Breast cancer aberrantly expresses tissue factor (TF) in cancer tissues and cancer vascular endothelial cells (VECs). TF plays a central role in cancer angiogenesis, growth, and metastasis and, as such, is a target for therapy and drug delivery. TF is the cognate receptor of factor VIIa (fVIIa). We have coupled PTX (paclitaxel, also named Taxol) with a tripeptide, phenylalanine-phenylalanine-arginine chloromethyl ketone (FFRck) and conjugated it with fVIIa. The key aim of the work is to evaluate the antiangiogenic effects of PTX-FFRck-fVIIa against a PTX-resistant breast cancer cell line. Matrigel mixed with VEGF and MDA-231 was injected subcutaneously into the flank of athymic nude mice. Animals were treated by tail vein injection of the PTX-FFRck-fVIIa conjugate, unconjugated PTX, or PBS. The PTX-FFRck-fVIIa conjugate significantly reduces microvessel density in matrigel () compared to PBS and unconjugated PTX. The breast cancer lung metastasis model in athymic nude mice was developed by intravenous injection of MDA-231 cells expressing luciferase. Animals were similarly treated intravenously with the PTX-FFRck-fVIIa conjugate or PBS. The conjugate significantly inhibits lung metastasis as compared to the control, highlighting its potential to antagonize angiogenesis in metastatic carcinoma. In conclusion, PTX conjugated to fVIIa is a promising therapeutic approach for improving selective drug delivery and inhibiting angiogenesis. 1. Introduction Scope. The objective is to selectively deliver a highly toxic drug to tumor cells and the related tumor vascular endothelium. The target is tissue factor (TF), a membrane bound protein aberrantly expressed on cancer cells and their endothelia. The drug carrier is factor VIIa (fVIIa), the natural ligand of TF. fVIIa is transformed to its competitive inhibitor by conjugation with paclitaxel-FFRck, resulting in paclitaxel- (PTX-) FFRck-fVIIa. The conjugation prevents thromboembolic complications associated with fVII administration and provides an effective antiangiogenic approach that targets TF-expressing endothelia and cancers. Tissue factor (TF) is a 47？kDa transmembrane glycoprotein receptor of factor VII/VIIa (fVIIa), a critical regulator of tissue hemostasis and one of the body’s most potent procoagulants [1]. Under normal conditions, TF is expressed by stromal cells, outer blood vessel layers (smooth muscle and adventitia), but not by vascular endothelial cells (VECs) (the inner most layer). Injury of the vascular wall causes TF to bind to its highly specific activating ligand, fVIIa from the plasma to
References
[1]
F. R. Rickles, “Mechanisms of cancer-induced thrombosis in cancer,” Pathophysiology of Haemostasis and Thrombosis, vol. 35, no. 1-2, pp. 103–110, 2006.
[2]
E. W. Davie, K. Fujikawa, and W. Kisiel, “The coagulation cascade: initiation, maintenance, and regulation,” Biochemistry, vol. 30, no. 43, pp. 10363–10370, 1991.
[3]
Y. Nemerson, “Tissue factor and hemostasis,” Blood, vol. 71, no. 1, pp. 1–8, 1988.
[4]
J. Folkman, “Angiogenesis: an organizing principle for drug discovery,” Nature Reviews Drug Discovery, vol. 6, no. 4, pp. 273–286, 2007.
[5]
M. Toi, K. Inada, H. Suzuki, and T. Tominaga, “Tumor angiogenesis in breast cancer: its importance as a prognostic indicator and the association with vascular endothelial growth factor expression,” Breast Cancer Research and Treatment, vol. 36, no. 2, pp. 193–204, 1995.
[6]
Y. Zhang, Y. Deng, T. Luther et al., “Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice,” Journal of Clinical Investigation, vol. 94, no. 3, pp. 1320–1327, 1994.
[7]
T. F. Zioncheck, S. Roy, and G. A. Vehar, “The cytoplasmic domain of tissue factor is phosphorylated by a protein kinase C-dependent mechanism,” The Journal of Biological Chemistry, vol. 267, no. 6, pp. 3561–3564, 1992.
[8]
K. Abe, M. Shoji, J. Chen et al., “Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 15, pp. 8663–8668, 1999.
[9]
M. Shoji, W. W. Hancock, K. Abe, et al., “Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor,” The American Journal of Pathology, vol. 152, no. 2, pp. 399–411, 1998.
[10]
J. Contrino, G. Hair, D. L. Kreutzer, and F. R. Rickles, “In situ detection of tissue factor in vascular endothelial cells: correlation with the malignant phenotype of human breast disease,” Nature Medicine, vol. 2, no. 2, pp. 209–215, 1996.
[11]
J. N. Zhou, S. Ljungdahl, M. C. Shoshan, J. Swedenborg, and S. Linder, “Activation of tissue-factor gene expression in breast carcinoma cells by stimulation of the RAF-ERK signaling pathway,” Molecular Carcinogenesis, vol. 21, pp. 234–243, 1998.
[12]
Y. W. van den Berg, L. G. van den Hengel, H. R. Myers, et al., “Alternatively spliced tissue factor induces angiogenesis through integrin ligation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 46, pp. 19497–19502, 2009.
[13]
M. Clauss, M. Gerlach, H. Gerlach et al., “Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration,” Journal of Experimental Medicine, vol. 172, no. 6, pp. 1535–1545, 1990.
[14]
S. Zucker, H. Mirza, C. E. Conner et al., “Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation,” International Journal of Cancer, vol. 75, pp. 780–786, 1998.
[15]
D. Mechtcheriakova, A. Wlachos, H. Holzmüller, B. R. Binder, and E. Hofer, “Vascular endothelial cell growth factor-induced tissue factor expression in endothelial cells is mediated by EGR-1,” Blood, vol. 93, no. 11, pp. 3811–3823, 1999.
[16]
A. L. Armesilla, E. Lorenzo, P. G. del Arco, S. Martínez-Martínez, A. Alfranca, and J. M. Redondo, “Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression,” Molecular and Cellular Biology, vol. 19, no. 3, pp. 2032–2043, 1999.
[17]
Z. Hu and A. Garen, “Targeting tissue factor on tumor vascular endothelial cells and tumor cells for immunotherapy in mouse models of prostatic cancer,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 21, pp. 12180–12185, 2001.
[18]
F. Lu, Z. Hu, J. Sinard, A. Garen, and R. A. Adelman, “Factor VII-verteporfin for targeted photodynamic therapy in a rat model of choroidal neovascularization,” Investigative Ophthalmology & Visual Science, vol. 50, no. 8, pp. 3890–3896, 2009.
[19]
G. Schoellmann and E. Shaw, “Direct evidence for the presence of histidine in the active center of chymotrypsin,” Biochemistry, vol. 2, pp. 252–255, 1963.
[20]
C. Kettner and E. Shaw, “[63] Inactivation of trypsin-like enzymes with peptides of arginine chloromethyl ketone,” Methods in Enzymology, vol. 80, pp. 826–842, 1981.
[21]
E. B. Williams, S. Krishnaswamy, and K. G. Mann, “Zymogen/enzyme discrimination using peptide chloromethyl ketones,” Journal of Biological Chemistry, vol. 264, no. 13, pp. 7536–7545, 1989.
[22]
B. B. Sorensen, E. Persson, P. O. Freskgard et al., “Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor,” The Journal of Biological Chemistry, vol. 272, no. 18, pp. 11863–11868, 1997.
[23]
J. M. Ndungu, Y. J. Lu, S. Zhu et al., “Targeted delivery of paclitaxel to tumor cells: synthesis and in vitro evaluation,” Journal of Medicinal Chemistry, vol. 53, no. 8, pp. 3127–3132, 2010.
[24]
A. Sun, M. Shoji, Y. J. Lu, D. C. Liotta, and J. P. Snyder, “Synthesis of EF24-tripeptide chloromethyl ketone: a novel curcumin-related anticancer drug delivery system,” Journal of Medicinal Chemistry, vol. 49, no. 11, pp. 3153–3158, 2006.
[25]
M. Shoji, A. Sun, W. Kisiel, et al., “Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa,” Journal of Drug Targeting, vol. 16, no. 3, pp. 185–197, 2008.
[26]
B. K. Adams, E. M. Ferstl, M. C. Davis et al., “Synthesis and biological evaluation of novel curcumin analogs as anti-cancer and anti-angiogenesis agents,” Bioorganic and Medicinal Chemistry, vol. 12, no. 14, pp. 3871–3883, 2004.
[27]
B. K. Adams, J. Cai, J. Armstrong et al., “EF24, a novel synthetic curcumin analog, induces apoptosis in cancer cells via a redox-dependent mechanism,” Anti-Cancer Drugs, vol. 16, no. 3, pp. 263–275, 2005.
[28]
S.-Z. Zhang, M. M. Lipsky, B. F. Trump, and I.-C. Hsu, “Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes,” Cell Biology and Toxicology, vol. 6, no. 2, pp. 219–234, 1990.
[29]
E. Erhardtsen, P. Nilsson, M. Johannessen, and M. S. Thomsen, “Pharmacokinetics and safety of FFR-rFVIIa after single doses in healthy subjects,” Journal of Clinical Pharmacology, vol. 41, no. 8, pp. 880–885, 2001.
[30]
T. S？derstr？m, U. Hedner, and B. Arnljots, “Active site-inactivated factor VIIa prevents thrombosis without increased surgical bleeding: topical and intravenous administration in a rat model of deep arterial injury,” Journal of Vascular Surgery, vol. 33, no. 5, pp. 1072–1079, 2001.
[31]
D. Torti and L. Trusolino, “Oncogene addiction as a foundational rationale for targeted anti-cancer therapy: promises and perils,” EMBO Molecular Medicine, vol. 3, no. 11, pp. 623–636, 2011.
[32]
T. M. Brand, M. Iida, and D. L. Wheeler, “Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab,” Cancer Biology & Therapy, vol. 11, no. 9, pp. 777–792, 2011.
[33]
M. De Palma and D. Hanahan, “The biology of personalized cancer medicine: facing individual complexities underlying hallmark capabilities,” Molecular Oncology, vol. 6, no. 2, pp. 111–127, 2012.
[34]
G. K. Dy and A. A. Adjei, “Understanding, recognizing, and managing toxicities of targeted anticancer therapies,” A Cancer Journal for Clinicians, vol. 63, no. 4, pp. 249–279, 2013.
