i(3)so3-galactosylceramide and Carcinoma

Carcinoma tumor cells express highly glycosylated mucins acting as ligands for selectin adhesion receptors and thus facilitating the metastatic process. Recently, a sulfated galactocerebroside SM4 was detected as solely P-selectin ligand on MC-38 colon carcinoma cells. Here we characterize the functionality of SM4 as selectin ligand using model membrane approaches. SM4 was found concentrated in lipid rafts of MC-38 cells indicating a local clustering that may increase the avidity of P-selectin recognition. To confirm this, SM4 was incorporated at various concentrations into POPC model membranes and lateral clustering was analyzed by fluorescence microscopy and found to be comparable to glycolipids carrying the sLe(x) epitope. SM4 containing liposomes were used as cell models, binding to immobilized P-selectin. Quartz crystal microbalance data confirmed SM4/P-selectin liposome binding that was inhibited dose-dependently by heparin. Comparable binding characteristics of SM4 and sLe(x) liposomes underscore the similarity of these epitopes. Thus, clustering of SM4 on tumor cells is a principle for binding P-selectin.

Topics: Animals; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Galactosylceramides; Heparin; Liposomes; Membrane Microdomains; Mice; P-Selectin; Protein Binding; Sulfoglycosphingolipids

Hematogenous carcinoma metastasis is associated with tumor cell emboli formation, which is now known to be facilitated by selectins. P-selectin-mediated interactions of platelets with cancer cells are based mostly on mucin- and glycosaminoglycan-type selectin ligands. We previously showed that mouse colon carcinoma cells (MC-38) carry P-selectin ligands of nonmucin origin, which were not identified. Here we show that P-selectin ligands recognized on MC-38 cells are sulfated glycolipids, thereby facilitating experimental metastasis in a syngeneic mouse model. Metabolic inhibition of sulfation by incubation of cells with sodium chlorate almost completely abrogated P-selectin binding. Metabolic labeling of MC-38 cells with (35)S sulfate revealed only a single band as detected by high-performance thin layer chromatography analysis of a total lipid extract. Matrix-assisted laser desorption/ionization tandem time-of-flight/time-of-flight analysis (MALDI-TOF-TOF) analysis of the purified sulfate-containing lipid fraction identified the selectin ligand to be a sulfated galactosylceramide SM4 (HSO(3)-3Galbeta-1Cer). Modulation of glycolipid biosynthesis in MC-38 cells altered P-selectin binding, thereby confirming sulfoglycolipids to be major P-selectin ligands. In addition, P-selectin was also found to recognize lactosylceramide sulfate SM3 (HSO(3)-3Galbeta-4Glcbeta-1Cer) and gangliotriaosylceramide sulfate SM2 [GalNAcbeta-4(HSO(3)-3)Galbeta-4Glcbeta-1Cer] in human hepatoma cells. Finally, the enzymatic removal of sulfation from the cell surface of MC-38 cells resulted in decreased P-selectin binding and led to attenuation of metastasis. Thus, SM4 sulfatide serves as a native ligand for P-selectin contributing to cell-cell interactions and to facilitation of metastasis.

Topics: Animals; Carcinoma; Cell Communication; Disease Progression; Humans; Ligands; Mice; Neoplasm Metastasis; P-Selectin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfoglycosphingolipids; Tumor Cells, Cultured