Characterization of peptides and phage that bind galectin-3 selected from bacteriophage display libraries: A study of the role of galectin-3 in metastasis-associated cancer cell adhesion.

摘要

Intravascular cancer cell adhesion plays a significant role in the metastatic process. Studies indicate that galectin-3, a member of the galectin family of soluble animal lectins, is involved in carbohydrate-mediated metastatic cell heterotypic (between carcinoma cells and endothelium) and homotypic (between carcinoma cells) adhesion via interactions with the tumor-specific Thomsen-Friedenreich glycoantigen (TFAg). The adhesion may also be attributed to galectin-3 multivalent binding of carbohydrate ligands via formation of di/oligomeric galectin-3. We hypothesized that the specific molecular inhibition of the interaction between galectin-3 and its ligands would reduce metastasis-associated carcinoma cell adhesion. To test this hypothesis, we identified peptide antagonists of galectin-3 using combinatorial bacteriophage (phage) display technology. The peptides bound with high affinity to purified galectin-3 protein (K d ≅ 17∼80 nM) and to cell surface galectin-3. Experiments with a series of recombinant serially truncated galectin-3 mutants indicated that the peptides bound the CRD of galectin-3. Furthermore, the peptides did not bind the CRD of other galectins and plant lectins. Synthetic galectin-3 CRD-specific peptides blocked the interaction between galectin-3 and TFAg and significantly inhibited rolling and stable heterotypic adhesion of human MDA-MB-435 breast carcinoma cells to endothelial cells under flow conditions, as well as homotypic tumor cell aggregation. Moreover, our biochemical studies demonstrated that one of our peptides, G3-C12, inhibited the dimerization of galectin-3 and, thus, blocked aggregation of latex bead cross-linked by dimeric or oligomeric galectin-3, indicating that dimerization of galectin-3 also plays a crucial role in cancer cell adhesion. These results demonstrate that carbohydrate-mediated metastasis-associated tumor cell adhesion could be inhibited efficiently with short synthetic peptides, which do not mimic naturally occurring glycoepitopes, yet bind to the galectin-3 CRD with high affinity and specificity.;In addition to characterization of the galectin-3 binding peptide, we also investigated the possibility that galectin-3 specific phage would target to tumors highly expressing the protein in mice. The phage harboring galectin-3 binding peptide failed to exclusively target to human MDA-MB-435 breast tumor heterotransplanted in mice, perhaps due to the wide-spread distribution of the protein, especially in the reticuloendothelial system (RES) organ tissue. However, the pharmacokinetic properties of random phage peptide display fd-tet libraries, in vivo, were fully investigated in order to gain a better understanding of the in vivo behavior of phage (Chapter 3). This part of my study will be extremely useful in the design of in vivo application strategies, as more and more in vivo studies with phage display libraries have been conducted to isolate tumor-avid molecules with a priori optimum in vivo properties.