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| | | | | | Cell adhesion molecules (CAM) are multi-functional proteins involved in a number of regulatory processes, including cell growth, differentiation and proliferation, migration, and regeneration (Figure 1). Cell adhesion is crucial in the formation and maintenance of coherent multicellular structures. Two major types of cell adhesion processes are seen in multicellular organisms: cell-cell adhesion, where physical bonds are formed between adjacent cells; and cell-matrix adhesion, where cells bind to adhesive proteins in the extracellular matrix.
Cells detect their extracellular milieu via interactions with cell adhesion molecule (CAM) components via integrins and syndecan molecules and with adjacent cells via cadherins, selectins, and the members of the Ig-CAM family. Several adhesion molecules share similar structural features. The adhesive domains in fibronectins and immunoglobin (Ig) type adhesion proteins are structurally related and serve as building blocks in many adhesion proteins. Fibronectins also serve as ligands for the integrin family of adhesion receptors. They contain multiple repeats of 90 amino acid domains known as fibronectin type III (FN III) domains which possess the Arg-Gly-Asp (RGD) cell attachment site. Ig domains that are commonly found in cell-cell adhesion molecules are often found together with FN III domains. Some adhesion molecules, such as N-CAM, may contain both Ig and FN III domains.
The main families of adhesion receptors connect to the cytoskeleton inside the cell. Following their interaction with the ligand, adhesion receptors cluster in regions called focal adhesions. Adhesion receptors also function as signaling molecules. Focal adhesions are rich in tyrosine phosphorylated proteins that couple cell adhesion to signal transduction pathways in the cell. Various adhesion receptors are closely linked to protein kinases and phosphatases.
Integrins are transmembrane heterodimeric receptors composed of noncovalently associated a- and b-subunits. About 20 integrins have been identified thus far. A variety of integrins are shown to support adhesion-dependent growth factor-activation of MAP kinase. Ligand binding to integrin leads to the assembly of focal adhesion which induces tyrosine phosphorylation of a number of cytoskeletal components and signaling molecules. A number of protein tyrosine kinases are activated in focal adhesion as a result of integrin ligation. Focal adhesion kinase (FAK) is activated through autophosphorylation when cells attach through integrin. It is then phosphorylated by src kinase. A number of other signaling molecules subsequently bind to FAK and are phosphorylated, including Grb2, which links FAK to the Ras pathway, and the 85 kDa subunit of the PI 3-kinase (Figure 2). Thus, FAK is a key component in the assembly of focal contact structures that can influence cytoskeletal organization and signal transduction.
Cell adhesion receptors cooperate closely with growth factor receptors through physical linkages between them. For example, integrin avb3 is reported to associate closely with insulin receptor substrate-1, which is a cytoplasmic signal transduction mediator of insulin and IGF receptors. C-CAM, a member of the Ig superfamily, becomes phosphorylated on tyrosine in insulin-stimulated hepatocytes. N-CAM and L-1 and N-cadherin can activate FGF receptor and serve as pseudoligands for the FGF receptor, binding to it at recognition sequences that resemble their own recognition sites.
Cell migration is dependent upon a delicate balance between cell adhesion and cell detachment. Cell adhesion receptors and their ligands also provide traction and stimulus for cell migration. A cell can either adhere to a surface in such a way that it becomes immobilized or it can use the surface to migrate. The outcome depends largely on the effectiveness of attachment. Fibronectin is generally a favorable substrate for cells to migrate on both in vivo and in vitro. Cell-cell adhesion molecules also participate in cell migration. For example, PECAM, an Ig type cell-cell adhesion protein, is necessary for the penetration of leukocytes into capillary walls, and N-Cad and N-CAM mediate the outgrowth of neurites.
A detailed knowledge of mechanisms involved in cell adhesion is of considerable significance in studying cancer metastasis. Alterations in several classes of adhesion molecules have been implicated in the progression of various forms of cancers. An essential step in tumor progression is the interaction of tumor cells with extracellular matrix (ECM) and basement membrane (BM) leading to the destruction of these components. Transformed cells are shown to express higher levels of integrin receptors than their progenitor cells. Function of integrin receptors is under the control of N-glycosylation. De-N-glycosylation of a5b1 results in dissociation of a5 and b1 subunits and less binding to fibronectin. Ganglioside GM3 also affects integrin receptor function. It enhances fibronectin binding of a5b1. CD44 is another receptor involved in matrix-dependent cell adhesion and motility. Expression of a splicing variant, CD44-E, is strongly correlated with metastatic potential. N-Glycosylation of CD44, induced by transfection of a1→2 fucosyl-transferase gene, enhances tumor cell motility and tumorigenicity in rat colon carcinoma cells.
The N-linked glycosylation of E-cadherins is implicated in the modulation of cadherin-dependent tumor cell adhesion and release of tumor cells from tumor mass. An increase of bisecting b1→ 4GlcNAc to mannose core induced by GlcNAc -T III gene transfection reduces b1→ 6GlcNAc antenna resulting in overall structural change, enhanced E-cadherin activity, and reduced malignancy. An opposite effect is seen when GlcNAc-T V gene activity is enhanced. This causes b1→ 6GlcNAc antenna to form multi-antennary structures without bisecting GlcNAc. This decreases E-cadherin activity and a reduction in adhesion between tumor cells occurs resulting in release of tumor cells from tumor tissue mass, thereby producing metastasis.
Tumor cell-induced activation of platelets is also an important step in metastatic process. Expression of P-selectins in activated platelets of both P- and E-selectins in endothelial cells (EC) are well known. SLex, SLea, and their analogs have been regarded as epitopes for these selectins. These are well-established tumor antigens. When tumor cells come in contact with platelets, they stimulate them to express P-selectin and form platelet-tumor cell aggregates that leads to the adhesion of aggregates to EC, which in turn begins metastatic deposition.
Some glycosphingolipids (GSLs) at the tumor cell surface are anti-adhesive to each other and may induce the release of tumor cells from tumor mass, thereby promote metastasis. GM3 and GD3 at optimal concentrations enhance integrin-dependent adhesion, which promotes tumor cell invasion. Shedding of gangliosides from tumor cells is much higher than that from normal cells. Serum of cancer patients generally shows much higher levels of gangliosides. Shedding of gangliosides is also believed to reduce immune response both in in vitro and in vivo, which may lead to tumor growth by allowing tumor cells to “escape” the normal immune response.
Blocking of carbohydrate binding proteins (e.g., selectins) or inhibiting a carbohydrate-processing enzyme (glycosidase or glycosyltransferae) are some of the approaches available for cancer therapy, however, they are yet to reach any clinical stage. Tumor associated carbohydrate antigens (TACA) are functionally identified as adhesion molecules that bind lectins or selectins expressed on ECs or on other target cells. Complimentary carbohydrates expressed on target cells can also recognize TACAs. If carbohydrate- dependent tumor cell adhesion triggers or promotes invasion and/or metastasis, use of GSL or oligosaccharides (encapsulated in liposomes) can be used to block metastasis.
All selectins contain a carbohydrate-binding motif. E- and P-selectins bind to sialyl Lex present as a minor component in neutrophils. This binding slows down the movement of neutrophils along the endothelial wall, which then allows leukocytes to have adequate access to chemokines. Once chemokines bind to their receptors on leukocytes, a signal is transmitted through the G-proteins, which activates integrins on leukocyte surface, establishing a firm attachment to endothelia. Tumor cells, particularly carcinoma and leukemic cells, are enriched with sialyl Lex and sialyl Lea structures.
Highly metastatic colonic carcinoma cells bind more strongly to E-selectin expressed on activated endothelial cells than their low metastatic counter parts. The amount of sialyl Lex is directly correlated to the prognosis of patients. Those with higher levels of sialyl Lex display poor prognosis.
Aggressive social behavior of tumor cells is dependent upon enhanced b1→ 6GlcNAc antenna to form multi-antennary N-linked glycosylation expressed on cadherin, integrin, CD44, LAMP-1 and other receptors. Reducing or eliminating any modifications of N-glycosylations can reduce this aggressive behavior of tumor cells. Several inhibitors of N-glycosylation, such as such as Castanospermine (a a-glycosidase inhibitor; Cat. No. 218775), and Swainsonine (Cat. No. 574775), have been studied. However, castanospermine exhibits far greater toxicity compared to swainsonine. Swainsonine is shown to reduce a-mannosidase activity and N-glycosylation processing in MDAY-D2 mouse tumors. Another compound 1,6-epi-cyclophellitol inhibits a-glucosidase activity and is less toxic than castanospermine. Administration of D-PDMP (Cat. No. 513100) to tumor bearing mice is shown to inhibit GSL synthesis and reduce tumor growth and metastasis. It inhibits synthesis and shedding of GSLs. To be effective anti-tumor drug should be able to inhibit glucosylation of ceramide, which is the initial step in GSL synthesis. | | | | | | | |
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