Complexation of c6-ceramide with cholesteryl phosphocholine - a potent solvent-free ceramide delivery formulation for cells in culture

Abstract

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca(2+) influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca(2+) homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides.

Figure 1. Chemical structure of CholPC.

Figure 2. Cellular incorporation of [³H]C6-Cer or [³H]CholPC.

FRTL-5 cells were exposed for the indicated time to 0.05 mM of either [³H]C6-Cer/CholPC, [³H]C6-Cer/DMSO, or C6-Cer/[³H]CholPC. The uptake of lipids was normalized to cell protein, and each value is an average from 6 dishes±SEM. The zero time labeling was about 1300 cpm/dish (with each label), and was substracted from later time point values.

Figure 3. Effect of C6-Cer on cell proliferation.

A. FRTL-5 cells were preincubated for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. [³H]Thymidine incorporation into cellular DNA during the last 4 h was determined. B. Cell proliferation measurement using cell count. Effect of C6-Cer on cell proliferation on FRTL-5 was measured by counting the cells after preincubation for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. DMSO alone was used as control. Each value gives the amount of cells per plate. C. HeLa cells were incubated with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO for 12 h. [³H]Thymidine incorporation into cellular DNA during the last 4 h was determined. D. FRTL-5 cells were exposed to 0.05 mM L-erythro-C6-Cer/CholPC or C6-dihydroCer/CholPC for 24 h after which [³H]thymidine incorporation into cellular DNA during the last 4 h was determined. E. FRTL-5 cells were exposed for 48 h to 0.05 mM Chol/CholPC, Chol/DMSO, or Chol/m-β-cyclodextrin after which [³H]thymidine incorporation into cellular DNA during the last 4 h was determined. Each value is the mean ± SEM of at least 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 4. C6-Cer/CholPC disrupts cytosolic calcium homeostasis in HeLa cells.

The cells (panel A) were preincubated for 90 min with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO, and changes in intracellular Ca²⁺ levels were measured using Fura 2-AM. DMSO alone was used as control. The cells were stimulated as indicated by the arrows (150 µM EGTA, 1 mM Ca²⁺, and 10 µM histamine). The Ca²⁺ traces are averages from 33 individually measured cells that were randomly selected. In panel B, cells were instead treated with Chol/CholPC or DMSO (control) and stimulated with 10 µM histamine in calcium containing HBSS buffer. The Ca²⁺ traces are averages from 34 individually measured cells that were randomly selected. Panel C shows the quantification of the change in F340/F380 from basal to maximal values upon 10 µM histamine stimulation in panel A. The data were analyzed using one-way Anova and Bonferroni’s multiple comparison test (***p<0.001 compared with DMSO; ¤¤¤p<0.001 compared with C6-Cer).

Figure 5. C6-Cer/CholPC and C6-dihydroCer reduce mitochondrial calcium uptake.

HeLa cells were preincubated for 180 min with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO (panels A and B), and changes in intracellular Ca²⁺ levels were measured using mtAEQ. DMSO was added to control cells. Panel A shows kinetics of changes in mitochondrial Ca²⁺ after challenge with histamine. In panel B, the change in Ca²⁺ response was quantitated. The cells were challenged with 100 µM histamine as indicated by the arrow. Panel C shows kinetics of the Ca²⁺ response after180 min exposure of cells to C6-dihydroCer. Traces (panel A and C) are averages of 3 measurements, each representing the average luminescence from a cell population of 150 000–200 000 cells. In panel B, the bar shows the average change in [Ca²⁺]_mito during histamine-induced Ca²⁺ release (± SEM, n = 3). The data were analyzed using one-way Anova and Bonferroni’s multiple comparison test (***p<0.001 compared with DMSO; ¤¤¤p<0.001 compared with C6-Cer).

Figure 6. Induction of apoptosis in FRTL-5 cells by C6-Cer.

A. The cells were exposed for 48 hrs to C6-Cer/CholPC or C6-Cer/DMSO (0.05 mM), and the fraction of apoptotic cells was measured. Each bar value is the mean±SEM of 3 different experiments. *p<0.05, ***p<0.001. B. Induction of apoptosis in FRTL-5 cells by Chol/Chol-PC. The cells were exposed for 48 hrs to Chol/Chol-PC (0.05 mM), and the fraction of apoptotic cells was measured. Each bar value is the mean±SEM of 3 different experiments (NS = no significance).