Dorsal Air Sac Assay Research Articles

NDRG1 activates VEGF-A-induced angiogenesis through PLC\u03b31/ERK signaling in mouse vascular endothelial cells

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1−/− mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling.

Abstract 1374: Lenvatinib, tri-specific targeted therapy to VEGFR/FGFR/RET, suppresses angiogenesis through the inhibition of both VEGFR and FGFR signaling pathways

Abstract Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of vascular endothelial growth factor receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Recently, lenvatinib showed a highly statistically significant improvement of progression free survival compared to placebo control in subjects with radioiodine-refractory differentiated thyroid cancer in a Phase 3 trial. To elucidate the mechanism of action in anti-tumor effect by lenvatinib, we examined the antiangiogenic activity of lenvatinib in the VEGF and FGF driven angiogenesis models both in vitro and in vivo. First, in in vitro angiogenesis model, lenvatinib inhibits both VEGF-driven and FGF-driven tube formation of HUVEC at almost similar dose (IC50 values are 2.1 nmol/L and 7.3 nmol/L, respectively). Next, the effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF4 transfectants) was significantly suppressed with oral treatments of lenvatinib at 10 and 30 mg/kg. In addtion, lenvatinib significantly inhibited bFGF-induced angiogenesis in matrigel plug assay, which is driven by recombinant human bFGF and stromal-derived mouse VEGF, at even 3 mg/kg. Increase of plasma FGF23 level is PD biomarker for an inhibition of FGFR1 signaling and it has been known that plasma FGF23 level is up-regulated by administration of FGFR inhibitors in mice. Lenvatinib at 10 mg/kg significantly elevated mice FGF23 levels 24 hours after lenvatinib treatment in a dose dependent manner. In conclusion, lenvatinib has potent both VEGF and FGF driven-antiangiogenic activity in vitro and in vivo. Inhibition of FGFR signaling pathway with lenvatinib was also supported by increase of plasma FGF23 levels at the same dose to show an inhibition in FGF-driven in vivo angiogenesis model. Therefore, the significant clinical outcome might be based on the unique antiangiogenesis activity of lenvatinib, in particular VEGFR and FGFR inhibition, which should be further investigated. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yusuke Adachi, Yuji Yamamoto, Yoichi Ozawa, Yasuhiro Funahashi, Kiyoshi Okamoto, Kenichi Nomoto, Junji Matsui. Lenvatinib, tri-specific targeted therapy to VEGFR/FGFR/RET, suppresses angiogenesis through the inhibition of both VEGFR and FGFR signaling pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1374. doi:10.1158/1538-7445.AM2015-1374

Cardioprotective effects on ischemic myocardium induced by SVVYGLR peptide via its angiogenic-promoting activity

In ischemic heart disease, cardiomyocyte death results from both necrosis and apoptosis, and the area of infarction gradually increases. The induction of therapeutic angiogenesis is a promising therapy for this condition. In the present study, we investigated the actions of SV peptide—a 7-amino-acid sequence with angiogenic properties derived from osteopontin in the extracellular matrix—in a rat model of myocardial ischemia. We examined the angiogenesis activity of SV ex vivo using ring assay and dorsal air sac assay and in ischemic myocardium. We also conducted histological evaluations of left-ventricle remodeling and evaluated left ventricular (LV) function by echocardiography. The SV peptide had strong angiogenic effects both ex vivo and in vivo. In the experimental rats, this peptide stimulated the formation of new blood vessels in ischemic myocardium. Histological evaluation of the left ventricle in the SV-treated group showed a significant decrease in the size of the infarction, rate of myocardial fibrosis, and cardiomyocyte hypertrophy. Moreover, administration of the SV peptide significantly improved LV function. These results indicate that SV peptide induced angiogenesis in ischemic myocardium and improved cardiac function. Thus, it could serve as a potentially useful new therapy for ischemic heart disease.

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage.

BackgroundLenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study.MethodWe evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors.ResultLenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score.ConclusionThese results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

N-myc Downstream-regulated Gene 1 Promotes Tumor Inflammatory Angiogenesis through JNK Activation and Autocrine Loop of Interleukin-1α by Human Gastric Cancer Cells

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.

Resveratrol derivative‐rich melinjo (Gnetum gnemon L.) seed extract suppresses multiple angiogenesis‐related endothelial cell functions and tumor angiogenesis

Angiogenesis is a promising target for cancer prevention and treatment. This study aimed to determine the antiangiogenic effects of melinjo (Gnetum gnemon L.) seed extract and its resveratrol derivative components, such as gnetin C (GC), gnetin L (GL), gnemonoside A (GMA), gnemonoside C (GMC), and gnemonoside D (GMD). An ethanol extract of melinjo seeds (EEMS) and the two gnetins markedly inhibited the proliferation and tube formation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor and basic fibroblast growth factor. The inhibitory effects of GC and GL were much stronger than those of resveratrol. GMC and GMD inhibited only proliferation, whereas GMA had almost no effect on the two endothelial cell functions. The EEMS and GC also reduced the cell viability of tube-forming HUVEC, with accompanying ERK1/2 inactivation, and suppressed the migration of HUVEC. Furthermore, dietary intake of EEMS significantly inhibited tumor angiogenesis in a mouse dorsal air sac assay. In conclusion, we found that the EEMS and its resveratrol derivatives, particularly GC, suppress multiple angiogenesis-related endothelial cell functions and/or tumor angiogenesis, indicating that the melinjo seeds and the natural resveratrol derivatives may be useful for cancer prevention and treatment.

Abstract 3468: MMP-9 inhibition abolishes hypoxia-induced angiogenesis in medulloblastoma

Abstract Hypoxia-inducible factor-1 (HIF-1) transcription factor is a critical mediator of the hypoxic response and upregulates expression of various proteins that promote angiogenesis and many other survival pathways in cancer. Overexpression of HIF-1α and matrix metalloproteinase-9 (MMP-9) is common in human cancers, including medulloblastoma, and is correlated with a malignant phenotype. Therefore, we investigated the effects of hypoxia-induced MMP-9 on angiogenesis in medulloblastoma cells. Western blotting and immunohistochemical analysis showed that hypoxia enhanced stabilization of HIF-1α and expression of VEGF and MMP-9 in medulloblastoma cells in vivo and in vitro. Further, endothelial tube formation assay in vitro and dorsal air sac assay in vivo showed that hypoxia enhanced angiogenesis in medulloblastoma cells. Transcriptional inactivation of MMP-9 using siRNA (pMMP-9-Si) inhibited hypoxia-induced stabilization of HIF-1α, and induced expression of VEGF and prolyl hydroxylase. Moreover, supplementation of human recombinant MMP-9 in MMP-9-Si-transfected cells restored VEGF expression and angiogenesis. In summary, the results of the present study indicate that MMP-9 upregulation could be an important mediator of tumor hypoxia-induced angiogenesis. Therefore, inhibition of MMP-9 could be an effective therapeutic strategy for the treatment of hypoxic medulloblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3468. doi:10.1158/1538-7445.AM2011-3468

Antitumor effects of 2‐oxoglutarate through inhibition of angiogenesis in a murine tumor model

Hypoxia-inducible factor 1 (HIF-1) plays essential roles in tumor angiogenesis and growth by regulating the transcription of several key genes in response to hypoxic stress and growth factors. HIF-1 is a heterodimeric transcriptional activator consisting of inducible alpha and constitutive beta subunits. In oxygenated cells, proteins containing the prolyl hydroxylase domain (PHD) directly sense intracellular oxygen concentrations. PHDs tag HIF-1alpha subunits for polyubiquitination and proteasomal degradation by prolyl hydroxylation using 2-oxoglutarate (2-OX) and dioxygen. Our recent studies showed that 2-OX reduces HIF-1alpha, erythropoietin, and vascular endothelial growth factor (VEGF) expression in the hepatoma cell line Hep3B when under hypoxic conditions in vitro. Here, we report that similar results were obtained in Lewis lung cancer (LLC) cells in in vitro studies. Furthermore, 2-OX showed potent antitumor effects in a mouse dorsal air sac assay and a murine tumor xenograft model. In the dorsal air sac assay, 2-OX reduced the numbers of newly formed vessels induced by LLC cells. In a murine tumor xenograft model, intraperitoneal injection of 2-OX significantly inhibited tumor growth and angiogenesis in tumor tissues. Moreover, 5-fluorouracil combined with 2-OX significantly inhibited tumor growth in this model, which was accompanied by reduction of Vegf gene expression and inhibited angiogenesis in tumor tissues. These results suggest that 2-OX is a promising anti-angiogenic therapeutic agent.

Anti-angiogenic and Anti-metastatic Effects of .BETA.-1,3-D-Glucan Purified from Hanabiratake, Sparassis crispa

Sparassis crispa (SC), Hanabiratake in Japanese, is an edible mushroom with medicinal properties, that contains more than 40% beta-D-glucan. It was concluded from results of the methylation study that beta-D-glucan from SC (SBG) was composed of a backbone of beta-(1-->3)-linked D-glucopyranosyl residues, and had beta-D-glucopyranosyl groups joined through O-6 and O-2 of D-glucose of the backbone. We purified SBG and investigated its anti-angiogenic functions and anti-metastatic effects on neoplasm using different animal models. The oral administration of the purified SBG suppressed B16-F10 cell-induced angiogenesis in the dorsal air sac assay using female ICR mice as well as vascular endothelial growth factor induced neovascularization in the Matrigel plug assay using female C57BL/6J mice. Furthermore, it suppressed the growth and numbers of the metastatic tumor foci in lung, along with the primary tumor growth in the spontaneous metastatic model using female C57BL/6J mice. From these results, it is apparent that the oral administration of SBG results in suppressive effect on tumor growth and metastasis in lung through the inhibition of tumor induced-angiogenesis. These effects are not a result of direct action on the endothelial cells because cell growth, migration and capillary-like tube formation were not affected in the human umbilical vein endothelial cells by SBG application. This is the first report showing that the oral administration of SBG is capable of suppressing angiogenesis and metastasis.

Anti Tumor Effect of 2-Oxoglutarate through Inhibition of Angiogenesis in a Murine Tumor Model.

Hypoxia-inducible factor 1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of several genes in response to hypoxic stress and changes in growth factors. HIF-1 is a heterodimeric transcriptional activator and consists of inducible α and constitutive β subunits. In oxygenated cells, intracellular oxygen concentrations are directly sensed by proteins containing the prolyl hydroxylase domain (PHD), which tag HIF-1α subunits for polyubiquitination and proteasomal degradation by prolyl hydroxylation using 2-oxoglutarate (2-OX) and dioxygen. Our recent studies have shown that 2-OX reduces HIF-1α, erythropoietin, and vascular endothelial growth factor (VEGF) expression by hypoxia in the hepatoma cell line Hep3B (Matsumoto K et al.; J. Cell. Physiol., 2006). Similar results were obtained in Lewis lung cancer (LLC) cells in in vitro studies. Here, to address the clinical usefulness of 2-OX, we investigated its antitumor effect using a mouse dorsal air sac (DAS) assay and a murine tumor xenograft model. For the DAS assay, LLC cells suspended in PBS with 0, 7.5, or 15 mM 2-OX per Millipore chamber were implanted subcutaneously into C57BL/6J mice. The values of blood vessel area by LLC cells were 100 ± 20.8, 64.0 ± 9.4, and 45.6 ± 4.4%, respectively, by quantitative analysis with angiogenesis-measuring software. This result indicated that 2-OX clearly inhibited the growth of subcutaneous tumors. To elucidate the effect of 2-OX on tumor growth, 2-OX was administrated to C57BL/6J mice inoculated with LLC cells. LLC cells (1 × 106) in PBS were implanted into the right flank region of 7-week-old mice. Daily intraperitoneal (i.p.) injections of 2-OX were started on the next day after implantation. From 6 to 12 days after implantation, we measured tumors with calipers and calculated volumes as (length × width2) × 0.5. LLC tumors were removed from mice 12 days after implantation for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical studies. Tumor volumes and weights 12 days after implantation were as follows: PBS alone, 330.8 ± 108.1 mm3 and 192.6 ± 66.4 mg; 50 mg/kg 2-OX, 128.8 ± 16.4 mm3 and 111.0 ± 64.5 mg; and 100 mg/kg 2-OX, 78.7 ± 43.7 mm3 and 88.8 ± 57.5 mg. Quantitative RT-PCR revealed that 2-OX treatment (100 mg/kg) decreased the expression levels of the VEGF gene 0.67-fold in tumor tissues compared with control. We observed quantitative differences in microvessel density (PBS alone, 100 ± 16.5%; 100 mg/kg 2-OX, 46.0 ± 13.5%) using immunostaining for the endothelial cell marker CD31. Intraperitoneal injection of 2-OX significantly inhibited tumor growth and angiogenesis in tumor tissues. Combination therapy is necessary for anti-angiogenic therapy in the human. To examine the effect of 2-OX in combination with 5-fluorouracil (5-FU) chemotherapy, we injected 5-FU i.p. on day 6 and/or 2-OX i.p. on each of days 6–15 in this mouse model. Tumor volumes and weights 15 days after implantation were as follows: PBS alone, 531.2 ± 144.9 mm3 and 306.3 ± 186.6 mg; 2-OX alone, 324.0 ± 156.5 mm3 and 308.7 ± 299.0 mg; 5-FU alone, 287.1 ± 155.2 mm3 and 204.7 ± 108.9 mg; and 5-FU + 2-OX, 98.7 ± 64.9 mm3 and 130.3 ± 113.5 mg. 5-FU combined with 2-OX significantly inhibited tumor growth in this model, which was accompanied by 53% reduction of VEGF gene expression in tumor tissues removed from mice 15 days after implantation, using quantitative RT-PCR analysis. These results suggest that 2-OX is a promising anti-angiogenic therapeutic agent. To examine whether the inhibitory effect of 2-OX is specific for PHD-containing proteins, an RNAi study will be performed.

Oxaliplatin targeting to angiogenic vessels by PEGylated cationic liposomes suppresses the angiogenesis in a dorsal air sac mouse model

Oxaliplatin (trans-l-diaminocyclohexane oxalatoplatinum, l-OHP) is a third-generation platinum analogue with proven anti-tumor activity against many tumor cell lines, however it does not show sufficient anti-tumor activity in vivo when used alone. In order to overcome this problem and to achieve an anti-angiogenic therapy with l-OHP, the drug was encapsulated into PEG-coated cationic liposomes, which were designed to target the newly formed vessels, and its anti-angiogenic activity was evaluated in an in vivo mouse dorsal air sac (DAS) assay. For the DAS assay, chambers filled with tumor cells were implanted underneath the dorsal skin. l-OHP encapsulated in PEG-coated cationic liposomes (5 mg/kg mice) was intravenously injected once on day 1, 2, 3 or 4 after chamber implantation. On the fifth day after chamber implantation, animals were sacrificed and tumor-angiogenesis was evaluated. Liposome-encapsulated l-OHP completely suppressed angiogenesis in the skin when it was administered day 3 after chamber implantation. Under similar experimental conditions, neither l-OHP encapsulated in PEG-coated neutral liposomes, nor free l-OHP, nor “empty” (no drug containing) PEG-coated cationic liposomes showed such strong suppressive effect. The present study suggests that the liposomal formulation of l-OHP, which targeted to angiogenic vessels, has a remarkable in vivo anti-angiogenic activity and the formulation may become a promising novel approach to achieve anti-angiogenic therapy.

Anti-angiogenic function of tocotrienol.

Angiogenesis means the formation of new blood vessels from preexisting vascular, is of fundamental importance in several pathological states such as tumor growth, rheumatoid arthritis, and diabetic retinopathy. Angiogenesis involves a set of steps, including activation and movement of endothelial cells and tube formation. Control of these steps by drugs or dietary food components is a hopeful approach for the prevention of angiogenic disorders. Based on these backgrounds, we searched the anti-angiogenic food components. As a result, we found that tocotrienol (T3), especially delta, beta, and gamma-T3 has the potent anti-angiogenic activity in vitro and in vivo experiments. T3, which is rich in rice bran and palm oil, inhibited growth factor-induced proliferation, migration and tube formation in human umbilical vein endothelial cells. T3 showed inhibition of tumor cell-induced angiogenesis in mouse dorsal air sac (DOS) assay. These results indicated that T3 is a potent anti-angiogenesis compound. Tocopherol (Toc) did not inhibit angiogenesis. The anti-angiogenic mechanism of T3 and Toc was evaluated by western blotting. T3 inhibited activation of growth factor-induced extracellular signal-regulated kinase, Akt (protein kinase B), and endothelial nitric oxide synthase (eNOS), which are located downstream of the various growth factor receptors. T3 suppressed phosphorylation of vascular endothelial growth factor (VEGF) receptor 2. These effects were dose-dependent manner. Anti-angiogenic mechanism of T3 mediates inhibition of growth factor induced survival, migration and angiogenesis signals. These findings suggested that T3 may have potential for preventing angiogenic disorders in humans.

CS‐706, a novel cyclooxygenase‐2 selective inhibitor, prolonged the survival of tumor‐bearing mice when treated alone or in combination with anti‐tumor chemotherapeutic agents

The potent chemopreventive activity of cyclooxygenase-2 (COX-2) inhibitors has been demonstrated in a number of preclinical studies, but their potency in antitumor activity is still in dispute. In this report, we demonstrate the potent antitumor activity of a novel COX-2 inhibitor, CS-706 in mouse colorectal adenocarcinoma colon 26 tumor-bearing mice treated with or without antitumor chemotherapeutic agents. Daily oral administration of CS-706 at doses of 3-100 mg/kg from the day of tumor inoculation (Day 0) inhibited tumor growth dose-dependently, and the maximal inhibition was 67% at a dose of 100 mg/kg. In contrast, celecoxib, a well-known COX-2 inhibitor, did not inhibit tumor growth at doses up to 100 mg/kg. Furthermore, CS-706 at a dose of 1 mg/kg or above markedly prolonged the survival time of tumor-bearing mice. Administration of 30 mg/kg CS-706 from Day 7 combined with a single intravenous treatment of 10 mg/kg cisplatin on Day 7 completely regressed the tumors in all tumor-bearing mice examined, whereas only in 1 of 10 mice tumor was regressed with cisplatin treatment. Similar combination effects were observed with 10 mg/kg CS-706 and 60 mg/kg 5-fluorouracil (5-FU). Moreover, 10 mg/kg CS-706 significantly inhibited angiogenesis induced by implanted chambers with colon 26 cells in a dorsal air sac assay in mice. Collectively, these results suggest that CS-706 is a potent antitumor agent, especially in combination with conventional chemotherapeutic agents, and that the anti-angiogenic activity of CS-706 may contribute at least in part to its marked antitumor activity.

Inhibition of Tumor Angiogenesis by Targeting Endothelial Surface ATP Synthase with Sangivamycin

Sangivamycin, an antibiotic with anti-tumor and anti-herpes virus activities by inhibiting both DNA/RNA synthesis and protein kinase C activity, was reported to suppress selectively DNA synthesis and growth of human umbilical vein endothelial cells and their tube formation in vitro. Here, to address the potential clinical use of sangivamycin in future, we investigated its anti-angiogenic effect in in vivo chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays, and investigated underlying mechanism. The effect of sangivamycin on blood vessel formation in CAM was observed under the microscope after treating for two days. For DAS assays, chambers fulfilled with tumor cells were implanted beneath mouse dorsal skin. After the mice were administered with sangivamycin, tumor-induced angiogenesis was observed under the microscope. The effect of sangivamycin on ATP synthesis on the endothelial cell surface was assayed by measuring ATP production with bioluminescence assay. Sangivamycin suppressed angiogenesis within CAM down to 94-71%, which was partially blocked by simultaneous addition of a 40-fold excess of adenosine. Sangivamycin also inhibited tumor-angiogenesis in the DAS assay by 61%, and suppressed ATP production on the endothelial cell surface by 75%. Sangivamycin inhibits the in vivo angiogenesis within CAM and tumor-induced angiogenesis within mouse dorsal skin, at least in part via inhibiting endothelial cell surface ATP metabolism in addition to inhibition of DNA/RNA synthesis and/or protein kinase C activity, suggesting a potential clinical use of sangivamycin as a novel anti-cancer reagent capable of targeting not only cancer cells but also endothelial cells.

In Vivo Angiogenesis Is Suppressed by Unsaturated Vitamin E, Tocotrienol ,

Antiangiogenic therapy using drugs and food components is a recognized strategy for the prevention of various angiogenesis-mediated disorders such as tumor growth, diabetic retinopathy, and rheumatoid arthritis. Our preliminary cell culture studies, using both bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVEC) on screening for food-derived antiangiogenic compounds, showed tocotrienol (T3), an unsaturated version of vitamin E, to be a potential angiogenic inhibitor. We therefore investigaged the in vivo antiangiogenic properties of T3 using 2 well-characterized angiogenic models [mouse dorsal air sac (DAS) assay and the chick embryo chorioallantoic membrane (CAM) assay]. In the DAS assay, the increased neovascularization (angiogenesis index, 4.8 α 0.6) in tumor cell–implanted mice was suppressed (angiogenesis index, 2.7 α 0.6) by dietary supplementation of 10 mg T3-rich oil/d (equivalent to 4.4 mg T3/d). In the CAM assay, T3 (500–1000 μg/egg) inhibited new blood vessel formation on the growing CAM and increased the frequency of avascular zone (36–50%). To evaluate the antiangiogenic mechanism, we conducted cell-culture studies and found that T3 significantly reduced fibroblast growth factor -induced proliferation, migration, and tube formation in HUVEC (P < 0.05), with δ-T3 having the highest activity. Western blot analysis revealed that δ-T3 suppressed the phosphorylation of phosophoinositide-dependent protein kinase (PDK) and Akt, and increased the phosphorylation of apoptosis signal-regulating kinase and p38 in fibroblast growth factor-treated HUVEC, indicating that the antiangiogenic effects of T3 are associated with changes in growth factor-dependent phosphatidylinositol-3 kinase /PDK/Akt signaling as well as induction of apoptosis in endothelial cells. Our findings suggest that T3 has potential as a therapeutic dietary supplement for preventing angiogenic disorders, and therefore future clinical study will be required to evaluate the efficacy and safety of T3.

Interferon-b Inhibits Liver Metastases from Murine Colon 26 Carcinoma and its Highly Metastatic Variant

The antitumor effects of Interferon-beta (IFN-beta) are due to its direct inhibition of cell proliferation, immunostimulatory activity, and the inhibition of angiogenesis. We investigated the mechanism of the effects of IFN-beta on a murine colon 26 cell line (CT 26) and its highly metastatic variant (L5). We examined its inhibitory effects on cell proliferation in vitro and the development of liver metastases in vivo. The proliferation of CT 26 in vitro was inhibited by IFN-beta in a dose- and time-dependent manner. The number of metastases was reduced in mice inoculated with CT 26 (P<0.01) and L5 (P<0.01) on Day 14 after treatment with IFN-beta. The median survival rate of the mice inoculated with L5 administered IFN-beta every other day, or every day was higher than in the control group (P<0.05). A dorsal air sac assay demonstrated that IFN-beta inhibited angiogenesis in mice inoculated with CT 26, but the effects disappeared with aminoguanidine, an inducible nitric oxide synthase inhibitor. These results showed that IFN-beta directly inhibits the proliferation of CT 26. In addition, the in vivo experiments suggested that IFN-beta might effectively inhibit liver metastases.

Dipalmitoylation of radicicol results in improved efficacy against tumor growth and angiogenesis in vivo

Tumor-related angiogenesis is likely to be a potential target for the treatment of cancer. One key to develop this angiostatic strategy would be to find useful angiogenesis inhibitors. Here we report the effects of radicicol, a microbial angiogenesis inhibitor that we previously identified using the chorioallantoic membrane assay, and its novel analog, 14,16-dipalmitoyl-radicicol, on tumor angiogenesis and growth. As expected for agents containing a penolic hydroxyl group, systemic administration of radicicol had little or no effect on neovascularization triggered by a M5076 mouse tumor cell line or a RMT-1 rat mammary carcinoma cell line established from autochthonous rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene in a mouse dorsal air sac assay system. The agent did not show growth-inhibitory activity against either transplantable M5076 tumors or autochthonous 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. In contrast, 14,16-dipalmitoyl-radicicol potently suppressed tumor angiogenesis and growth in these experimental models. Furthermore, the analog significantly prolonged the survival rate of M5076-implanted mice. Although not stronger than radicicol, it dose-dependently inhibited embryonic angiogenesis in the chorioallantoic membrane assay, the dose required for half-maximal inhibition (ID(50)) value being 23 microg (27 nmol) per egg, and showed concentration-dependent antiproliferative activity against microvascular endothelial cells in vitro. These data suggest that 14,16-dipalmitoyl-radicicol is a promising antitumor agent with antiangiogenic activity.

Anthraquinone derivative emodin inhibits tumor-associated angiogenesis through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation

An anthraquinone derivative, emodin, suppresses tumor development both in vitro and in vivo. In this study, we examined the anti-angiogenic activity of emodin and its modifying effect on the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In cell cultures, emodin inhibited endothelial cell proliferation, migration, and tube formation in a dose-dependent manner. In addition, the mouse dorsal air sac assay revealed the vivo anti-angiogenic potential of emodin. Matrix metalloproteinase-9 (MMP-9) expression, which is critical for the angiogenic process, including migration and tube formation, decreased after exposure to emodin, as determined by polymerase chain reaction with reverse transcription (RT-PCR) and gelatin zymography. Moreover, the phosphorylation of ERK 1/2 decreased after exposure to emodin in a dose-dependent manner. These observations suggest that emodin has the potential to inhibit several angiogenic processes and that these effects may be related to suppression of the phosphorylation of ERK 1/2.

Inhibition of in vivo angiogenesis by N-β-alanyl-5- S-glutathionyl-3,4-dihydroxyphenylalanine

N-β-alanyl-5- S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from the flesh fly, inhibits human tumor growth in the nude mice model; however, the mechanism of its action is unclear. The in vivo antitumor effect includes the inhibition of tumor cell proliferation and suppression of angiogenesis. Angiogenesis is essential for tumor growth in vivo. In this study, we examined whether 5-S-GAD inhibits tumor cell-induced angiogenesis by performing the mouse dorsal air sac assay. We found that intraperitoneal administration of 5-S-GAD inhibited the angiogenesis induced by S180 mouse sarcoma cells. Furthermore, 5-S-GAD also inhibited vascular endothelial growth factor-induced angiogenesis in the Matrigel plug assay and embryonic angiogenesis in the chick embryo chorioallantoic membrane assay. However, 5-S-GAD did not show any effect on the proliferation, migration, and tube formation of vascular endothelial cells. These results provide the first evidence that a bioactive substance derived from the flesh fly has antiangiogenic activity in vivo, although the mechanisms involved could not be explained.

Antitumor Effect of Kigamicin D on Mouse Tumor Models

Kigamicin D is a novel anticancer agent that was identified using a new screening strategy that targets the tolerance of cancer cells to nutrient starvation [1, 2]. Oral administration of kigamicin D was previously described to show a strong antitumor effect in human tumor xenograft models of pancreatic tumors [2]. In this paper we describe that kigamicin D shows the same selective cytotoxicity against normal human cells such as lung fibroblast and prostate stromal cells under nutrient starved condition as against cancer cells. Kigamicin D inhibited tumor cell-induced angiogenesis in a dorsal air sac assay. On the basis of these results we tested other human tumor xenograft models and transplantable syngeneic tumor models in order to determine the spectrum of activity of kigamicin D against various cancers. Kigamicin D showed a weak antitumor effect against LX-1 and DMS-273 lung cancers, but had no effect on DLD-1 colon cancers. When tested against syngeneic tumors, kigamicin D showed a weak antitumor effect against colon26, but showed augmentation of tumor growth on IMC carcinoma at a broad dosage level. Kigamicin D does not show good antitumor activity against human xenograft tumors except pancreatic tumors and murine syngeneic tumors. We found that kigamicin D has excellent antitumor effect specific to pancreatic cancers. Surprisingly, high dosage of kigamicin D increased tumor growth of IMC carcinoma by than 200%. The phenomenon suggests that kigamicin D may cause some immunological response to the tumor.