eased by approximately 1.4 fold in belinostat treated HT29 cells relative to controls, as shown by 1H MRS, indicating that the de novo formed PC was used to supply PtdCho synthesis. This finding concurs with the previously reported role of HDAC inhibition in inducing the expression of CTP PC cytidylyltransferase, the rate Hedgehog Pathway limiting enzyme in PtdCho biosynthesis . Interestingly, levels of GPC, which is a PtdCho breakdown product, fell post belinostat treatment in cells , suggesting that HDAC inhibition probably also inhibited PtdCho degradation, which together with activation of the synthetic pathway led to net augmentation in this membrane phospholipid. However, this effect did not translate to increases in cell volume , suggesting that PtdCho was not used to synthesize new outer cell membrane, and that it was probably deployed to other cellular compartments.
More work Bicalutamide is required to establish the significance of the rise in PtdCho following belinostat treatment, assess the effect of therapy on other lipid species, and define the involvement of PtdCho metabolizing enzymes in the MRS changes observed here. The induction in ChoKa observed following HDAC inhibition and the ensuing increase in PC are unusual and, paradoxically, more commonly associated with malignancy . Indeed ChoKa expression is linked to oncogene activation and correlates with poor patient prognosis . Further studies are required to elucidate the significance of the choline metabolism effects observed here in relation to HDAC inhibitor– induced antitumor activity.
Finally, and to assess whether any of the changes observed in cancer cells could serve as potential noninvasive biomarkers of HDAC inhibition, surgery we investigated the effect of belinostat in tumor xenografts derived from the same HT29 cells used in vitro. In vivo 1H MRS revealed increased tCho/ water ratio following belinostat treatment. Although therapy induced changes in water content can contribute to a rise in tCho/water, increased PME/TotP was also observed by in vivo 31P MRS following treatment with belinostat. Furthermore, ex vivo MRS revealed increased PC and GPC levels in belinostat treated tumors relative to controls. This indicates that the in vivo MRS detectable changes are primarily due to increased PC and GPC content in the drug treated group.
The rise in PC concurs with our in vitro cell observations and with our previous findings with LAQ824 and SAHA , indicating that this effect is likely to be associated with the mechanism of action of HDAC inhibitors. We also found an increase in tumor GPC following belinostat treatment, but a decrease in GPC was observed in belinostat treated cells and previously in LAQ824 treated tumor extracts . Interestingly, although HDAC inhibition led to significant increases in cellular AA levels and previously in HT29 tumors in vivo post LAQ824 treatment , no significant effects on AA content were recorded in the belinostat treated tumors. The basis for this difference is unclear and may relate to druginduced physiologic effects that may vary under the different treatment conditions. In line with our previous findings with LAQ824 , glucose concentration fell in the belinostat treated tumors compared with controls albeit to a lesser extent compared with LAQ824 .