Choline plasmalogens isolated from swine liver inhibit hepatoma cell proliferation associated with caveolin-1/Akt signaling

Abstract

Plasmalogens play multiple roles in the structures of biological membranes, cell membrane lipid homeostasis and human diseases. We report the isolation and identification of choline plasmalogens (ChoPlas) from swine liver by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC)/MS. The growth and viability of hepatoma cells (CBRH7919, HepG2 and SMMC7721) was determined following ChoPlas treatment comparing with that of human normal immortal cell lines (HL7702). Result indicated that ChoPlas inhibited hepatoma cell proliferation with an optimal concentration and time of 25 μmol/L and 24 h. To better understand the mechanism of the ChoPlas-induced inhibition of hepatoma cell proliferation, Caveolin-1 and PI3K/Akt pathway signals, including total Akt, phospho-Akt(pAkt) and Bcl-2 expression in CBRH7919 cells, were determined by western blot. ChoPlas treatment increased Caveolin-1 expression and reduced the expression of phospho-Akt (pAkt) and Bcl-2, downstream targets of the PI3K/Akt pathway. Further cell cycle analysis showed that ChoPlas treatment induced G1 and G1/S phase transition cell cycle arrest. The expression of essential cell cycle regulatory proteins involved in the G1 and G1/S phase transitions, cyclin D, CDK4, cyclin E and CDK2, were also analyzed by western blot. ChoPlas reduced CDK4, cyclin E and CDK2 expression. Taken together, the results indicate that swine liver-derived natural ChoPlas inhibits hepatoma cell proliferation associated with Caveolin-1 and PI3K/Akt signals.

Figure 1. Purification and Identification of ChoPlas.

A, Chromatogram of PC compositions. Kromasil C18 column; mobile phase, methanol-hexane-0.05 mol/L ammonium acetate-glycerol (V/V); column temperature, 35°C; injection, 20 μL; detection wavelength, 206 nm; flow rate, 1.0 ml/min; B, High Performance Thin Layer Chromatogram of ChoPlas. ChoPlas separated by HPLC was applied to the TLC plate and developed with chloroform/methanol/water (60:25:4, v/v, plate 1 bands were visualized by I₂ vapor and plate 2 bands were visualized by 5 nmol/L HgCl.

Figure 2. Structure Identification of ChoPlas.

A, Molecular characterization of ChoPlas on the MS/MS spectrum. The mass spectrum of swine liver ChoPlas in positive mode. Elution was performed as in Figure 1. Observed ions correspond to [M+H]+ (m/z 793.95) and ions resulting from fragmentation at the sites indicated in the structure formula of the [M+H]+ anion; B, Structure of the identified ChoPlas.

Figure 3. Average inhibition rate following treatment with ChoPlas as determined by MTT assay (24h and 48h).

CBRH-7919 cells (1×10⁴ cells/well) were seeded into a 96 well plate with medium supplemented with 20 % FBS and incubated for 24 h. Cells were then treated with 25 μmol/L of ChoPlas for 24 and 48 h. Viable cells were stained with MTT and the resulting formazan crystals were dissolved in DMSO. Absorbances at 490 nm were measured with a multiscan plate reader. Data are presented as the mean±S.D (n=3).

Figure 4. Effect of ChoPlas on Caveolin-1.

CBRH7919 cells were treated with 25 μmol/L ChoPlas for 24 h and then harvested. (A)Total protein was extracted and then blotted with caveolin-1antibody; (B) Graph depicting the semi-quantization of caveolin-1expression computed using Image J software. Data are presented as the mean±S.D(n=3).

Figure 5. Effect of ChoPlas on Akt/PI3K pathway proteins.

CBRH7919 cells were treated with 25 μmol/L of ChoPlas for 24 h and then harvested. (A)Total protein was extracted and then blotted with phosphor-Akt(Thr³⁰⁸), total Akt and Bcl-2 antibodies; (B) Graph depicting the semi-quantization of phosphor-Akt(Thr³⁰⁸), total Akt and Bcl-2 expression computed using Image J software. Data are presented as the mean±S.D (n=3).

Figure 6. Effect of ChoPlas on the cell cycle of cultured CBRH-7919 cells.

Cells were incubated with 25 μmol/L ChoPlas for 24 h. The cell cycle was analyzed by flow cytometry. A: Control. B: Vehicle . C: In the presence of ChoPlas.

Figure 7. Cell cycle regulator protein expression following ChoPlas treatment.

CBRH7919 cells were treated with 25 μmol/L ChoPlas for 24 h and then harvested. (A) Total protein was extracted and then blotted with Cyclin D1, CDK4, Cyclin E and CDK2 antibodies; (B) Graph depicting the semi-quantization of Cyclin D1, CDK4, Cyclin E and CDK2 expression computed using Image J software. Data are presented as the mean±S.D (n=3).

Figure 8. Hypothesized model for the proliferation inhibition mechanism of ChoPlas on hepatoma cells.

Exogenous natural ChoPlas activates caveolin-1, affecting PI3K/Akt pathway signals and decreasing cell cycle regulatory proteins and Bcl-2 expression.