E7389, a macrocyclic ketone analog of the marine natural product halichondrin B, currently is undergoing clinical trials for cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via tubulin-based antimitotic mechanisms, which are similar or identical to those of parental halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G 2 -M phase of the cell cycle, beginning as early as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on acridine orange/ethidium bromide staining, and cell surface annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria, proteolytic activation of caspase-3 and -9, and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). In LNCaP human prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389.

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Borrelidin, an antibiotic with anti-angiogenic activity, not only suppresses new capillary tube formation, but also collapses formed capillary tubes in a rat aorta culture model. Since it selectively inhibits threonyl-tRNA synthetase, we examined the effect of threonine on its antiangiogenic activity. We found that a high concentration of threonine (1mM) attenuated the ability of borrelidin to inhibit both capillary tube formation in the rat aorta culture model and human umbilical vein endothelial cells (HUVEC) proliferation, yet did not affect the ability of borrelidin to collapse formed capillary tubes or to induce apoptosis in HUVEC. Borrelidin activated caspase-3 and -8, and inhibitors of both caspase-3 and -8 suppressed borrelidininduced apoptosis in HUVEC. Taken together, these data suggest that the anti-angiogenic effects of borrelidin are mediated through at least two mechanisms, i.e. one threoninedependent and the other threonine-independent, and borrelidin induces apoptosis in endothelial cells via the caspase-8/-3 pathway.Angiogenesis is a complicated multistep process, which includes proliferation, adhesion, invasion, migration, and tube formation of endothelial cells1). One in vitro model that seems to accurately represent the biological phenomena is the rat thoracic aorta tube formation (RATF) model. In this model, a small piece of aorta is cultured in a matrix basement membrane. Using this model, screening of crude natural extracts and microbial metabolites for antiangiogenic activity has led to the discovery of borrelidin in a culture broth of Actinomycetes2). Borrelidin showed antiangiogenic and anti-metastatic effects in animal models3).The anti-angiogenic activity of borrelidin is unique in involving dual modes of action; inhibition of new capillary tube formation and collapse of formed capillary tubes via the cell death of capillary-forming endothelial cells2).Borrelidin was originally isolated as an antibiotic4), and is produced by a variety of Streptomyces5). Its antibiotic effect is due to the specific inhibition of threonyl-tRNA synthetase; other aminoacyl-tRNA synthetases are not inactivated5,6). Borrelidin has been shown to inhibit the growth of mammalian cells as well as bacteria, and increased levels of threonyl-tRNA synthetase result in borrelidin resistance in Chinese hamster ovary (CHO) cells7), suggesting that borrelidin inhibits cell proliferation in mammalian cells via the inhibition of threonyl-tRNA synthetase, as it does in bacteria. In contrast, the growth inhibition of CHO cells by borrelidin is antagonized by threonine in a dose-dependent manner7). These findings suggested that the involvement of threonyl-tRNA synthetase inhibition could be tested by using culture media containing a range of threonine concentrations.In this paper, we present the results of our studies to examine the threonine dependence of borrelidin's anti-angiogenic effects, and to explore the mechanisms

Abstract-An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH 2 -terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor-signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter-induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (PϽ0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (PϽ0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG-treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury. (Circ Res. 1998;82:980-987.)Key Words: smooth muscle cell proliferation Ⅲ thrombin Ⅲ thrombin receptor Ⅲ restenosis Ⅲ angioplasty T hrombin, a serine protease derived from its precursor, prothrombin, plays an important role in cellular responses.1,2 Stimulation of vascular endothelial cells by thrombin leads to the release of several vasoactive substances, such as prostacyclin, platelet-activating factor, and PDGF, increases [Ca 2ϩ ] i and also induces translocation of the neutrophiladhesive molecule (P-selectin) to the endothelial cell surface.3,4 Thrombin also has a strong mitogenic effect on vascular SMCs.5 These diverse cellular responses to thrombin may orchestrate the hemostatic, inflammatory, and proliferative responses to vascular injury. 6 In vitro and in vivo studies have implicated bFGF and PDGF as well as thrombin in these responses.7-12 However, thrombin may be especially important as a mediator of vascular lesion formation, since it is present after balloon catheter injury. 10The thrombin receptor has been cloned, and it has been proposed that thrombin binds to its receptor and cleaves it after Arg41 in the receptor's NH 2 -terminal portion, thereby exposing a new NH 2 terminus, which functions as a tethered ligand for the receptor. 13 The thrombin receptor can mediate the mitogenic effect of thrombin, 5 and increased thrombin receptor expression was observed in neointimal cells throughout vascular lesion formation. 12 However, the role of...

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that play crucial roles in the adaptation of cancer cells to hypoxia. HIF-1α overexpression has been associated with poor prognosis in patients with various types of cancer. Here, we describe ER-400583-00 as a novel HIF-1 inhibitor. ER-400583-00 suppressed the production of HIF-1α protein in response to hypoxia, with a half-maximal inhibitory concentration value of 3.7 nM in human U251 glioma cells. The oral administration of 100 mg/kg ER-400583-00 to mice bearing U251 tumor xenografts resulted in a rapid suppression of HIF-1α that persisted for 24 h. Immunohistochemical analysis revealed that ER-400583-00 suppressed the proliferation of cancer cells most prominently in areas distal to the region of blood perfusion, where HIF-1α-expressing hypoxic cancer cells were located. These hypoxic cancer cells were resistant to radiation therapy. ER-400583-00 showed a synergistic interaction with radiation therapy in terms of antitumor activity. These data suggest that HIF-1 blockade by small compounds may have therapeutic value in cancer, especially in combination with radiation therapy.