CDP-choline accumulation in breast and colorectal cancer cells treated with a GSK-3-targeting inhibitor

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Poole Dorset). Cancer cell lines were purchased from ATCC (LGC Standards, Teddington, UK) and maintained in Dulbecco’s Modified Eagles medium (Gibco, Life Technologies ltd, UK) supplemented with 10% foetal bovine serum and penicillin (100µU/ml)/streptomycin (100 µg/ml) in 75 cm² tissue culture flasks until confluent. MDA-MB-468 breast cancer cells and HCT-8 colorectal cancer cells were obtained from the American Tissue Culture Collection (ATCC) (LGC standards UK).

The sensitivity of cancer cells to the GSK3 inhibitor SB216763 was determined using the MTT [(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazonium Bromide] assay. Briefly, cells were seeded in 96-well plates by adding 100 µl of cells per well at cell densities of 5 × 10⁴ per ml. The cells were left overnight at 37 °C in a CO₂ incubator, and then treated with a range of concentrations of SB216763 (1–200 µM) for 72 h. Cells were then quantified by addition of MTT (0.5 mg/ml in medium) and incubation for 2 h followed by removal of medium and addition of DMSO (200 µl). The absorbance was then measured at 570 nm using a scanning multi-well spectrophotometer (Dynatech MR 5000, Dynatech Laboratories, Chantilly,VA, USA) after 10-s agitation and the readings analysed using Ascent software. IC₅₀s were calculated using CompuSyn software (ComboSyn, Paramus, NJ, USA).

Cells (10⁶) in 5 ml of medium were seeded in eight 25 cm² tissue culture flasks and left overnight in a CO₂ incubator at 37 °C. The cells were then treated for 4, 24 or 72 h with SB216763 (40 µM) after which the medium was removed and fresh media (0.5 ml) containing 37KBq/ml of [³H-methyl] choline were added and incubated at 37 °C for 15 min. The cells were then washed 4 × by the rapid rinsing of each flask with ice-cold phosphate-buffered saline (5 ml). The cells were then incubated with medium (1 ml) for 1 h at 37 °C after which the medium was retained for measurement of radioactivity and the cells detached and transferred into a microfuge tube. The cells were pelleted by centrifuging at 400g for 5 min and the supernatant retained for radioactive counting. The cells were then lysed by addition of 0.375 ml of a 1:2 mixture of chloroform and methanol, and periodic agitation on a vortex mixer for 1 h. Then, 0.125 ml of TRIS: HCl buffer (1 mM pH 7.4) and 125 ml of chloroform were added. After shaking the mixture was centrifuged at 10,000g for 10 min and the upper (aqueous phase) and lower (lipid phase) were collected. The cell residue at the interface was dissolved in 0.5 ml of NaOH (1 M) and after neutralising with HCl (1 M) the protein content in the cell residue determined. The lipid phase was added to scintillation fluid (5 ml). The volume of the aqueous phase was determined and 100 µl was added to a scintillation vial containing 5 ml of scintillation fluid. A further 100 µl was subject to phosphate precipitation [13] by addition of 100 µl each of Ba(OH)₂ (0.3 M) and ZnSO₄ (5%) followed by vigorous shaking for 5 min. The precipitate was pelleted and 150 µl was added to a scintillation vial with 5 ml of scintillation fluid.

As described previously [14], cells were prepared as for ‘[³H] choline incorporation assay’ and cells were incubated with [¹⁴C-U] glucose in medium (0.5 ml) (3.7 KBq/ml) for 2 h. The cells were then washed 4 × with ice-cold PBS, detached with trypsin and transferred to a 1-ml microfuge tube and pelleted by centrifugation at 400g for 5 min. The supernatant was removed and radioactivity was determined by adding to a scintillation vial containing 5 ml of scintillation fluid and measuring in a scintillation counter. The pelleted cells were suspended in PBS (1 ml), pelleted and radioactivity determined in the wash. The lipid and aqueous components were then fractionated as described for ‘[³H-methyl] choline incorporation assay’. The lipid phase volume was measured and radioactivity determined in a 50-µl sample. The remainder was dried at 37 °C and the complex glycerides were subject to saponification by dissolving in a 1:10 water: ethanol solution of KOH (10 M) and heating to 70 °C for 20 min [15]. The glycerol and fatty acids were separated by fractionation into aqueous and lipid phases as described above [16] and the radioactivity was determined in the two phases.

Cells (2 × 10⁶) were seeded in each of four 75 cm² tissue culture flasks and allowed to achieve 80% confluence in a CO₂ incubator at 37 °C. Media were then removed and 10 ml of medium containing SB216763 (40 µM) was added to 2 flasks and 10 ml of medium containing the same volume of DMSO (vehicle—typically 10 µl) was added to the two control flasks. After the respective treatment period (24 or 72 h), the cells were detached with trypsin (5 ml) and the trypsin neutralised with ice-cold medium decanted into 15 ml tubes and placed on ice for 10 min. The cells were then centrifuged at 400g for 5 min and after removal of the medium, the cells were suspended in ice-cold saline (0.9%) (1 ml) transferred to a microfuge tube, centrifuged at 400g for 5 min. This step was repeated twice to remove extracellular phosphate. The pellet was suspended in 0.375 ml of chloroform: methanol (1:2) and 10 µl EDTA (10 mM) and 20 μl of 1-aminopropyl phosphoric acid (0.3 µmol) as an internal standard and left on ice for 1 h with occasional mixing. The aqueous and lipid phases were then separated by addition of 125 ml each of chloroform and TRIS buffer (10 mM pH7.4) followed by centrifugation at 12,000g for 10 min. The aqueous phase was then made up to 0.6 ml by addition of 60 µl of D₂O and water. The NMR measurement was carried out on a Bruker ADVANCE III 400 NMR spectrometer (400 MHz) operating at 161.98 MHz for ³¹P with continuous broadband ¹H decoupling (WALTZ16) and at least 20,000 acquisitions. The parameters for each acquisition were: acquisition time 1.258 s; relaxation delay 2 s (inter-pulse time = 3.258 s) (metabolite concentrations were the same when spectra were obtained with a 1-s relaxation delay, indicating that metabolites were fully relaxed in this solvent system after a 1-s delay [17]); pulse angle 45°. All acquisitions were made at 25 °C. Metabolite concentrations were determined by comparison with the internal standard (0.3 µmol) signal at 11.8 ppm. The reference for 0 ppm is 85% H₃PO₄.

Cells were seeded (10⁶ per flask) in 25 cm² flasks, then treated in triplicate when 70% confluent with SB216763. Glycogen synthesis was assessed by measuring conversion of [¹⁴C-(U)]-glucose into [¹⁴C-(U)]-glycogen in the cells treated with SB216763 for 4 h. The cells were incubated with 0.25 ml of [¹⁴C]-glucose (55 KBq/ml) for 90 min, washed rapidly with ice-cold PBS, then dissolved in 150 µl of KOH 20% (w/v) for 1 h at 70 °C. Glycogen was precipitated by addition of 50 µl of glycogen (2 mg/ml) as carrier followed by 0.5 ml of ice-cold ethanol and leaving for 2 h at − 20 °C. After centrifugation at 10,000g for 10 min, the supernatant was removed, and the precipitated glycogen dissolved in 150 µl of hot water, and transferred to scintillation vials containing 5 ml of scintillation fluid. The radioactive glycogen value was normalised to protein content carried out on 50 µl of the supernatant after neutralising the KOH with HCl (1 M).

Data are expressed as mean ± SEM and statistical differences determined by the student t test and values expressed as (t = x, p < y), where t values and probability are unitless values. All experiments were carried out 2 or more times.

The effect of 3-day treatment with different concentrations of SB216763 (0–100 µM) on the growth of SKBr3 cells is shown in Fig. 1. Subsequent experiments were carried out using 40 µM concentrations of SB213673.

Control cells and cells treated with SB216763 were subject to ³¹P NMR spectroscopy. Figure 2a–c shows an example of ³¹P NMR spectra from a chemical extract of control and SB216763-treated SKBr3 cells. A peak corresponding with CDP-choline which was at very low levels or undetectable levels in the controls was present at significantly higher levels (p < 0.05) in the cells treated with SB216763 for 24 h [control 0.002 µmol/mg protein (± 0.001), SB-treated 0.012 µmol/mg protein (± 0.003)] and was also present in cells treated for 72 h (Fig. 2c). The CDP-choline peak was also present in HCT8 colorectal cancer cells treated with SB216763 for 24 h (Fig. 2e) but not controls (Fig. 2d). CDP-choline is formed by reaction of phosphocholine (PCho) with cytidine triphosphate (CTP). The content of PCho was similar in control [0.1 (± 0.012) µmol/mg protein] and SB-treated [0.085 (± 0.03) µmole/mg protein] SKBr3 cells. The next step is the reaction of CDP-choline with diacylglycerol (DAG). As DAG can be formed, de novo, from the glycolysis intermediate glycerol 3 phosphate, the effect of SB216763 on carbon flux from glucose was examined.

Glucose incorporation into lipids was determined in SKBr3 and HCT8 cells. Figure 3 shows the incorporation of glucose into lipid (Fig. 3a) and fatty acids (Fig. 3b) and glycerol (Fig. 3c) in cells treated with SB216763 relative to untreated cells. In both cell lines, the incorporation of ¹⁴C- from glucose into lipid was decreased (SKBr3: t = 11.7, p < 0.001; HCT8 t = 8.2, p < 0.001) and this decrease was found to be in both the glycerol (derived from dihydroxyacetone phosphate during glycolysis) (SKBr3: t = 3.1, p < 0.01; HCT8: 7.3, p < 0.001) and fatty acid (derived from intermediates of the Krebs cycle) (SKBr3: t = 11, p < 0.001; HCT8: t = 9.3, p < 0.001) fractions.

Figure 4 shows the total uptake (a and b) and proportion of phosphorylated [³H-methyl] choline (c and d) and the incorporation into lipids (e and f) by SKBr3 and HCT8 cells treated with SB216763 for 4, 24 and 72 h compared with untreated cells. In each cell line, [³H-methyl] choline incorporation into lipids was decreased after 4 h (HCT8: t = 5.6, p < 0.001; SKBr3: t = 16, p < 0.001), 24 h (HCT8: t = 5.8, p < 0.001; t = 3.5, p < 0.01) and 72 h (HCT8: t = 6.6, p < 0.001; SKBr3: t = 28, p < 0.001) treatment with SB216763. The uptake of [³H-methyl] choline was unaffected by treatment of cells with SB216763 by each cell line at each time point except HCT8 cells at 72 h which showed a significant (t = 3.3 p < 0.05) increase in incorporation. The proportion of [³H-methyl] choline that was phosphorylated was unchanged in cells treated for 4 h, decreased in HCT8 (t = 4.1, p < 0.01) and SKBr3 (t = 18 p < 0.001) cells treated for 24 h and unchanged in cells treated for 72 h with SB216763.

The effect of treatment with SB216763 on glycogen synthesis cells is shown in Fig. 5. Conversion of [¹⁴C] glucose into [¹⁴C] glycogen was found to be increased by HCT8 cells treated with SB216763.