Dihydrotanshinone I inhibits angiogenesis both in vitro and in vivo

Dihydrotanshinone I （DI）, a naturally occurring compound extracted from Salvia miltiorrhiza Bunge, has been reported to have cytotoxicity to a variety of tumor cells. In this study, we investigated its anti-angiogenic capacity in human umbilical vein endothelial cells. DI induced a potent cytotoxicity to human umbilical vein endothelial cells, with an IC50 value of approximately 1.28 μg/ml.At 0.25-1μg/ml, DI dose-dependently suppressed human umbilical vein endothelial cell migration, invasion, and tube formation detected by wound healing, Transwell invasion and Matrigel tube formation assays, respectively. Moreover, DI showed significant in vivo anti-angiogenic activity in chick embryo chorioailantoic membrane assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 μg/egg. Taken together, our results showed that DI could inhibit angio-genesis through suppressing endothelial cell proliferation, migration, invasion and tube formation, indicating that DI has a potential to be developed as a novel anti-angiogenic agent.     

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.     

Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

A novel RGDS-analog inhibits angiogenesis in vitro and in vivo

In this study the anti-angiogenic action of a novel non-peptide RGDS-analog named RAM was tested in vitro and in vivo. RAM inhibited FGF-2-induced chemotaxis by 80% in an adhesion-independent way. Further, it induced HUVEC-apoptosis in collagen-seeded HUVEC, indicating that such pro-apoptotic effect was adhesion-independent. In vivo studies revealed that RAM inhibited FGF-2 induced angiogenesis by 60% in the mouse Matrigel-assay and in the chicken-egg chorion-allantoic membrane assay. Finally, RAM was markedly more stable in serum as compared to the template RGDS and after 24 h incubation in 100% serum was significantly more active than RGDS. Taken together these results show that RAM exerts anti-chemotactic and pro-apoptotic effects, by an unexpected adhesion-independent mechanism, as we have recently shown for the template RGDS molecule [Blood 103 (2004) 4180], and has in vivo relevant anti-angiogenic properties, with marked stability in serum; therefore, RAM represents a novel promising anti-angiogenic molecule.     

Embellistatin, a microtubule polymerization inhibitor, inhibits angiogenesis both in vitro and in vivo

The efficient inhibition of angiogenesis is considered as a promising strategy for the treatment of angiogenesis-related diseases including cancer. Herein, we report that embellistatin, a bicyclic ketone compound known as a microtubule polymerization inhibitor, exhibits anti-angiogenic activity. Embellistatin inhibited in vitro angiogenesis of bovine aortic endothelial cells (BAECs) such as bFGF-induced invasion and tube formation as well as bFGF-induced mouse corneal angiogenesis in vivo. Notably, embellistatin exhibited stronger inhibition activity for the growth of BAECs than that of normal and cancer cell lines. Cell cycle analysis revealed that the compound arrests cell cycle at G2/M phase, which is associated with the increased expression of p21(WAF1) and p53 partly. These results demonstrate that embellistatin may serve the basis for the development of new anti-angiogenic agents.     

Homocysteine inhibits angiogenesis in vitro and in vivo

Homocysteine has been reported to inhibit endothelial cell proliferation, which is closely related to angiogenesis. However, the relationship between homocysteine and angiogenesis is unknown. To clarify whether homocysteine would inhibit angiogenesis in vitro and in vivo, we examined the effect of homocysteine on tube formation by bovine aortic endothelial cells (BAECs) and by human microvessel endothelial cell-1 (HMEC-1) in vitro, and on angiogenesis in vivo using the chorioallantoic membrane (CAM) assay, as well as on BAEC proliferation and migration. Homocysteine, but not cysteine, inhibited BAEC proliferation, migration, and tube formation in a dose-dependent manner at concentrations from 0 to 10 mM. Homocysteine also inhibited tube formation by HMEC-1s. In these assay, 50% inhibition was induced by about 1 mM homocysteine. In the in vivo CAM assay, 0, 10, 100, 500, and 1000 microgram homocysteine induced an avascular zone by 0, 0, 16.7, 53.3 and 76.5%, respectively, also showing a dose-dependent effect. It was suggested that homocysteine inhibited angiogenesis by preventing proliferation and migration of endothelial cells.     

Lebectin, a Macrovipera lebetina venom-derived C-type lectin, inhibits angiogenesis both in vitro and in vivo

Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin-ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express alphavbeta3, alphavbeta5, and alpha5beta1 integrins, as well as the alpha2, alpha3, alpha6, and beta4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC(50) approximately 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the alpha5beta1 and alphaV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel trade mark (IC(50) = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel trade mark Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases.     

The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo

Angiogenesis consists of multistep pathways such as the degradation of the matrix, proliferation of the endothelial cells, motility of the endothelial cells, formation of the cord structure and network formation of microvessels. The small GTPase Rho participates in cell motility through actin fiber polymerization. The role of the small GTPase Rho signal transduction pathway in regulating angiogenesis, however, is still unknown. In this study, we investigated the role of the small GTPase Rho signal transduction pathway in angiogenesis in vitro and in vivo using the exoenzyme, Clostridium botulinum C3 transferase, which specifically suppresses Rho and a compound, Y-27632, which suppresses p160ROCK (Rho-associated coiled-coil containing protein kinase). In this paper, we showed that the small GTPase Rho-p160ROCK signal transduction pathway played an important role in angiogenesis both in vitro and in vivo. These results suggest that inhibition of the small GTPase Rho signal transduction pathway by the p160ROCK inhibitor could be a possible new strategy for angiogenic diseases.     

A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) β(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) β(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) β(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) β(3) integrin.     

DTD, an anti-inflammatory ditriazine, inhibits angiogenesis in vitro and in vivo

The ditriazine derivative DTD (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3,2-d]-1,2,3-ditriazine) has been previously reported to reduce the degree of granulomatous inflammation and vascular density in a murine air pouch granuloma model. The aim of this study was to test whether DTD affects angiogenesis. Our results show that DTD inhibits in vivo angiogenesis in the chorioallantoic membrane (CAM) assay at doses equal or lower than 0.3 nmol/egg. Different in vitro assays were used to study the potential effects of this compound on key steps of angiogenesis, namely, a colorimetric assay of cell proliferation/viability, a morphogenesis on Matrigel assay, zymographic assays for gelatinases and nuclear morphology and cell cycle analysis for apoptosis induction. Our data indicate that DTD inhibits proliferation but does not induce apoptosis in endothelial cells in vitro. DTD suppresses the endothelial capillary-like chord formation at concentrations lower than those required to inhibit proliferation. DTD treatment inhibits the matrix metalloproteinase-2 production in endothelial and fibrosarcoma cells, but does not affect the cyclooxygenase-2 expression in endothelial cells, as assessed by western blot analysis. Taken together, results here presented indicate that DTD exhibits an anti-angiogenic activity that is independent of inflammatory processes and make it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12 h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.     

Matriptase-2 inhibits HECV motility and tubule formation in vitro and tumour angiogenesis in vivo

Ezrin, primarily acts as a linker between the plasma membrane and the cytoskeleton, is involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion, motility, and modulation of signaling pathways. Although ezrin is now recognized as a key component in tumor metastasis, its roles and the underlying mechanisms remain unclear. In the present study, we chose highly metastatic human lung carcinoma 95D cells, which highly express the ezrin proteins, as a model to examine the functional roles of ezrin in tumor suppression. An ezrin-silenced 95D cell line was established using lentivirus-mediated short hairpin RNA method. CCK-8 assay and soft agar assay analysis showed that downregulation of ezrin significantly suppressed the tumorigenicity and proliferation of 95D cells in vitro. cell migration and invasion studies showed that ezrin-specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. In parallel, it also induced rearrangements of the actin cytoskeleton. Flow cytometry assay showed that changes in the ezrin protein level significantly affected the cell cycle distribution and eventual apoptosis. Furthermore, further studies showed that ezrin regulated the expression level of E-cadherin and CD44, which are key molecules involved in cell growth, migration, and invasion. Meanwhile, the suppression of ezrin expression also sensitized cells to antitumor drugs. Altogether, our results demonstrated that ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will benefit the development of therapeutic strategy for lung cancer.     

Valproic acid inhibits the growth of cervical cancer both in vitro and in vivo

Valproic acid (VPA), a well-known anti-convulsant, is currently under extensive evaluation as an anti-cancer agent. It is known to exert its anti-cancer effect mainly by inhibiting the enzyme histone deacetylase I. In our study, we investigated the effects of VPA on cervical cancer both in vitro and in vivo cancer models. We examined the effects of acute VPA (0, 1.2, 2.4, 5.0 mM) treatment on cell proliferation in cervical cancer cell lines HeLa, SiHa and Ca Ski and histone acetylation, p21 and p53 gene expression in HeLa cell line. We also investigated the effect of chronic VPA administration in tumour xenograft growth studies. Our results show that with acute treatment, VPA can increase the expression of net histone H3 acetylation and up-regulate p21 expression with no effect on p53 expression. Chronic administration of VPA had a net cytostatic effect that resulted in a statistically significant reduction of tumour growth and improved survival advantages in tumour xenografts studies. Furthermore, we also demonstrated that VPA has a direct anti-angiogenic effect in tumour studies and could potentially be a promising candidate for further cervical cancer trails.     

Streptochlorin, a marine natural product, inhibits NF-kappaB activation and suppresses angiogenesis in vitro

Angiogenesis is an essential step in tumor progress and metastasis. Accordingly, small molecules that inhibit angiogenesis would appear to be a promising way to cure angiogenesis-related diseases, including cancer. In the present study, we report that streptochlorin, a small molecule from marine actinomycete, exhibits a potent antiangiogenic activity. The compound potently inhibited endothelial cell invasion and tube formation stimulated with vascular endothelial cell growth factor (VEGF) at low micromolar concentrations where it showed no cytotoxicity to the cells. In addition, streptochlorin inhibited TNF-alpha-induced NF-kappaB activation in the newly developed cell-based reporter gene assay. These data demonstrate that streptochlorin is a new inhibitor of NF-kappaB activation and can be a basis for the development of novel anti-angiogenic agents.