostat treated cells relative to controls. The amplitude of this increase is similar to that observed by 1H and 31P MRS indicating that the elevation in steady state PC is driven primarily by increased de novo synthesis. To further delineate the molecular processes driving this effect, we assessed the expression of ChoKa. qRT PCR analysis revealed an induction in ChoKa gene expression in HT29 cells which correlated strongly with the rise in PC levels measured by MRS and which was also confirmed by ChoKa protein expression in these cells. A similar increase in ChoKa expression was also observed in PC3 cells, albeit to a lesser extent. These findings point to the induction of ChoKa expression as a key driver of the rise in PC observed by MRS following belinostat treatment in both HT29 and PC3 cells.