Lipid metabolism in large T47D human breast cancer spheroids: 31P- and 13C-NMR studies of choline and ethanolamine uptake

Abstract

31P- and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300 μm) spheroids. Spheroids were perfused inside the spectrometer with 1,2- 13C-labelled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4±1 fmol/cell compared to 8±1 fmol/cell in small (150 μm) proliferating spheroids ( P < 0.0002). The average PC pool size at steady state was reduced to 11±6 fmol/cell compared to 22±8 ( P < 0.007). This could be correlated with an overall reduction of choline uptake in the non-proliferating spheroid fraction. The rate of the enzyme choline kinase was 0.3 fmol/(cell h) compared to 1.0 fmol/(cell h) ( P < 0.0001) for proliferating cells. The rate constant of CTP: phosphocholine cytidyltransferase (0.05 h −1) was not significantly altered, but the rate of the enzyme was reduced from 1.3 to 0.2–0.5 fmol/(cell h). The pool size of PE in medium containing serum ethanolamine (1.7 μM) was approximately the same (15 fmol/cell) in small and large spheroids. In the presence of high ethanolamine (0.028 mM) the average PE level decreased slightly (11 fmol/cell) and the rate of the enzyme ethanolamine kinase in the non-proliferating fraction was 0.7 fmol/(cell h) versus 1.0 fmol/(cell h) in the proliferating cells ( P < 0.07). The rate constant of CTP: phosphoethanolamine cytidyltransferase (0.07 h −) was not significantly altered but the corresponding reaction rate was reduced from 1.4 to 0.2–0.8 fmol/(cell h). The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine.