ganglioside--gd1b and Breast-Neoplasms

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.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 7; Caspase 8; Caspase Inhibitors; Female; Galactosyltransferases; Gangliosides; Humans; MCF-7 Cells; Microscopy, Fluorescence; Oligopeptides; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le(x) biosyntheses [Basu, S (1991) Glycobiology, 1, 469-475; and ibid, 427-435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of (14)C-L-Serine. At lower concentrations (0-20 microM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both (14)C-sphingolipid and (14)C-ceramide was higher. However, at higher concentrations (20-100 microM), wherein apoptosis occurred in high frequency, the (14)C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Caspases; Cattle; Cell Line, Tumor; Enzyme Activation; Gangliosides; Humans; Microscopy, Fluorescence; Phosphatidylserines; Serine; Sphingolipids