Effect of surface modified liposomes on the aggregation of platelets and tumor cells

Summary

Metastasis is still the most serious reason for the high mortality of cancer patients. It is a complex process in which platelets play a crucial role. Several attempts have been performed to inhibit the metastatic process, some of these using modified liposomes. The aggregation behaviour of human platelets and HT29 colon carcinoma cells in the presence of liposomes with a modified surface has been investigated in the present study. Liposomes (PC/CH/DMPE) were unmodified, sterically stabilized by polyethylene glycol (PEG-DSPE), or equipped with the carbohydrate ligand sialyl Lewis^X (conjugated to PEG-DMPE or DMPE as anchor) intended to specifically compete with ligands expressed by HT29 cells. We found in vitro that an addition of surface modified liposomes to human platelets in plasma caused an up to 2.9-fold increase in platelet aggregation. In addition, when HT29 tumor cells were mixed with platelets and surface modified lipo somes, the number of tumor cells found in aggregates increased significantly from 8.3 % (only tumor cells) to 30.2 %. This result was supported by fluorescence micrographs demonstrating a strong association of platelets and liposomes around the tumor cells. In addition, a clear decrease in number and a change in the distribution of metastases after intravenous injection of HT29 cells in combination with liposomes was observed in vivo. While in control mice metastases in lung, liver and in intestine were prevailing, liposomal treatment resulted in a new localization of metastases in muscles. Taking together, the ability of surface modified liposomes to enhance aggregate formation of platelets and tumor cells has been demonstrated for the first time. The capability of these vesicles to interfere with the metastatic process might have implications for the use of such liposomes for therapeutic applications.

Keywords

Liposomes - sialyl Lewis X - platelets - metastasis

^* Present address: Department of Molecular Biotechnology, Anhalt University of Applied Sciences, Köthen, Germany

• References

• 1 Kannagi R. Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer. Glycoconj J 1997; 14: 577-84.
• 2 Saiki I, Koike C, Obata A. et al. Functional role of sialyl Lewis X and fibronectin-derived RGDS peptide analogue on tumor-cell arrest in lungs followed by extravasation. Int J Cancer 1996; 65: 833-9.
• 3 Honn KV. Platelets and cancer metastasis: a causal relationship?. Cancer Metastasis Rev. 1992: 325-51.
• 4 McCarthy JB. Tumor cell adhesive mechanisms and their relationship to metastasis. Semin Cancer Biol 1991; 2: 155-67.
• 5 Zhou J. The role of adhesive proteins in the hematogenous spread of cancer. In Vivo 2000; 14: 199-208.
• 6 Borsig L, Wong R, Feramisco J. et al. Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis. Proc Nat Acad Sci USA 2001; 98: 3352-7.
• 7 Taucher S, Salat A, Gnant M. et al. Impact of pretreatment thrombocytosis on survival in primary breast cancer. Thromb Haemost 2003; 89: 1098-106.
• 8 Amirkhosravi A, Mousa SA. et al. Amaya,M. Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454. Thromb Haemost 2003; 90: 549-54.
• 9 Bakewell SJ, Nestor P, Prasad S. et al. Platelet and osteoclast beta3 integrins are critical for bone metastasis. Proc Nat Acad Sci USA 2003; 100: 14205-10.
• 10 Koike C, Watanabe M, Isoai A. et al. How platelet aggregation affects B16BL6 melanoma cell trafficking. FEBS Lett 1998; 427: 286-90.
• 11 Borsig L, Wong R, Hynes RO. et al. Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis. Proc Nat Acad Sci USA 2002; 99: 2193-8.
• 12 Metzig C, Grabowska E, Eckert K. et al. Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo. In Vivo 1999; 13: 7-12.
• 13 Brown JR, Fuster MM, Whisenant T. et al. Expression patterns of alpha 2,3-sialyltransferases and alpha 1,3-fucosyltransferases determine the mode of sialyl Lewis X inhibition by disaccharide decoys. J Biol Chem 2003; 278: 23352-9.
• 14 Fuster MM, Brown JR, Wang L. et al. A disaccharide precursor of sialyl Lewis X inhibits metastatic potential of tumor cells. Cancer Res. 2003; 63: 2775-81.
• 15 Bloemen PG, Henricks PA, vanBloois L. et al.. Adhesion molecules: a new target for immunoliposomemediated drug delivery. FEBS Lett 1995; 357: 140-4.
• 16 Kessner S, Krause A, Rothe U. et al. Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells. Biochim Biophys Acta 2001; 1514: 177-90.
• 17 Mastrobattista E, Storm G, vanBloois L. et al.. Cellular uptake of liposomes targeted to intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelial cells. Biochim Biophys Acta 1999; 1419: 353-63.
• 18 Vodovozova EL, Moiseeva EV, Grechko GK. et al.. Antitumoractivity of cytotoxic liposomes equipped with selectin ligand SiaLe(X), in a mouse mammary adenocarcinoma model. Eur J Cancer 2000; 36: 942-9.
• 19 Stahn R, Schafer H, Kernchen F. et al. Multivalent sialyl Lewis x ligands of definite structures as inhibitors of E-selectin mediated cell adhesion. Glycobiology 1998; 8: 311-9.
• 20 Zeisig R, Stahn R, Wenzel K. et al. Effect of sialyl Lewis X-glycoliposomes on the inhibition of E-selectin- mediated tumorcell adhesion in vitro. Biochim Biophys Acta 2004; 1660: 31-40.
• 21 Kaila Thomas BE. 4^th. Design and synthesis of sialyl Lewis(x) mimics as E- and P-selectin inhibitors. Med Res Rev 2002; 22: 566-601.
• 22 Zeisig R, Müller K, Maurer N. et al. The composition- dependent presence of free (micellar) alkylphosp holipid in liposomal formulations of octadecyl-1,1-dimethyl- piperidino-4-yl-phosphate affects its cytotoxic activity in vitro. J Membr Biol 2001; 182: 61-9.
• 23 Walkowiak B, Kesy A, Michalec L. Microplate reader–a convenient tool in studies of blood coagulation. Thromb Res 1997; 87: 95-103.
• 24 Puri RN. ADP-induced platelet aggregation and inhibition of adenylyl cyclase activity stimulated by prostaglandins: signal transduction mechanisms. Biochem Pharmacol 1999; 57: 851-9.
• 25 Engers R, Gabbert HE. Mechanisms of tumor metastasis: cell biological aspects and clinical implications. J Cancer Res Clin Oncol 2000; 126: 682-92.
• 26 Sliva D. Signaling pathways responsible for cancer cell invasion as targets for cancer therapy. Curr Cancer Drug Targets 2004; 4: 327-36.
• 27 Awada A, Cardoso F, Atalay G. et al. The pipeline of new anticancer agents for breast cancer treatment in 2003;. Crit Rev Oncol Hematol 2003; 48: 45-63.
• 28 Easton EW, Schiphorst WE, vanDrunen E. et al. Human myeloid alpha 3-fucosyltransferase is involved in the expression of the sialyl-Lewis(x) determinant, a ligand for E- and P-selectin. Blood 1993; 81: 2978-86.
• 29 Zogakis TG, Libutti SK. General aspects of anti-angiogenesis and cancer therapy. Expert Opin Biol Ther 2001; 1: 253-75.
• 30 Ross EL, Barker JN, Allen MH. et al. Langerhans’ cell expression of the selectin ligand, sialyl Lewis x. Immunology 1994; 81: 303-8.
• 31 McEver RP. Selectin-carbohydrate interactions during inflammation and metastasis. Glycoconj J 1997; 14: 585-91.
• 32 Moss MA, Zimmer S, Anderson KW. Role of metastatic potential in the adhesion of human breast cancer cells to endothelial monolayers. Anticancer Res 2000; 20: 1425-33.
• 33 Vodovozova EL, Gayenko GP, Razinkov VI. et al. Saccharide-assisted delivery of cytotoxic liposomes to human malignant cells. Biochem Mol Biol Int 1998; 44: 543-53.
• 34 Shibata K, Hiruma K, Kanie O. et al. Synthesis of 1,1-linked galactosyl mannosides carrying a thiazine ring as mimetics of sialyl Lewis X antigen: investigation of the effect of carboxyl group orientation on P-selectin inhibition. J Org Chem 2000; 65: 2393-8.
• 35 Shirota K, Kato Y, Irimura T. et al. Anti-metastatic effect of the sialyl Lewis-X analog GSC-150 on the human colon carcinoma derived cell line KM12-HX in the mouse. Biol Pharm Bull 2001; 24: 316-9.
• 36 Stahn R, Zeisig R. Cell adhesion inhibition by glycoliposomes: Effects of vesicle diameter and ligand density. Tumor Biology 2000; 21: 176-86.
• 37 Chambers AF, MacDonald IC, Schmidt EE. et al.. Steps in tumor metastasis: new concepts from intravital videomicroscopy. Cancer Metastasis Rev 1995; 14: 279-301.
• 38 Mannaioni PF, Di Bello MG, Masini E. Platelets and inflammation: role of platelet-derived growth factor, adhesion molecules and histamine. Inflamm Res 1997; 46: 4-18.
• 39 Price ME, Cornelius RM, Brash JL. TI: Protein adsorption to polyethylene glycol modified liposomes from fibrinogen solution and from plasma. Biochim Biophys Acta 2001; 1512: 191-205.
• 40 Chiu GN, Bally MB, Mayer LD. Effects of phosphatidylserine on membrane incorporation and surface protection properties of exchangeable poly(ethylene glycol)-conjugated lipids. Biochim Biophys Acta 2002; 1560: 37-50.
• 41 Potts JR, Campbell ID. Structure and function of fibronectin modules. Matrix Bio 1996; 15: 313-20.
• 42 Hiller E. Hämostase und maligne Erkrankungen. Target Forum 2000; 3: 31-40.
• 43 Stensrud G, Monkkonen J, Karlsen J. Toxicity of gamma irradiated liposomes. 2. In vitro effects on cells in culture. Int J Pharm 1999; 178: 47-53.