The Molecular Mechanisms Of Modified Heparin Inhibits P-selectin-mediated Adhesion Of Breast Carcinoma Cells

Selectin is one of the cell adhesive molecular families, and it mediates the adhesion of leukocytes to vascular endothelial cells, leukocytes to leukocytes, and leukocytes to platelets in inflammation. Recently, accumulating evidence indicate that selectins are involved in the interaction among tumor cells, platelets and endothelial cells in the hematogenous metastasis. The interaction among tumor cells, platelets and endothelial cells is a key step during the metastatic process.It is well known that hematogenous metastasis is a highly regulated process and a cascade of dynamic events involving many factors. A large amount of adhesion molecules play crucial roles in metastasis such selectins and integrins. Selectins are primarily responsible for the first step in cell adhesion at the beginning of the metastssis, however, their roles to the overall metastatic process are essential. P-selectin, one member of the selectin family, was reported to involve in the interaction between tumor cells and platelets or endothelial cells. Therefore, the interference of the interaction between selectins and their ligands may lead to inhibiting metastasis to some extend. So it is envisaged that P-selectin may be a potential target for tumor therapy.P-selectin plays its physiologic role by interacting with its counterpart ligand. To our know, all high-affinity ligands to selectin almost have specific carbohydrate structures, including Lewis tetrasaccharide, P-selectin glycoprotein ligand-1, heparan sulfate, chondroitin sulfate and sulfated glycoproteins or glycolipid. At present, a large amount of studies focused on agonists to P-selectin. These agonists included antibodies and mimics of ligands for P-selectin. Of the P-selectin antagonists, heparins have shown promising results in inhibiting the adhesion of P-selectin to tumor cells in vitro and in vivo. However, the mechanism by which agonists inhibit the interaction between tumor cells and platelets remains to be elucidated.Heparin is a highly sulfated member of the glycosaminoglycan family and has been an excellent anticoagulant in clinical uses. However, its anticoagulant properties and the potential of bleeding complications may contraindicate applications of heparin in anti-metastasis therapy. Thus, chemically modified heparins with diminished anticoagulant activities were prepared and proved to inhibit metastasis in vitro and in vivo. Though the mechanisms by which heparin inhibit metastasis remains ambiguous, chemically modified heparins with diminished anticoagulant activities are still one kind of potential therapeutic compounds considering the studies results from the animal model of anti-metastasis and the clinical therapy.Breast cancer is one of malignant tumor in women, and the mobility in increasing progressively by rate of 2% each year. It is common that the invasion and metastasis of breast cancer is the main reason for poor prognosis. As has been documented, P-selectin binds to human breast cancer cells. Whether this interaction can be blocked by chemically modified heparins with diminished anticoagulant activities or what kinds of P-selectin ligands are expressed on breast cancer cells remains to be further studied. In this study, we prepared 4 heparin derivatives (RO-Heparin, CR-Heparin, 2/3ODS-Heparin and N/2/3DS-Heparin) by means of chemical modifications. We selected human breast cancer cell lines ZR-75-30 and analyzed the interaction between the cell lines and P-selectin, CHO cells expressing a transfected human P-selectin cDNA and platelets expressing P-selectin under static and flow conditions. Furthermore, heparin and various modified heparins were evaluated for their ability to inhibiting P-selectin-dependent interactions. The results indicate that P-selectin binds to the breast cancer cell under static and flow conditions. Heparin and chemically modified heparins could significantly inhibit the interaction between these cell lines and P-selectin, CHO cells expressing a transfected human P-selectin cDNA and platelets expressing P-selectin under static and flow conditions. Then, to further investigate the mechanism of the interaction, we explored the carbohydrate structure of P-selectin ligand on the tumor cell surface by using specific mAb, treatment with heparinase and flow cytometric analysis. Flow cytometric analysis showed that ZR-75-30 cells expressed the moderate level of heparan sulfate proteoglycans on their cell surfaces. However, treatment with heparinase had no effect on P-selectin binding indicating that the P-selectin ligands on the ZR-75-30 cell surface are not heparan sulfate-like proteoglycans structure. To further analyze the P-selectin ligands on the ZR-75-30 cell surface, treatment assay with neuraminidase and typsin indicated that P-selectin binds to unsialylated ligands on ZR-75-30 cells. Inhibition of sulfate biosynthesis assay indicated that sulfate content of membrane protein had effects on the interaction of P-selectin and ZR-75-30 cells. In view of sulfate content of the modified heparins compared with heparin control, the degree of sulfation of modified heparin is positively correlated with inhibition abilities of the interaction between P-selectin and ZR-75-30 cells. Whether modified heparins have more extensive applications in blocking breast cancer metastasis involving P-selectin requires to be further exploited.In conclusion, the results in this study indicated that 4 chemically modified heparins (RO-Heparin, CR-Heparin, 2/3ODS-Heparin and N/2/3DS-Heparin) could inhibit P-selectin-mediated adhesion to breast cancer cells. However, the P-selectin ligands on the ZR-75-30 cell surface are not heparan sulfate-like proteoglycans structure. The study will provide a good target for applications of chemically modified heparins in clinical therapy of blocking breast cancer metastasis and provide new evidence for further insight into mechanisms of the interaction between P-selectin and its counterpart ligand.