the altered metabolic features of tumors relative to normal tissues, including increased lipid synthesis and aerobic glycolysis . These metabolic changes are increasingly being investigated as diagnostic as well as treatment Hedgehog Pathwy response biomarkers, with techniques such as magnetic resonance spectroscopy being of particular value for translating findings from preclinical models to humans . MRS allows the detection of many metabolites and in preclinical studies has shown that response to molecularly targeted therapeutics is often associated with altered metabolism . For example, inhibitors of HSP90 , phospholipase Cg1 , mitogen activated protein kinase , or phosphoinositide 3 kinase have all been shown to alter choline phospholipid metabolism in human cancer cells.
In the case of the HDAC inhibitor LAQ824, in vitro and in vivo MRS showed increased phosphocholine levels both in human colon cancer cells and tumors posttreatment . A similar effect was also observed in human colon and prostate Acadesine cancer cells treated with the HDAC inhibitor SAHA or its fluoroanalogue , respectively. Furthermore, LAQ824 treatment caused a substantial reduction in tumor bioenergy related metabolites that was observed in vivo but not in vitro . This effect was attributed to the antiangiogenic action of LAQ824, whereas the rise in PC was likely to relate to the effect of HDAC inhibition on tumor cell metabolism although the molecular and biochemical mechanisms behind this change remain unclear.
Here, we assess whether similar metabolic effects would be observed with the alternative chemotype HDAC inhibitor and probe compound belinostat and the molecular and biochemical processes underlying the observed metabolic alterations. We show that HDAC inhibition with belinostat in human cancer cells leads to increased alanine and branched chain farriers amino acid content that was associated with altered glucose utilization. Belinostat also increased PC levels, thus confirming our previous finding with the alternative chemotype agent LAQ824. Importantly, we show for the first time that this effect is associated with induction of choline kinase a gene and protein expression. The increase in PC is also observed in belinostat treated tumors in vivo, thus supporting the role of PC as a potential noninvasive metabolic imaging biomarker of HDAC inhibition.
Cell culture Human HT29 colon and PC3 prostate carcinoma cells were grown in Dulbecco’s Modified Eagle’s Medium or RPMI, respectively, containing 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in a 37C humidified 5% CO2 atmosphere. Cells were preserved and propagated according to ATCC’s protocols, screened monthly for mycoplasma and passaged for no longer than 3 months. All cell culture materials were from Life Technologies. Western blot Analysis of the molecular effects of HDAC inhibition was conducted by Western blotting as previously described . The primary antibodies rabbit antiacetyl histone 3 , rabbit anti ChoKa , mouse anti GAPDH , and rabbit a tubulin were used. The secondary anti rabbit and anti mouse antibodies were from GE Healthcare Life Sciences . Growth inhibition and cell cycle analysis Cell counts were carried out on a Beckman Coulter Vi Cell Cell Viability Analyzer. The impact of belinostat on cell .