Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction.

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.

Tissue factor isoforms in cancer and coagulation: may the best isoform win.

Tissue factor (TF), the trigger of blood coagulation, is a 47 kDa membrane protein that also impacts on non-hemostatic processes, such as atherosclerosis, primary tumor growth and metastasis. TF binding to its ligand FVIIa induces activation of protease-activated receptor-2 and this event is thought to considerably influence atherosclerosis and tumor angiogenesis. TF-dependent activation of the coagulation cascade, rather than PAR-2 activation, then leads to the potentiation of metastasis. Importantly, a soluble alternatively spliced isoform of TF (asTF) has been discovered, but the function of asTF in hemostatic and non-hemostatic events is poorly understood. In this review, we aim to present a side-by-side evaluation of normally-spliced, full length TF (flTF) and asTF with regard to coagulant function, atherosclerosis, tumor progression and malignancy-associated thrombosis.

Synthetic peptide analogs of tissue factor and factor VII which inhibit factor Xa formation by the tissue factor/factor VIIa complex.

Factor VII (FVII) and tissue factor (TF) form a binary complex which initiates the extrinsic pathway of the blood coagulation cascade. The infrequent tripeptide motif Trp-Lys-Ser (WKS) is found three times in TF. It has been suggested that the motif is involved in binding of TF to FVII(a). Also. Lys165 and Lys166 of TF have been reported to be important for factor X activation. To elucidate the molecular interactions between TF and FVIIa, and the interactions between the binary complex and FX, we examined the inhibitory effect of synthetic TF and FVII peptide analogs. One- and two-stage chromogenic assays were employed, as well as one-stage coagulation assay. The peptide analogs of TF possessed the WKS motif, the double lysine residues or other regions of TF. Synthetic peptides of FVII encompassing sequences of the FVII285-305 region were included for comparative purposes. TF154-167 and FVII300-305 significantly inhibited both FX activation and plasma coagulation. FVII285-294 acted synergistically, increasing that effect observed by FVII300-305 on FX activation. However, TF163-175 possessing the double lysine residues did not inhibit FX activation, indicating that inhibition of FXa formation and coagulation by TF154-167 is due to the region 154-162 of TF. None of the peptides, including the WKS tripeptide, interfered with the FVIIa activity of the TF/FVIIa complex. Thus, the results do not suggest that the WKS motifs are necessary for binding of TF to FVIIa but that the third WKS motif may be of importance for the activation of FX.