Atsushi Mamiya

Coagulation factor IX regulates cell migration and adhesion in vitro.

Coagulation factor IX is thought to circulate in the blood as an inactive zymogen before being activated in the coagulation process. The effect of coagulation factor IX on cells is poorly understood. This study aimed to evaluate the effects of intact coagulation factor IX and its cleavage fragments on cell behavior. A431 cells (derived from human squamous cell carcinoma), Pro5 cells (derived from mouse embryonic endothelial cells), Cos7 cells, and human umbilical vein endothelial cells were utilized in this study. The effects of coagulation factor IX and its cleavage fragments on cell behavior were investigated in several types of experiments, including wound‐healing assays and modified Boyden chamber assays. The effect of coagulation factor IX depended on its processing; full‐length coagulation factor IX suppressed cell migration, increased adhesion to matrix, and enhanced intercellular adhesion. In contrast, activated coagulation factor IX enhanced cell migration, suppressed adhesion to matrix, and inhibited intercellular adhesion. An activation peptide that is removed during the coagulation process was found to be responsible for the activity of full‐length coagulation factor IX, and the activity of activated coagulation factor IX was localized to an EGF domain of the coagulation factor IX light chain. Full‐length coagulation factor IX has a sedative effect on cells, which is counteracted by activated coagulation factor IX in vitro. Thus, coagulation factor IX may play roles before, during, and after the coagulation process.

Efficient nonviral gene therapy with FasL and Del1 fragments in mice

The expression of FasL in cancer cells is currently being explored as a potential cancer therapy. Because high levels of FasL are necessary for effective treatment, current methods typically rely on the use of highly efficient viral vectors. However, because viral vector‐based gene therapy is associated with certain risks, the development of effective nonviral routes for gene delivery would be useful. The present study aimed to improve FasL gene therapy with a nonviral vector by taking advantage of the E3 and C1 domains of Del1 protein, which induces apoptosis and localizes to the extracellular matrix.

The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis

Activated coagulation factor IX (FIX) attenuated cell adhesion to the extracellular matrix (ECM) and induced apoptosis. This activity was localized to the first epidermal growth factor (EGF) domain, EGF-F9. Experiments with caspase-3 inhibitors revealed that attenuation of adhesion and apoptosis by EGF-F9 were dependent on caspase-3.

Improvement of FasL Gene Therapy In Vitro by Fusing the FasL to Del1 Protein Domains

Gene delivery, transfection, cytotoxicity, and many other factors influence the ability of gene therapy to treat cancer. In addition, as with pharmacologic agents, longer exposure to higher concentrations of gene products should intensify their effects (Wada et al., 2007). Cytotoxic gene products, such as FasL and TRAIL, may remain in tissues after the death of the transfected cells, and they are known to induce apoptosis in both transfected cells and neighbouring cells (Hyer et al., 2003; Kagawa et al., 2001). They have been examined for use in cancer gene therapy and its effects have been examined in vitro and in vivo (Elojeimy et al., 2006; Griffith et al., 2009). FasL delivered via a viral vector can reduce tumour size and improve prognosis in an explanted tumour model.

Long-term gene therapy with Del1 fragment using nonviral vectors in mice with explanted tumors

Cancer gene therapy using nonviral vectors is useful for long periods of treatment because such vectors are both safe and inexpensive, and thus can be used repeatedly. It has been reported that gene therapy with an E3C1 fragment of Del1 in a mouse explanted tumor model improved prognosis. The present study aimed to analyze the long-term effects of repeated non-viral gene transfer of E3C1. Mice with explanted tumors of SCCKN cells, a human squamous carcinoma, were treated with a plasmid encoding E3C1. Plasmids were injected locally every week using a transfection reagent. Control mice treated with mock DNA started to be euthanized on day 18, because the tumors had grown to over 15% of the body weight, and all of them had died by day 43. On the other hand, the tumors in two of ten mice treated with E3C1 had disappeared. The other eight mice started to be euthanized on day 46 and eight of ten mice had been euthanized by day 197. After 18 days of therapy, the tumor volume of control mice was 2,804±829 mm3 and that of the E3C1 mice was 197±159 mm3. Histochemical studies showed enhanced apoptosis in the E3C1-treated tumors, as compared with controls. Changes in cell morphology and decreased polymerized actin induced by E3C1 indicated disturbed cell adhesion to the matrix. In in vitro studies of SCCKN cells, prolonged administration of an E3C1 recombinant protein to cultured cells reduced adhesion-independent growth of cancer cells, as compared with control cells. These data suggest that E3C1 treatment induces anoikis.