Anthracycline Treatment of the Monocytic Leukemia Cell Line THP-1 Increases Phosphatidylserine Exposure and Tissue Factor Activity.

Abstract

1136 Introduction: Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. While malignancy alone conveys an increased risk for thrombosis, treatment with cytotoxic chemotherapeutics further elevates this risk. However, the pathogenic mechanisms underlying this process remain poorly defined. We hypothesized that treatment of tumor cells with cytotoxic chemotherapeutic drugs leads to phosphatidylserine (PS) exposure on the cell surface, as well as increased cellular tissue factor (TF) activity and release of TF positive microparticles (MPs). Aim: The aim of this study was to determine the effect of anthracyclines on TF activity and MP release from the human monocytic leukemia cell line THP-1. Materials and Methods: THP-1 cells were treated with different concentrations of doxorubicin (0-10μg/ml) or daunorubicin (0-1μg/ml) for 0–24 hours. These concentrations are similar to those found in plasma of cancer patients. The intact cells were analyzed for TF activity using a two stage chromogenic assay. TF antigen was evaluated by ELISA and TF mRNA by realtime PCR. In addition, TF activity of MPs released into the culture supernatant was measured using a two stage chromogenic assay. PS exposure on cells and MPs were analyzed by flow cytometry and PS exposure on MP was also evaluated in an annexin V capture assay. Cell viability was assayed by a dye exclusion assay as well as by flow cytometry. Results: We found that doxorubicin and daunorubicin increased PS exposure on the cell surface. Treatment with anthracyclines increased both cellular TF and MP TF activity in a concentration-dependent manner that also resulted in concentration dependent increase in apoptosis. Furthermore, this increased TF dependent activity was abolished with the addition of annexin V or lactadherin supporting a role of PS exposure in this elevated activity. Conclusion: These results support our hypothesis that treatment of THP-1 cells with chemotherapeutic agents induces apoptosis and increases cellular TF activity. This increased activity appears primarily mediated by an increase in cell surface expression of PS. Additionally, the anthracyclines increase the release of TF positive MPs from the THP-1 cells. We propose that this increase in cellular TF activity in tumor cells and increased numbers of TF positive MPs may contribute to thrombosis in cancer patients treated with chemotherapeutic drugs. Disclosures: No relevant conflicts of interest to declare.