Tissue Factor Regulation, RhoA Localization, and Induction of Apoptosis in Human Cancer Cell Lines upon Simvastatin Treatment

Abstract number: OR038

Åberg¹ M, Tammik² O, Tenno^1,2 T, Wickström³ M, Siegbahn¹ A

^{1,2 11}Department of Medical Sciences, Clinical Chemistry, Uppsala University Hospital, Sweden ²²Department of Hematology and Oncology, University of Tartu, Estonia ³³Department of Medical Sciences, Clinical Pharmacology, Uppsala University Hospital, Sweden

Tissue factor (TF) is the main initiator of the coagulation cascade. Cancer confers a prothrombotic state and some malignant cells express TF, while others produce TF up-regulating cytokines. RhoA is primarily related to cytoskeletal regulation but also to tumorigenesis. We investigated whether some commonly used drugs affected human cancer cell lines regarding TF regulation and cell survival.

Methods:  Breast (MDA-MB-231, MCF-7), prostate (DU145, PC3), and glioma (U410-MG) cancer cell lines were incubated for 96 h together with simvastatin (5 µM), enalapril (20 µg/ml), or dalteparin (10 U/ml). TF mRNA and surface expression were determined using RT-PCR and flow cytometry. For apoptosis, the cell lines were incubated for 8, 24, 48, and 72 h with the same drugs and with staurosporine (1 µM) as positive control. Caspase-3 and nuclear fragmentation, size, and shape were measured using an Arrayscan II HCS reader. Finally, cells were incubated for 4 and 24 h with the drugs and RhoA delocalization was estimated by confocal microscopy.

Results:  Simvastatin significantly down-regulated TF mRNA and surface expression in U410-MG, MDA-MB-231, and PC3, however, a trend towards down-regulation could also be noted in MCF-7 and DU145. Highly significant changes in caspase-3 activity, shrinking of the nucleus and nuclear fragmentation could be detected in U410-MG, MDA-MB-231, and PC3 after 48 h of incubation with simvastatin. A clear delocalization of active RhoA from the cell surface was observed in MDA-MB-231 and PC3 cells already 4 h after addition of simvastatin. Dalteparin and enalapril had little or no effect at all upon TF, apoptosis and RhoA.

Conclusion:  Simvastatin down-regulated TF in all cell lines. However, only the best responders, MDA-MB-231, U410-MG, and PC3 went into apoptosis and had delocalized RhoA. These results indicate there are several different mechanisms involved.