Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells

Abstract

Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: β1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis.

Keywords: Gal-T2); apoptosis; caspase; disialyl-ganglioside GD1b; human breast cancer MCF-7 cells; human β1,3-galactosyltransferase-2 (GD1b synthase.

Figure 1

Structures of gangliosides and biosynthetic pathway of disialo GD1b.

Figure 2

Effect of various gangliosides on MCF-7 cell growth. (A) The cytotoxicity of various gangliosides on the MCF-7 cells has been examined using an XTT kit for cell growth assay. The cultured cells (approximately 1 × 10⁴ cells) in 96-well microplates (volume, 100 μL/well) for 24 h with various gangliosides were checked for the cytotoxicity; (B,C) The cytotoxicity of the GD1b on the MCF-7 cells was evaluated using an XTT cell proliferation assay kit. Cells were exposed to GD1b at various concentrations (0 to 80 μM) and also incubated with time course (1, 3, 6, 12 and 24 h). Control was treated with methanol (8 μL/100 μL) only. The absorbance at a wavelength of 490 nm was then measured using a digitalized ELISA reader (Molecular Devices, Sunnyvale, CA, USA). Data are reported as the percentage change in comparison with the control group, which were arbitrarily assigned as 100% viability. Data represent five experiments (means ± SD). * p < 0.01 vs. control.

Figure 3

Apoptosis of MCF-7 cells by GD1b treatment. (A) Double staining of the MCF-7 cells treated GD1b using annexin V and PI. Control (left) was treated with 80 µL/mL of methanol and right phannal was treated with 80 µM of GD1b for 24 h and stained with FITC-conjugated Annexin V (top) and PI (bottom) and then analyzed by flow cytometry; (B) Effect of GD1b on MCF-7 cells morphological changes (left). Morphological changes of control cells and cells treated GD1b were observed under a phase-contrast microscope with 100×. Apoptosis of MCF-7 cells treated with GD1b was stained with DAPI and Annexin V. Control (upon) was treated with 80 µL/mL of methanol. Cells sown in the lower pannel was treated with 80 µM of GD1b for 24 h and stained with FITC-conjugated Annexin V and DAPI and then analyzed by immunocytostaining (magnification 63×).

Figure 3

Apoptosis of MCF-7 cells by GD1b treatment. (A) Double staining of the MCF-7 cells treated GD1b using annexin V and PI. Control (left) was treated with 80 µL/mL of methanol and right phannal was treated with 80 µM of GD1b for 24 h and stained with FITC-conjugated Annexin V (top) and PI (bottom) and then analyzed by flow cytometry; (B) Effect of GD1b on MCF-7 cells morphological changes (left). Morphological changes of control cells and cells treated GD1b were observed under a phase-contrast microscope with 100×. Apoptosis of MCF-7 cells treated with GD1b was stained with DAPI and Annexin V. Control (upon) was treated with 80 µL/mL of methanol. Cells sown in the lower pannel was treated with 80 µM of GD1b for 24 h and stained with FITC-conjugated Annexin V and DAPI and then analyzed by immunocytostaining (magnification 63×).

Figure 4

Effects of GD1b treatment on cell cycle arrest and apoptosis. The lysates containing equal protein content were separated by SDS-PAGE and analyzed by western blotting. Expression of cell cycle arrest- (A) and apoptosis- (B) related molecules were examined of protein extracts of MCF-7 cells treated without (negative control with methanol) or with various concentrations of GD1b for 24 h. MCF-7 cells were treated with 50 µM GD1b for 24 h in the presence or absence of 20 µM Z-VAD and then analyzed by immunoblotting (C) with antibody PARP and capase-7. GAPDH was used as the internal control.

Figure 5

Establishment of GD1b synthase-transfected MCF-7 cell line. (A) Effect of GD1b synthase transfection on MCF-7 cells morphological changes. Morphological changes of MCF-7, vector- (pcDNA 3.1 vector) and GD1b (pcDNA 3.1 vector/GD1b synthase)-transfectant cells were observed under a phase-contrast microscope with 100×; (B) Expression levels of GD1b synthase of vector- and GD1b-transfectant cells were analyzed by RT-PCR. β-Actin was used as the internal control; (C) Gangliosides contents in vector- and GD1b-trnasfectant cells were analyzed by TLC; (D) Comparison of ganglioside contents in Mock (vector-transfected) MCF-7 cells and GD1b synthase-transfected MCF-7 cells. Percentage of each ganglioside was measured by densitometry with total gangliosides of each lane as 100%.

Figure 6

Effects of GD1b overexpression on the cell growth. The growth of vector- and GD1b-transfectant cells was evaluated using an XTT cell proliferation assay kit. Vector- and GD1b-transfectant cells were serum (+) incubated for 0, 12, 24 and 48 h. All data are reported as the percentage change in comparison with the control group, which were arbitrarily assigned 100% viability. Data represent five experiments (means ± sd). * p < 0.01 and ** p < 0.001 vs. vector (mock) transfectants, at each incubation time.

Figure 7

Effects of GD1b overexpression on cell cycle arrest and apoptosis. The cell lysates with equal protein content were subjected to SDS-PAGE and the separated proteins were analyzed by Western blotting using each antibody. Expression of cell cycle arrest- (A) and apoptosis- (B) related molecules were examined of protein extracts of Vector- and GD1b-transfectant cells. GAPDH was used as the internal control.

Figure 8

Schematic illustration of b-series gangliosides such as GD1b and GD3 for the apoptotic activities in breast cancer cells. The red square indicates b-series ganglioside during biosynthesis.