Abstract 673: Use of membrane-tethered anti-thrombin compounds in prostate cancer therapy

Abstract

Abstract The tissue factor-thrombin pathway is known to mediate proliferation, adhesion, invasion and metastasis of many types of cancer cells. These effects are additional to the thromboembolic processes commonly occurring in cancer, particularly in patients on hormonal therapies. In prostate cancer, there is an overexpression of the thrombin receptor PAR1 correlating with tumor progression. Anticoagulants such as heparin and hirudin are known to inhibit metastasis of tumors and improve survival rates in prostate cancer patients with localized disease. However, anticoagulants given systemically have an associated risk of bleeding which is higher in cancer patients and can be compounded by an increased risk of thrombocytopenia. Anticoagulants which can be localized to a tumor lesion are therefore highly favorable. The aim of the following study was to determine the efficacy of membrane-tethered anticoagulants on thrombin mediated migration and proliferation of prostate cancer cells in vitro. The hirudin cytotopic analogs PTL004 and PTL006, previously designed for use with anti-complement therapeutics in transplantation, were assessed as inhibitors of proliferation and migration of prostate cancer cells, in comparison to non-tailed hirudin-derived peptide, a PAR-1 antagonist and hirudin itself. Methods: For proliferation assays, 2000 PC3 prostate cancer cells were seeded in 96 well plates in serum free culture medium. Cells were preincubated for 30 mins with anticoagulant inhibitors at concentrations from 1 µM to 0.001 µM, and then 0.5 nM thrombin was added. Proliferation was measured after 48 hrs using MTT. To measure effects on cell migration, 10,000 PC3 cells were seeded onto porous membrane inserts in 24 well plates in serum free medium and then incubated with the inhibitors at 1 µM for 30 mins followed by 0.5 nM thrombin. Migration of cells through the membrane was measured after a 24 hr period. Results: Thrombin increased proliferation of PC3 cells in serum-free medium with a 1.5 fold increase in cell number after 48h. The tailed derivative PTL004 inhibited proliferation with an IC50 of 1 µM, equivalent to hirudin and the PAR1 antagonist. Liposomes prepared with PTL004 on the surface were more active than the tailed preparation in free solution with an IC50 10-50 fold lower. However, the non-tailed hirudin-derived peptide, and the derivative PTL006, which is known to have a more rapid dissociation rate when used with other cell types, were not active under these assay conditions. In the migration assays, PTL004 was also most active among the derivatives we tested. Conclusions: The membrane localizing anti-coagulant PTL004 has comparable anti-migratory and anti-proliferative properties to hirudin, and liposomes bearing this compound are even more effective at inhibition of proliferation of PC3 cells. The results demonstrate the potential of a cytotopic approach using tailed anticoagulants in localized prostate cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2011-673