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The thrombin inhibitor Argatroban reduces breast cancer malignancy and metastasis via osteopontin-dependent and osteopontin-independent mechanisms

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Osteopontin (OPN) has been clinically and experimentally associated with breast cancer metastasis. Proteolytic cleavage of OPN by thrombin has been reported to increase its biologic activity. The purpose of this study was to determine if inhibition of thrombin could reduce the malignancy-promoting effects of OPN on breast cancer cell behavior in vitro and in vivo. MDA-MB-468 human breast cancer cells were stably transfected to overexpress OPN (468-OPN) or a control vector (468-CON) and compared for functional differences in malignant/metastatic behavior in response to treatment with the thrombin-specific inhibitor Argatroban. Western blot analysis revealed that both 468-CON and 468-OPN cells produce thrombin and the thrombin-related protein tissue factor, and express very low levels of thrombin receptor (PAR-1). In vitro assays demonstrated that Argatroban treatment (25 μg/ml) of 468-OPN cells resulted in decreased cell growth, colony-forming ability, adhesion, and migration relative to untreated controls (P < 0.05), but did not have a significant effect on 468-CON cells. Following mammary fat pad injection, treatment with Argatroban (9 mg/kg/day) increased the in vivo tumor latency of both 468-CON and 468-OPN cells, and reduced primary tumor growth of 468-OPN cells (relative to untreated controls; P < 0.05). Furthermore, Argatroban treatment significantly decreased lymphatic metastasis of both 468-CON (P < 0.04) and 468-OPN (P < 0.01) cells relative to untreated controls. These novel findings indicate that inhibition of thrombin can reduce malignant and metastatic behavior of MDA-MB-468 breast cancer cells using both OPN-dependent and OPN-independent mechanisms, and suggest that thrombin inhibitors such as Argatroban may hold potential as therapeutic agents to combat breast cancer progression.

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Analysis of variance


Bovine serum albumin


Ethylenediaminetetraacetic acid


Epidermal growth factor receptor


Fetal bovine serum


Fluorescein isothiocyanate


Hematoxylin and eosin


Hepatocyte growth factor


High-powered field


Horseradish peroxidase


Lymph node


Low molecular weight heparin


Lymphovascular invasion


Minimum essential media




Protease-activated receptor




Phosphate-buffered saline


Phenylmethylsulfonyl fluoride


Red green blue




Sodium dodecyl sulfate


Tris-buffered saline + 0.05% Tween-20


Tissue factor


Transforming growth factor α




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We thank Michael Keeney and the London Health Sciences Centre Flow Cytometry Unit for their help in carrying out and analyzing the flow cytometry studies. We also thank Michael Keeney, Ian Chin-Yee, and Anargyros Xenocostas for helpful discussions and intellectual input. This work was supported in part by grants from the London Regional Cancer Program and the National Research Council of Canada (to A.L.A.) and by grant #04-MAY-00089 from the Ontario Cancer Research Network (to A.F.C. and A.B.T.). B.D.H. is the recipient of a Translational Breast Cancer Scholarship through the London Regional Cancer Program. A.F.C. is the recipient of a Canada Research Chair in Oncology. A.L.A. is supported by the Imperial Oil Foundation.

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Correspondence to Alison L. Allan.

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Schulze, E.B., Hedley, B.D., Goodale, D. et al. The thrombin inhibitor Argatroban reduces breast cancer malignancy and metastasis via osteopontin-dependent and osteopontin-independent mechanisms. Breast Cancer Res Treat 112, 243–254 (2008).

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