New Vandetanib analogs: fused tricyclic quinazolines with antiangiogenic potential

The antiangiogenic effects of three novel anilinoquinazoline derivatives were studied with the aim to find new multi-kinase inhibitors as anticancer agents. The compounds are characterized by dioxolane, dioxane and dioxepine rings and bear the same aniline substituent in 4 position as Vandetanib, known antiangiogenic agent. The in vitro assays were carried out on human umbilical vein endothelial cells (HUVECs), whereas in vivo angiogenesis was evaluated by means of Matrigel plug assay. The results showed that these compounds exert, even though to different extents, antiangiogenic activity affecting the various step of the process that leads to the formation of new blood vessels. At high concentrations they induced antiproliferative effects, whereas at non-cytotoxic concentrations they inhibited cell migration and the formation of tubular structures in Matrigel. In in vitro assays the dioxolane derivative 1 was more effective than Vandetanib. Indeed, it inhibited the effects induced by exogenous VEGF and FGF-2 on both cell proliferation and morphogenesis, whereas Vandetanib was completely ineffective. Moreover, all the compounds, as Vandetanib, counteracted the FGF-2-induced increase in the hemoglobin content in the Matrigel plugs. Our results showed that all the three novel derivatives possess both in vitro and in vivo antiangiogenic activity, with compound 1 more effective than Vandetanib to inhibit in vitro angiogenesis induced by exogenous cytokines.     

Bp5250 inhibits vascular endothelial growth factor-induced angiogenesis and HIF-1α expression on endothelial cells

Angiogenesis plays a critical role in many physiological and pathological phenomena. A number of anti-angiogenesis drugs have been used in the clinical treatment of diseases such as malignant tumors and macular degeneration. Vascular endothelial growth factor (VEGF), the major pro-angiogenesis factor, is known to stimulate various steps of endothelial angiogenic activity, such as proliferation, migration, and differentiation into vessel-like tubes. In this study, we tested the effects of bp5250 on the angiogenesis of human umbilical endothelial cells (HUVECs). Bp5250 suppressed VEGF-induced endothelial cell proliferation by triggering apoptosis, and reduced endothelial cell migration toward VEGF. Bp5250 also decreased VEGF-stimulated tube formation and rat aortic ring sprouting on Matrigel in a concentration-dependent manner. In the VEGF-activated signaling pathways, bp5250 decreased the phosphorylation of ERK, p38, PI3K-AKT, Src, and FAK and also reduced the activation of the cytoskeleton-associated Rho family, all in a concentration-dependent manner. Bp5250 also attenuated the hypoxia-inducible factor-1α (HIF-1α) and VEGF-stimulated mRNA expression of HUVECs under the hypoxic condition. In vivo, angiogenesis was restrained by a daily intraperitoneal administration of bp5250 in a dose-dependent manner (1–3 mg/kg/d) in the Matrigel plug implantation assay. These results indicate that bp5250 is a potential candidate for developing anti-angiogenic agents.     

Valproic acid inhibits angiogenesis in vitro and in vivo

Valproic acid (VPA) is a widely used antiepileptic agent that is undergoing clinical evaluation for anticancer therapy. We assessed the effects of VPA on angiogenesis in vitro and in vivo. In human umbilical vein endothelial cells, therapeutically relevant concentrations of VPA (0.25 to 1 mM) inhibited proliferation, migration, and tube formation. VPA 1 mM inhibited endothelial cell proliferation by 51 +/- 5%, migration by 86 +/- 11%, and tube formation by 82 +/- 3%. These changes were preceded by the hyperacetylation of histone H4, indicating the inhibition of histone deacetylase (HDAC), and a decreased expression of the endothelial nitric-oxide synthase (eNOS). The inhibition of endothelial cell tube formation by VPA was prevented by addition of the nitric oxide donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate). The anticonvulsive active VPA derivative 2-ethyl-4-methylpentanoic acid, which does not inhibit HDAC, did not affect endothelial cell proliferation, tube formation, or eNOS expression. VPA was also found to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay and in a Matrigel plug assay in mice. Embryos from VPA-treated mice showed disturbed vessel formation. These results indicate that therapeutic plasma levels of VPA inhibit angiogenesis by a mechanism involving a decrease in eNOS expression preceded by HDAC inhibition.     

Dose-dependent effect of dehydroepiandrosterone, but not of its sulphate ester, on angiogenesis

Although dehydroepiandrosterone (DHEA) is widely used in the elderly to prevent some adverse effects of ageing, possible deleterious side effects have not been fully assessed. We evaluated the direct actions of DHEA and DHEA sulphate on angiogenesis, a critical event in pathologies that are common in the elderly (cancer, atherosclerosis, inflammation... etc.). At physiological concentrations found in human plasma following DHEA therapy (1-50 nM), DHEA had no action on angiogenesis in vitro. In contrast, higher concentrations of DHEA (10-100 microM), which can be found in tissues after local administration or storage, inhibited in vitro endothelial cell proliferation (blockage in G2/M), migration and capillary tube formation and in vivo angiogenesis in the Matrigel plug assay. This inhibition might be due to a decreased glucose-6-phosphate dehydrogenase activity and to a modification of the tubulin network involved in cell proliferation and migration. The sulphate ester form of DHEA had no effect on angiogenesis.     

XR5967, a novel modulator of plasminogen activator inhibitor-1 activity, suppresses tumor cell invasion and angiogenesis in vitro

Recent reports suggest that elevated levels of plasminogen activator inhibitor (PAI)-1 may contribute to tumor progression. We have recently shown that antibodies to PAI-1 block the invasive and migratory potential of human fibrosarcoma cells and suppress angiogenesis in vitro. Here we report the in vitro evaluation of a low-molecular-weight modulator of PAI-1, XR5967, on invasion, migration and angiogenesis. XR5967, a diketopiperazine, dose-dependently inhibited the activity of human and murine PAI-1, towards urokinase plasminogen activator (uPA), with IC50 values of 800 nM and 8.3 microM, respectively. This was confirmed by SDS-PAGE, revealing that XR5967 inhibited complex formation between PAI-1 and uPA. This suppression may be caused by XR5967 promoting insertion of the reactive center loop within PAI-1. XR5967 dose-dependently inhibited the invasion of human HT1080 fibrosarcoma cells through Matrigel. Their invasion was reduced by 57% (p<0.001) at 5 microM. HT1080 cell migration was inhibited in a similar manner, indicating that PAI-1 may play an additional role in invasion, which is distinct to its role in the regulation of proteolysis. The potential of XR5967 to inhibit the invasion/migration of human endothelial cells was investigated in an in vitro model of angiogenesis. In this model XR5967 reduced tubule formation by 77% at 5 microM (p<0.001), highlighting a crucial role for PAI-1 in angiogenesis. These data stress the importance of a balanced proteolysis in the processes of invasion, migration and angiogenesis. Our results support the clinical findings and indicate that modulation of PAI-1 activity, with low-molecular-weight inhibitor of PAI-1 activity, may be of therapeutic benefit for the treatment of cancer.     

OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt–nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to study the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt–NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt–NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9's clinical promise as a component of anticancer therapy.     

siRNA沉默Twist基因对鼻咽癌细胞CNE-2增殖的影响

目的探讨RNA干扰沉默Twist基因对人鼻咽癌细胞株CNE-2的迁移、侵袭和增殖等生物学活性的影响。方法构建Twist-shRNA表达载体,脂质体法将重组质粒分别转染入鼻咽癌细胞株CNE-2中,RT-PCR法检测Twist mRNA、Western blot法检测Twist蛋白表达量的变化。MTT法检测细胞的增殖,细胞创伤愈合试验观察细胞迁移能力,Tran-swell小室试验检测细胞侵袭能力的变化。结果沉默Twist基因可减慢CNE-2细胞生长速度(与对照组比较P<0.05),降低CNE-2细胞的迁移速度,抑制CNE-2的体外侵袭能力。结论靶向封闭Twist基因的表达,可明显抑制CNE-2细胞的增殖、迁移及侵袭能力,提示Twist可作为鼻咽癌防治的一个新的靶点。     

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

N-beta-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.     

Rhodostomin, a snake venom disintegrin, inhibits angiogenesis elicited by basic fibroblast growth factor and suppresses tumor growth by a selective alpha(v)beta(3) blockade of endothelial cells

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, although angiogenic factor and integrin-extracellular matrix interaction modulate this process. We report here that a snake venom-derived disintegrin, rhodostomin, inhibited distinct steps in angiogenesis elicited by basic fibroblast growth factor (bFGF), and also suppressed in vivo murine melanoma tumor growth. Rhodostomin dose-dependently inhibited bFGF-induced human umbilical vein endothelial cell (HUVEC) proliferation as examined by cell number count, metabolic activity, and BrdU incorporation assays with submicromolar IC(50) values. However, it apparently did not affect the viability of murine B16F10 melanoma cells, even up to 50 microM. Rhodostomin also inhibited HUVEC migration and invasion evoked by bFGF, and tube formation of bFGF-treated HUVECs in Matrigel. Moreover, rhodostomin selectively inhibited bFGF-, but not vascular endothelial growth factor-associated angiogenesis in the chick chorioallantoic membrane model. Furthermore, rhodostomin blocked both bFGF- and B16F10-induced neovascularization in murine Matrigel plug model and suppressed the growth of subcutaneously inoculated B16F10 solid tumor, leading to a prolonged survival of the rhodostomin-treated C57BL/6 mice. The antiangiogenic effect of rhodostomin on bFGF-treated HUVECs is related to the integrin alpha(v)beta(3) blockade, as evidenced by its selective inhibition on the binding of 7E3, a monoclonal antibody (mAb) raised against alpha(v)beta(3,) but not that of P1F6, an alpha(v)beta(5) mAb toward both naive and bFGF-primed HUVECs. Moreover, 7E3 specifically blocked fluorescein isothiocyanate-conjugated rhodostomin binding to HUVEC, whereas P1F6 and anti-integrin alpha(2), alpha(3), alpha(4), or alpha(5) mAbs did not.     

羧甲基壳聚糖对人高转移肺癌细胞——95-D增殖能力和体外转移能力的抑制作用

目的评价羧甲基壳聚糖对人高转移肺癌细胞——95-D增殖能力和体外转移能力的抑制作用.方法用软琼脂克隆形成实验、创伤实验、黏附实验和transwell小室实验来评价羧甲基壳聚糖对95-D细胞的影响.结果羧甲基壳聚糖浓度在0～150 μg·ml-1范围内,剂量依赖性地抑制95-D细胞的增殖能力、移动能力、浸润能力、黏附能力和克隆形成能力.结论羧甲基壳聚糖能有效抑制95-D细胞的增殖能力和体外转移能力.     

Ginsenoside Rg3 alleviates the migration,invasion, and angiogenesis of lung cancer cells by inhibiting the expressions of cyclooxygenase-2 and vascular endothelial growth factor

Lung cancer (LC) is a common cancer with high incidence and mortality rates. In recent years, ginsenoside Rg3 (Rg3), a traditional medicine, is widely used for the treatment of LC. Herein, we concentrate on assessing the effect of Rg3 on LC cell migration and invasion. The effects of Rg3 (0, 25, 50, and 100 μg/ml) on the viability, migration, invasion, angiogenesis, and expressions of epithelial–mesenchymal transition (EMT)-related proteins, cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) of LC cell lines were evaluated by cell counting kit-8 (CCK-8), scratch, transwell, tube formation, and western blot assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to assess transfection efficiency. COX2 overexpression plasmid and short hairpin RNA for VEGF (shVEGF) were applied to evaluate whether the effect of Rg3 is related to COX2 and VEGF through rescue assay. In this study, Rg3 significantly dose-dependently suppressed the viability, migration, invasion, angiogenesis, and protein expressions of N-cadherin, vimentin, COX2, and VEGF in H1299 and A549 cells, while promoting the expression of E-cadherin protein. COX2 overexpression markedly reversed the effects of Rg3 on the viability, migration, invasion, angiogenesis, and EMT-related protein expression levels in LC cells; however, such effects of COX2 overexpression were offset by VEGF knockdown. In sum, Rg3 alleviates the migration, invasion, and angiogenesis of LC cells by inhibiting the expressions of COX2 and VEGF.     

Development of a Simple Cell Invasion Assay System

Cell invasion assay is important for studying various biological events, such as inflammation, cancer metastasis, and angiogenesis. In this study, we developed a simple method for the quantification of cell invasion by using a culture insert with fluorescence blocking micropore membrane (FBM). Fluorescence labeled cells were simply added to a culture insert with a 8 micrompore FBM precoated with Matrigel and incubated for an appropriate duration. Then, the FBM was examined under a fluorescence microscope to count the invaded cell number. By this method, accurate invasion assay is easily performed without the steps of fixation and staining of cells and removal of cells which do not invade.     

In vivo and in vitro antitumor effect of ascorbic acid, lysine, proline and green tea extract on human melanoma cell line A2058

BACKGROUND: Melanoma, a very serious form of skin cancer, causes the most skin cancer-related deaths, due to metastasis. Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling. MMPs, VEGF, Ki-67 (proliferative protein) and constituents of ECM play a critical role in angiogenesis, and are crucial in neoplastic invasion and metastasis. MATERIALS AND METHODS: The effect of a diet (NM) containing lysine, proline, arginine, ascorbic acid and green tea extract on the growth of tumors induced by implanting human melanoma A2058 cells in athymic nude mice was examined and, also, on the expression of MMPs, VEGF and Ki-67 in these tumors. The effect of NM in vitro on the melanoma A2058 cell line was tested by measuring: cell proliferation by the MTT assay, expression of MMPs by gelatinase zymography and invasion through Matrigel. RESULTS: Nutrient supplementation strongly suppressed the growth of tumors (by 57%)without adverse effects in nude mice. Histological studies supported these findings by showing inhibition of MMP-9 and VEGF secretion and mitotic index. In vitro, NM inhibited melanoma cell growth by 64% at 500 microg/ml and Matrigel invasion by 95% at 100 microg/ml NM. CONCLUSION: These results suggest that NM may have a therapeutic potential in melanoma.     

新生霉素抑制血管生成及其与长春新碱的协同作用

目的研究新生霉素的抑制血管生成作用及其机制。方法以鸡胚尿囊膜模型测定对血管生成的作用，并分别用MTT法、明胶酶谱法等观察新生霉素对于内皮细胞和肺癌PG细胞的影响。结果新生霉素200 μg/egg对鸡胚尿囊膜血管生成的抑制率为68.7％，且呈浓度依赖性抑制内皮细胞的增殖、运动、MMP-2分泌以及管腔的形成；新生霉素和长春新碱对血管生成及PG细胞的增殖均有明显的协同作用。结论本研究首次确定新生霉素具有抑制血管生成活性,与长春新碱联合可增强对血管生成的抑制作用。     

Protocatechuic Acid Induces Angiogenesis through PI3K‐Akt‐eNOS‐VEGF Signalling Pathway

In this study, we sought to elucidate whether protocatechuic acid contributes to induce angiogenesis as well as its mechanisms. To this end, we examined the role of protocatechuic acid on human brain microvascular endothelial cell line (HBMEC) proliferation, invasion and tube formation in in vitro. For the study of mechanisms involved, the phosphoinositide 3 kinase (PI3K)‐Akt inhibitor LY294002, the endothelial nitric oxide synthase (eNOS) inhibitor L ‐NAME, vascular endothelial growth factor (VEGF), antagonist sFlt‐1 and VEGF receptor blocker SU‐1498 were used. Proliferation of HBMEC was tested by MTT. Scratch adhesion test was used to assess the ability of invasion. A Matrigel tube formation assay was performed to test capillary tube formation ability. PI3K‐Akt‐eNOS‐VEGF pathway activation in HBMEC was tested by Western blot. Our data suggested that protocatechuic acid induces angiogenesis in vitro by increasing proliferation, invasion and tube formation. VEGF expression was increasing by protocatechuic acid and counteracted by VEGF antagonist sFlt‐1, LY294002 and L‐NAME in HBMEC. Tube formation was increased by protocatechuic acid and counteracted by VEGF receptor blocker‐SU1498, LY294002 and L‐NAME. These data suggest that protocatechuic acid may be a candidate therapy for stroke recovery by promoting angiogenesis via a programmed PI3K/Akt/eNOS/VEGF signalling axis.     

Toluquinol,a marine fungus metabolite,is a new angiosuppresor that interferes the Akt pathway

Toluquinol, a methylhydroquinone produced by a marine fungus, was selected in the course of a blind screening for new potential inhibitors of angiogenesis. In the present study we provide the first evidence that toluquinol is a new anti-angiogenic-compound. In a variety of experimental systems, representing the sequential events of the angiogenic process, toluquinol treatment of activated endothelial cells resulted in strong inhibitory effect. Toluquinol inhibited the growth of endothelial and tumor cells in culture in the micromolar range. Our results indicate that the observed growth inhibitory effect could be due, at least in part, to an induction of apoptosis. Toluquinol induced endothelial cell death is mediated via apoptosis after a cell cycle block and caspase activation. Capillary tube formation on Matrigel and migratory, invasive and proteolytic capabilities of endothelial cells were inhibited by addition of toluquinol at subtoxic concentrations. Inhibition of the mentioned essential steps of in vitro angiogenesis agrees with the observed inhibition of the in vivo angiogenesis, substantiated by using the chick chorioallatoic membrane assay and confirmed by the murine Matrigel plug, the zebrafish embryo neovascularization and the zebrafish caudal fin regeneration assays. Data here shown altogether indicate that toluquinol has antiangiogenic effects both in vitro and in vivo that are exerted partly by suppression of the VEGF and FGF-induced Akt activation of endothelial cells. These effects are carried out at lower concentrations to those required for other inhibitors of angiogenesis, what makes toluquinol a promising drug candidate for further evaluation in the treatment of cancer and other angiogenesis-related pathologies.     

Cooperation of urokinase plasminogen activator and matrix metalloproteinases in NK cell invasion

We have previously investigated the role of the urokinase plasminogen activator (uPA) system in NK cell invasion. We have also studied NK cell-derived matrix metalloproteinases (MMPs) in extracellular matrix (ECM) degradation. We now report that both enzyme systems cooperate in NK cell invasion. Zymographic analyses detected uPA in RNK-16 cell conditioned media (CM) with the same molecular weights as the uPA we have previously shown to be associated with the rat NK cell urokinase plasminogen activator receptor. The combination of aprotinin, an inhibitor of plasmin, and Batimastat (BB94), an inhibitor of MMPs, in Matrigel invasion assays showed a more potent inhibitory effect on NK cell invasion than either inhibitor alone. Finally, a down regulation of uPA mRNA was noted following RNK-16 stimulation with collagen IV, fibronectin, and laminin.     

Effect of nicotine on human umbilical vein endothelial cells (HUVECs) migration and angiogenesis

The effects of nicotine on vascular endothelial cells have not been completely elucidated. We performed this study to assess the changes in cellular behaviors of human umbilical vein endothelial cells (HUVECs) treated with nicotine. We examined changes in cell count and morphology and assayed cellular migration with Boyden chamber and microcapillary tube formation in a Matrigel matrix following treatment with various concentrations of nicotine. Compared to the control, nicotine stimulated cell proliferation, migration, and tube formation at concentrations similar to those found in smokers. Although there were no specific morphological changes in HUVECs treated with nicotine at the concentration similar to that in smokers, at high concentration (10(-4) M), morphological changes such as cytoplasmic vacuolization and irregular cell shape were observed, which were assumed to be the result of direct cytotoxicity of nicotine. In HUVECs, nicotine enhanced cellular proliferation, migration and angiogenesis in vitro, and thus caused a functional change, not a morphological change at a concentration similar to that in habitual smokers.     

Wf-536 prevents tumor metastasis by inhibiting both tumor motility and angiogenic actions

The signaling pathway of Rho and Rho-associated coiled-coil forming protein kinase (ROCK) is involved in tumor metastasis. In the present study, we investigated the suppressive effect of a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride], on spontaneous tumor metastasis in vivo and analyzed its action on tumor cell motility and angiogenesis to clarify its action mechanism. Wf-536 (0.3-3 mg/kg/day) was found to inhibit Lewis lung carcinoma (LLC) metastasis and LLC-induced angiogenesis in orally treated mice; in vitro, it inhibited both invasion and migration by LLC cells and invasion, migration, and formation of capillary-like tubes on Matrigel by endothelial cells, without cytotoxicity or anti-proliferative action in either cell type. We conclude that Wf-536 has tumor anti-metastatic activity which may depend on inhibition of tumor motility and angiogenesis. The findings support its further clinical development as an anti-metastatic agent.