No abstract

Summary Adhesion of cancer cells to vascular endothelium is an important step in haematogenous metastasis of cancer. A human hepatocellular carcinoma cell line, HepG2, strongly adheres to human umbilical vein endothelial cells (HUVECs) through the interaction of Eselectin and its carbohydrate ligand sialyl Lewis X. In this study, we investigated alteration in integrin expression on HepG2 cells, which follows the selectin-mediated initial adhesion of HepG2 cells to HUVECs. Expression of afil integrin was markedly increased when the HepG2 cells adhered to HUVECs. Among the tested cytokines that are known to be produced by endothelial cells, recombinant hepatocyte growth factor (rHGF) could replace the effect of HUVECs, and a similar increase in integrin expression was observed by the addition of 20 ng ml-' rHGF to HepG2. The increment of a2fil integrin expression was significantly inhibited by anti-HGF neutralizing antibody treatment. HepG2 cells expressed a2, a6, f, and 4 integrin subunits, but expression of integrins other than a2fi was not affected by the rHGF treatment.The rHGF treatment of HepG2 cells resulted in augmented adhesion to immobilized collagen. This augmentation in adhesion to collagen was completely blocked by the addition of anti-a2-or anti-,B-integrin antibody. In double-chamber chemoinvasion experiments, transmigration of the HepG2 cells through extracellular matrix (ECM) gel was significantly accelerated by co-cultivation with HUVECs. A similar level of enhancement in transmigration activity of the cancer cells was observed by the addition of rHGF. Our interpretation of the results described above is that the cancer cells received stimulation from cytokines, such as HGF, presented by vascular endothelial cells, following the initial adhesion of cancer cells via selectins. This resulted in the secondary increment in the expression of cell adhesion molecules, such as the a2f, integrin, and led to the augmented adhesive activities of cancer cells towards extracellular matrices at vascular walls. We suggest that this sequence of events is involved in the facilitated migration of some cancer cells to extravascular tissues.

The antisense cDNA approach was used to identify the endogenous fucosyltransferase species responsible for synthesis of the sialyl Lewis X (NeuAc␣233 Gal␤134[Fuc␣133]GlcNAc␤13 R) determinant in human lymphoid cells. The cultured human adult T-cell leukemia cell line, ED40515-N, expressed the message of ␣133 fucosyltransferase (Fuc-T) IV and VII, with a low level of the Fuc-T III and VI message, and manifested the sialyl Lewis X as well as Lewis X (Gal␤134 [Fuc␣133]GlcNAc␤13 R) determinant at the cell surface. Transfection of this cell line with the pRc/CMV vector containing an antisense human Fuc-T VII construct (pRc/CMV/5FT7AS) resulted in a significant decrease of endogenous Fuc-T VII message and a marked reduction in the cell surface expression of sialyl Lewis X determinant as well as a reduction in the enzymatic activity of ␣133 fucosyltransferase against sialylated type 2 chain substrate. This was accompanied by diminution of cell adhesive activity toward E-selectin on interleukin-1␤-treated endothelial cells. These results indicated that the synthesis of the sialyl Lewis X determinants that were functionally active as E-selectin ligands was mainly mediated by Fuc-T VII in these lymphoid cells. On the other hand, the message of Fuc-T IV showed no significant change in the transfectant clones, and the surface expression of the Lewis X antigen as well as the enzymatic activity of ␣133 fucosyltransferase against non-sialylated type 2 chain substrate was well preserved. The clear contrast between the diminished expression of sialyl Lewis X and the conserved manifestation of Lewis X in the transfectant clones suggested that the synthesis of sialyl Lewis X and that of Lewis X are independently regulated by different fucosyltransferases in human lymphoid cells. Fuc-T VII must be involved in the synthesis of sialyl Lewis X, while the synthesis of Lewis X is mediated by an enzyme other than Fuc-T VII, most probably Fuc-T IV.

The adhesion of cancer cells to the vascular endothelium is an important step in the hematogenous metastasis of cancer. Human breast cancer cells adhere to human umbilical vein endothelial cells (HUVECs) through the interaction of E selection on HUVECs and the carbohydrate ligand sialyl Lewisx on the cancer cells. We investigated the alteration of integrin expression on human breast cancer cells, following selectin-mediated initial adhesion to HUVECs. Four cell lines derived from human breast cancer expressed α₂-, α₃-, α₅ -, α₆-and β₁-integrins. The expression of (α₂β₁- and α₃β₁-integrins on BT-20 cells, strongly expressing epidermal growth factor (EGF) receptors, was markedly increased by addition of the heparin-binding EGF-like growth factor (HB-EGF). The expression of α₂β₁-integrin on SK-BR-3 cells also was increased by the addition of HB-EGF. However, no such effect of HB-EGF on the expression of integrins was observed in T-47D and MCF-7 cells, nor on expression of the EGF receptor. The increase of integrin expression in BT-20 cells was inhibited by the addition of the tyrosine kinase inhibitor genistein. HB-EGF treatment of BT-20 or SK-BR-3 cells resulted in the augmentation of cancer cell adhesion to immobilized collagen. When BT-20 cells were cocultured with HUVECs, a similar level of augmentation of cancer cell adhesion to collagen was observed. The augmentation of cancer cell adhesion to collagen was inhibited by addition of an anti-HB-EGF-neutralizing antibody. Our interpretation of the results described above is that the cancer cells receive stimulation from cytokines, such as HB-EGF, produced by vascular endothelial cells, following the initial adhesion of cancer cells via selectins. This results in a secondary increase in the expression of cell adhesion molecules, such as the Pi-integrin family, and leads to augmentation in the adhesive activities of cancer cells at the vessel walls. We postulate that these events are the ones involved in the enhanced transmigration of cancer cells to extravascular tissues following the selectin-mediated adhesion to the endothelium.

The adhesion molecules involved in the attachment of breast cancer cells to endothelial cells were investigated in vitro. All six human breast cancer cell lines expressed sialyl Lewis &supx; antigen(s-Le &supx;). Only two cell lines expressed sialyl Lewis&supa; antigen. A correlation was found between the degree of s-Le&supx; expression and the attachment of cancer cells to human umbilical vein endothelial cells (HUVECs) activated by IL-1beta. Monoclonal antibodies against s-Le &supx; or E-selectin inhibited this adhesion. These findings suggest that s-Le &supx; on the surface of cancer cells and E-selectin on the surface of endothelial cells play roles in adheision of breast cancer cells to vascular endothelium.