Abstract A72: Assessment of pyuvate dehydrogenase kinase inhibition by dichloroacetate in human colon carcinoma cells by dynamic hyperpolarized 13C MRS and steady state 1H MRS
Authors: Chung Yuen-Li , Troy Helen , Judson Ian R. , Griffiths John R. , Leach Martin O. , Eykyn Tom 
Published: June 2012

Abstract
Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), which causes the activation of pyruvate dehydrogenase and increases glucose oxidation by promoting the influx of acetyl-CoA into the mitochondria and the Krebs cycle [1].
The aim of this work was to use hyperpolarised 1-13C pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to study the mechanism of action of DCA and to develop a non-invasive biomarker for response following PDK inhibition. Inhibition of PDK by DCA should cause a drop in lactate production. The hyperpolarised 13C experiment measures the exchange of labeled pyruvate for labeled lactate which has been shown to be proportional to lactate dehydrogenase activity and the availability of substrates as well as cellular NADH, the enzyme co-factor [2]. Conversely, steady state measurements of eupolarised (i.e. unlabelled) lactate production report on the overall flux through glycolysis.
Dynamic nuclear polarization (DNP) was used to hyperpolarise labelled 1-13C pyruvate, and to measure lactate formation in real-time in intact cells by 13C-MRS. Intact human HT29 (colon) carcinoma cells were studied after 24 hours of DCA treatment (100mM). Culture media and extracts from the DCA-treated and control cells were analysed by 1H-MRS. Cell cycle analysis was also performed.
DCA treatment of HT29 cells caused a drop in cell number (∼40% of controls, p&lt;0.0001) and G1 arrest (p&lt;0.0001). A dramatic drop in lactate formation (0.69+/−0.06 nmol/s/106 cells in control versus 0.06+/−0.01 nmol/s/106 cells in treated cells; p&lt;0.0001) was measured in real-time by DNP 13C-MRS. This could be due to the combined effect of: i) a drop in cellular NADH due to apoptosis, as DCA is known to induce apoptosis [1] which causes NADH loss [2]; and/or ii) an increase in glucose oxidation causing a decrease in NADH available for further metabolism of pyuvate to lactate. 1H spectra of the culture media of DCA treated cells also showed a reduction in steady state eupolarized lactate production (38% of controls; p&lt;0.0001), increased alanine uptake (p&lt;0.0001) and no difference in glucose uptake when compared with controls.
Increases in alanine (p&lt;0.0001), glucose (p=0.02), free choline (p=0.02) and glycerophosphocholine (p&lt;0.0001) and decrease in phosphocholine (p&lt;0.0001) were found in DCA-treated cell extracts. The phospholipid changes could be associated with modulation in membrane turnover, as DCA is known to reduce proliferation in cancer cells [1].
DCA treatment resulted in reduced lactate formation/production in HT29 cells, as shown by both real time and steady-state measurements. These changes are consistent with the mechanism of action and cellular response to PDK inhibition. DCA treatment also altered phospholipid metabolism, which could also be used as biomarkers of response.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A72.