Novel oligosaccharide has suppressive activity against human leukemia cell proliferation

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with beta1-4, alpha1-6 and beta1-6 glycosidic bond were synthesized; Galbeta1-4GlcNH(2), Galalpha1-6GlcNH(2), Galalpha1-6GlcNAc, Galbeta1-6GlcNH(2), Galbeta1-4Galbeta1-4GlcNH(2) and Galbeta1-4Galbeta1-4GlcNAc. Galalpha1-6GlcNH(2) (MelNH(2)) and glucosamine (GlcNH(2)) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH(2). Adding Galalpha1-6GlcNH(2) or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galalpha1-6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galalpha1-6GlcNH(2) were stained. The difference in the stainability of the K562 cells by Galalpha1-6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galalpha1-6GlcNH(2) and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galalpha1-6GlcNH(2) binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (gamma) antibodies).