The long-term goal of the proposed study is to alter cell surface glycans that mediate cell-cell interactions and enhance cancer metastasis. We have described several modified disaccharides that permeate cell membranes, prime oligosaccharide synthesis by acting as alternate substrates (decoys), and alter the expression of sialyl Lewis X (sLe x) on cell surface glycoconjugates, sLe x constitutes part of the carbohydrate ligand recognized by cell adhesion molecules belonging to the family of selectin receptors on platelets, leukocytes, and endothelial cells, sLe x-containing ligands expressed on many types of carcinomas interact with selectins on these cells. This interaction is thought to facilitate tumor cell metastasis from primary tumors by promoting platelet-tumor cell aggregates, which lodge in the microvasculature of distant organs and form micrometastases. Patient survival studies after surgical resection of tumors indicate higher mortality for patients whose tumors type positive for sLe x and related antigens. Thus, the development of pharmacological agents to inhibit sLe x on tumor cells could improve patient survival. The hypothesis driving this research is that it should be possible to pharmacologically alter Lewis antigen presentation on tumor cells using disaccharide decoys. The specific objective of this proposal is to develop disaccharide analogs and to examine their therapeutic value for blocking cancer metastasis in mouse models. Positive results emerging from these studies will encourage us to translate our findings in model systems to phased trials in human cancer patients. Our five year goals include the following specific aims:
Aim 1. Synthesize disaccharide analogs of GIcNAcbetaa1,3GaI-R. We will make a series of analogs of N-acetyl-D-glucosaminea1,3-D-galactose (GlcNAcbeta3Gal) in which the 3', 4' and 6'-hydroxyl groups are missing, fluorinated, thiolated, alkylated, or aminated as potential inhibitors of sLe x formation. Analogs will be conjugated to various hydrophobic aglycones to determine the most effective derivative for cellular uptake and inhibitory activity. Radioactive forms of the disaccharides will be made to study the biodistribution of the compounds in mice.
Aim 2. Determine inhibitory activity of disaccharide analogs. Preliminary results show that peracetylated 4-deoxy- GlcNbetaca3Galbeta-O-naphthalenemethanol reduces the expression of sLex. The mode of action of this and other analogs will be examined in human LS 180 colon carcinoma cells and murine Lewis lung carcinoma (LLC) cells. Priming of oligosaccharides on the glycosides, alteration of endogenous glycoconjugate biosynthesis, and inhibition of selectin-mediated cell adhesion in vitro will be examined. Enzyme assays will establish if the compounds act as inhibitors of a4galactosyltransferase(s) involved in the biosynthesis of sLe/x.
Aim 3. Examine the therapeutic efficacy of disaccharide analogs on tumor metastasis and inflammation. Active compounds will be formulated and tested as inhibitors of experimental and spontaneous metastasis of LS 180 human colon carcinoma cells and syngeneic murine Lewis lung carcinoma. Solid tumor formation and inflammatory reactions will be analyzed to confirm the selective inhibitory action of the analog on tumor metastasis.
