Bovine lactoferrin inhibits tumor-induced angiogenesis

Recent studies have demonstrated that bovine lactoferrin (bLF) suppresses tumor growth and metastasis in the mouse and rat and moreover may inhibit angiogenesis. To determine whether angiogenesis inhibition might contribute to antitumor activity, we examined the influence of bLF on tumor-induced angiogenesis and endothelial cell functions as well as angiogenesis-related cytokine production. Bovine LF exhibited dose-dependent inhibition of angiogenesis on 4-6-day-old chick embryo chorioallantoic membranes (CAMs) that lack a mature immune response. This inhibition was reversed when bLF was simultaneously treated with basic fibroblast growth factor (bFGF). It also inhibited in vitro formation of tube-like structures of mouse endothelial KOP2.16 cells. Moreover, it potently suppressed bFGF- or VEGF-induced proliferation of mouse endothelial KOP2.16 cells, but not of mouse fibroblast A31 cells and Lewis lung carcinoma (3LL) cells. In mice, both orally and intraperitoneally administered bLF significantly and dose-dependently suppressed 3LL cell-induced angiogenesis in a dorsal air sac assay. As orally administered bLF was reported to exhibit antitumor activity through production of interferon (IFN)-gamma and interleukin (IL)-18 in intestinal mucosa (Kuhara T et al., Nutr Cancer 2000;38:192-9), production of these cytokines in mouse serum and peritoneal macrophages by bLF was examined. IFN-gamma was not detected in serum by bLF administration. However, bLF markedly elevated IL-18 concentration in serum by oral administration, but not by intraperitoneal administration. It also induced IL-18 in peritoneal macrophages in vitro. These results suggest that bLF participates as a regulator of angiogenesis, possibly explained by blocking endothelial function and inducing IL-18 production. Antitumor activity of bLF may thus be partly mediated by angiogenesis inhibition.     

Carboxymethyl benzylamide dextran inhibits angiogenesis and growth of VEGF-overexpressing human epidermoid carcinoma xenograft in nude mice

Vascular endothelial growth factor (VEGF) expression is elevated in a wide variety of solid tumours. Inhibition of VEGF activities is able to reduce angiogenesis and tumour growth. We have recently shown in vitro that carboxymethyl dextran benzylamide (CMDB7) prevents the binding of VEGF(165) to its cell surface receptors and thus inhibits VEGF activities on endothelial cells. In the present study, we explored the effects of CMDB7 on highly aggressive human epidermoid carcinoma A431 cells known to overexpress epidermal growth factor receptors (EGFRs) and produce a high amount of VEGF and a minor quantity of bFGF. In vitro, CMDB7 blocked the mitogenic activity of A431-conditioned medium on endothelial cells. Concerning A431 cells, CMDB7 inhibited their proliferation and the VEGF(165) binding to them. In vivo, administration of CMDB7 (10 mg kg(-1)) three times per week for 2 weeks inhibited the growth of A431 xenografts in nude mice by 73% as compared to the control group. Immunostaining of endothelial cells with mouse-specific GSL-1 lectin in tumour sections revealed that CMDB7 also inhibited the density of intratumour endothelial cells by 66%. These findings demonstrate that CMDB7 has an efficient antiangiogenic and antitumour action in vivo even when tumour cells produce a high level of VEGF and EGFRs.     

A novel hypoxia-dependent 2-nitroimidazole KIN-841 inhibits tumour-specific angiogenesis by blocking production of angiogenic factors

Tumour angiogenesis is initiated by angiogenic factors that are produced in large amounts by hypoxic tumour cells. The inhibition of this step may lead to tumour-specific antiangiogenesis because normal tissues are not usually hypoxic. On the other hand, blocking a biological function of endothelial cells is known to result in angiogenic inhibition. To produce a tumour-specific and powerful antiangiogenesis, we determined whether potent angiogenic inhibition could be achieved by inhibiting the production of angiogenic factors by hypoxic tumour cells and simultaneously blocking certain angiogenic steps in endothelial cells under normoxia. We focused on the 2-nitroimidazole moiety, which is easily incorporated into hypoxic cells and exhibits its cytotoxicity as hypoxic cytotoxin. We designed and synthesised 2-nitroimidazole derivatives designated as KIN compounds, and investigated their antiangiogenic activities under normoxia using a chick embryo chorioallantoic membrane. KIN-841 (2-nitroimidazole 1-acetylhydroxamate) showed a potent angiogenic inhibition in a dose-dependent manner. This compound inhibited the proliferation of bovine pulmonary arterial endothelial (BPAE) cells more strongly than that of tumour cells, such as Lewis lung carcinoma (3LL) cells, under normoxia. The inhibition of cell proliferation by KIN-841 under hypoxia increased about five-fold compared to that under normoxia. Moreover, under hypoxia, KIN-841 significantly decreased the excessive production of vascular endothelial cell growth factors induced by 3LL cells as determined by tritium-labelled thymidine ([(3)H]thymidine) incorporation into BPAE cells and by ELISA. Intraperitoneal administration of KIN-841 suppressed 3LL-cell-induced in vivo angiogenesis in the mouse dorsal air sac system. These results indicate that the regulation of the production of angiogenic factors by hypoxic tumour cells is a useful target for tumour-specific angiogenesis inhibition, and that KIN-841, which causes simultaneous direct inhibition of endothelial cell function and production of angiogenic factors by hypoxic tumour cells, is a very potent inhibitor of tumour-specific angiogenesis. Thus, the potential for clinical use of KIN-841 as an antitumour drug is very high.     

烟曲霉素抑制小鼠结直肠癌生长及转移的体内外研究

背景与目的：血管新生与肿瘤生长密切相关,烟曲霉素（Fumagillin）可特异性抑制血管内皮细胞的增殖,但其对结直肠癌的治疗作用尚不十分清楚。本研究旨在探讨Fumagillin对结直肠癌细胞系生长的抑制作用及其作用机制。方法：将WiDr或HT-29细胞以5×105/L的量分别接种于20只SCID小鼠背部皮下,接种4周后每2日腹腔注射Fumagillin（0.1mg/kg）或Cyclo（1mg/kg）（Arg-Gly-Asp-D-Phe-Val）持续4周,然后处死动物并测量原发瘤质量及原发瘤内微血管密度。在体外培养脐带静脉内皮细胞（HUVECs）,向培养基内加入Fumagillin（0.01mg/kg）或Cyclo（0.1mg/kg）,观察其对HUVECs增殖及微管形成的抑制作用。以基因芯片技术筛选Fumagillin处理后HUVECs的基因表达变化,并以定量PCR和免疫印迹技术对基因芯片的结果做进一步验证。结果：Fumagillin处理组小鼠的结直肠癌原发瘤质量及肿瘤内CD105阳性的微血管数量显著小于对照组小鼠（P〈0.05）,体外实验显示,Fumagillin处理后HUVECs的增殖和微管形成受到明显抑制,基因芯片检测显示Fumagillin处理后HUVECs有71个基因表达上调和143个基因表达下调,表达变化的基因多与细胞黏附、移动、增殖和基因转录有关。定量PCR和免疫印迹技术发现Fumagillin抑制HUVECs表达cyclinE2,白细胞活化黏附因子（activated leukocyte cell adhesion molecule,ALCAM）和细胞间黏附分子-1（intercellular adhesion molecule-1,ICAM-1）。结论：Fumagillin通过抑制新生血管的形成来抑制结直肠癌的生长。     

Caveolin-1在血管生成中的作用

Caveolin-1是细胞膜表面内陷结构Caveolae的标志性蛋白，识别并与多种信号蛋白分子相作用，在内皮细胞内表达量较高，在血管内皮细胞增殖、迁移和分化过程中具有重要作用，在小鼠动物模型中对血管生成、肿瘤生长起抑制作用，有可能成为抑制肿瘤血管生成的靶蛋白。     

Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis

Background:

Molecules that are highly expressed in tumour endothelial cells (TECs) may be candidates for specifically targeting TECs. Using DNA microarray analysis, we found that the lysyl oxidase (LOX) gene was upregulated in TECs compared with its expression in normal endothelial cells (NECs). LOX is an enzyme that enhances invasion and metastasis of tumour cells. However, there are no reports on the function of LOX in isolated TECs.

Methods:

TECs and NECs were isolated to investigate LOX function in TECs. LOX inhibition of in vivo tumour growth was also assessed using β-aminopropionitrile (BAPN).

Results:

LOX expression was higher in TECs than in NECs. LOX knockdown inhibited cell migration and tube formation by TECs, which was associated with decreased phosphorylation of focal adhesion kinase (Tyr 397). Immunostaining showed high LOX expression in human tumour vessels in vivo. Tumour angiogenesis and micrometastasis were inhibited by BAPN in an in vivo tumour model.

Conclusion:

LOX may be a TEC marker and a possible therapeutic target for novel antiangiogenic therapy.     

干扰异粘蛋白基因的表达抑制乳腺癌血管生成

目的:通过干扰乳腺癌细胞异粘蛋白基因（metadherin,MTDH）的表达水平,观察乳腺癌血管生成的变化,探讨MTDH对乳腺癌血管生成的调控作用及分子机制。方法:构建干扰MTDH表达的质粒,转染乳腺癌MDA-MB-231细胞系,建立低表达MTDH的prp-MTDH细胞系及空载体的prp-control对照组细胞系。收集各组乳腺癌细胞的条件培养基,通过小管形成实验和鸡胚绒毛尿囊膜实验,研究干扰MTDH表达对乳腺癌血管生成的影响;通过qRT-PCR和Western blot技术检测MTDH表达降低对血管生成相关因子ERK1/2、VEGF、MMP2和MMP9的影响。结果:通过qRT-PCR检测MTDH基因的表达,发现prp-MTDH细胞系的MTDH mRNA水平由1.01±0.04降低到0.26±0.02（t=19.86,P＜0.01）,Western blot检测MTDH在蛋白水平亦明显降低。通过小管形成实验和鸡胚绒毛尿囊膜实验,发现干扰MTDH表达后的prp-MTDH细胞系血管生成能力明显下降(t=18.43,P＜0.01;t=10.23,P＜0.05)。通过对血管生成相关因子的检测,发现干扰MTDH表达后,具有活性的p-ERK1/2表达降低,VEGF、MMP2、MMP9均明显降低,差异具有统计学意义（P＜0.05）。结论:通过干扰异粘蛋白基因表达可以抑制乳腺癌的血管生成,异粘蛋白基因可能会成为乳腺癌抗血管生成治疗的新靶点。     

Ezetimibe suppresses development of liver tumors by inhibiting angiogenesis in mice fed a high‐fat diet

Nonalcoholic steatohepatitis (NASH) is a common cause of liver cirrhosis and hepatocellular carcinoma (HCC). However, effective therapeutic strategies for preventing and treating NASH‐mediated liver cirrhosis and HCC are lacking. Cholesterol is closely associated with vascular endothelial growth factor (VEGF), a key factor that promotes HCC. Recent reports have demonstrated that statins could prevent HCC development. In contrast, we have little information on ezetimibe, an inhibitor of cholesterol absorption, in regards to the prevention of NASH‐related liver cirrhosis and HCC. In the present study, a steatohepatitis‐related HCC model, hepatocyte‐specific phosphatase and tensin homolog (Pten)‐deficient (Pten^Δhep) mice were fed a high‐fat (HF) diet with/without ezetimibe. In the standard‐diet group, ezetimibe did not reduce the development of liver tumors in Pten^Δhep mice, in which the increase of serum cholesterol levels was mild. Feeding of a HF diet increased serum cholesterol levels markedly and subsequently increased serum levels of VEGF, a crucial component of angiogenesis. The HF diet increased the number of VEGF‐positive cells and vascular endothelial cells in the tumors of Pten^Δhep mice. Kupffer cells, macrophages in the liver, increased VEGF expression in response to fat overload. Ezetimibe treatment lowered cholesterol levels and these angiogenetic processes. As a result, ezetimibe also suppressed inflammation, liver fibrosis and tumor growth in Pten^Δhep mice on the HF diet. Tumor cells were highly proliferative with HF‐diet feeding, which was inhibited by ezetimibe. In conclusion, ezetimibe suppressed development of liver tumors by inhibiting angiogenesis in Pten^Δhep mice with hypercholesterolemia.     

Tamoxifen inhibits endothelial cell proliferation and attenuates VEGF-mediated angiogenesis and migration in vivo

INTRODUCTION: Angiogenesis is fundamental to tumour growth and vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic cytokines known. We have previously demonstrated that tamoxifen reduces serum VEGF in certain cancer patients. We hypothesized that tamoxifen may attenuate the angiogenetic response to VEGF. METHODS: Human dermal microvessel endothelial primary cell cultures (HMEC) were incubated with tamoxifen (1.25-5.0 microg) or vehicle. Cell proliferation was quantified using 5-bromo-2'-deoxyuridine (BrdU) labelling endothelial cell proliferation assay. The effect of oral tamoxifen (20 mg/day) on VEGF-mediated angiogenesis in vivo was assessed using a Matrigel angiogenesis assay in the Sprague-Dawley rat. RESULTS: Tamoxifen (5.0 microg/ml) significantly reduced HMEC proliferation over 24 h when compared with cells treated with vehicle alone. Oral administration of tamoxifen in the rat (20 mg/day) significantly reduced endothelial cell proliferation and migration in response to VEGF. CONCLUSION: Tamoxifen (5.0 microg/ml) reduces proliferation of a VEGF-dependent endothelial cell line in vitro. In vivo, orally administered tamoxifen reduces VEGF-mediated angiogenesis in the rat. These findings indicate that tamoxifen may directly inhibit the effect of VEGF on the endothelial cell, in addition to its previously described effect of reducing serum VEGF levels. This data supports a role for tamoxifen in modulation of the VEGF-dependent angiogenic response to surgical trauma, particularly as an adjuvant therapy for VEGF-dependent tumours.     

低剂量紫杉醇联合沙利度胺抑制小鼠S-180肉瘤血管生成的研究

目的:探讨低剂量紫杉醇联合沙利度胺对小鼠S-180肉瘤血管生成的作用。方法:建立S-180移植瘤小鼠模型,将40只荷瘤小鼠随机分为4组。A组:生理盐水组(对照组);B组:紫杉醇组,腹腔内注射20 mg/kg,每周3次,连续2周;C组:沙利度胺组,200 mg/kg灌胃,每周3次,连续2周;D组:紫杉醇+沙利度胺组,腹腔内注射紫杉醇(20 mg/kg)和沙利度胺(200 mg/kg)灌胃,每周3次,连续2周。观察瘤重、抑瘤率、血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达和微血管密度(microvessel density,MVD)计数。结果:与对照组比较,B、C、D组均能降低瘤重、减少VEGF的表达、降低MVD计数(P<0.05);D组分别与B、C组比较,VEGF的表达及MVD计数均低于B、C组,且差异具有统计学意义(P<0.05)。结论:小剂量紫杉醇联合沙利度胺能一定程度上抑制S180肉瘤血管的生长,两者具有协同作用。     

Alpha1-antitrypsin inhibits angiogenesis and tumor growth

Disturbances of the ratio between angiogenic inducers and inhibitors in tumor microenvironment are the driving force behind angiogenic switch critical for tumor progression. Angiogenic inhibitors may vary depending on organismal age and the tissue of origin. We showed that alpha(1)-antitrypsin (AAT), a serine protease inhibitor (serpin) is an inhibitor of angiogenesis, which induced apoptosis and inhibited chemotaxis of endothelial cells. S- and Z-type mutations that cause abnormal folding and defective serpin activity abrogated AAT antiangiogenic activity. Removal of the C-terminal reactive site loop had no effect on its angiostatic activity. Both native AAT and AAT truncated on C-terminus (AATDelta) inhibited neovascularization in the rat cornea and delayed the growth of subcutaneous tumors in mice. Treatment with native AAT and truncated AATDelta, but not control vehicle reduced tumor microvessel density, while increasing apoptosis within tumor endothelium. Comparative analysis of the human tumors and normal tissues of origin showed correlation between reduced local alpha(1)-antitrypsin expression and more aggressive tumor growth.     

Neuropilin-1与肿瘤血管新生和生长转移

神经纤毛蛋白-1（Neuropilin-1）最早是作为轴突导向分子collapsin／semaphorin的受体被发现，在神经发育过程中引导轴突选择正确途径以成功到达靶区，与此同时又可作为血管内皮生长因子受体-2（VEGFR-2）的共受体表达于血管内皮细胞，在血管新生中发挥作用。近来的研究表明NRP-1可通过依赖于VEGF和独立于VEGF的方式参与调节肿瘤血管新生，在肿瘤生长转移中发挥作用。     

EGCG对胃癌血管新生中VEGF信号通路的多靶点作用

目的：探讨EGCG对胃癌血管生成抑制作用及其信号通路。方法：建立裸鼠异位胃癌模型,经腹腔注射EGCG,检测肿瘤生长及肿瘤组织微血管密度;不同浓度EGCG处理胃癌细胞24 h,检测胃癌VEGF蛋白和mRNA表达及VEGF分泌;同时不同浓度EGCG处理脐静脉内皮细胞,检测内皮细胞生长、迁移和体外小管形成。结果：EGCG显著抑制胃癌生长和肿瘤血管生成,平均肿瘤抑制率60.4%;EGCG显著抑制胃癌VEGF蛋白、mRNA表达和VEGF分泌;EGCG时间和剂量依赖性地抑制VEGF诱导的内皮细胞增殖,同时也剂量依赖性地抑制VEGF诱导的内皮细胞的迁移和小管生成。结论：EGCG多靶点作用于VEGF信号通路,抑制胃癌生长和血管生成。     

Study of puupehenone and related compounds as inhibitors of angiogenesis

Puupehenone, a sesquiterpene produced by certain sponges, was selected in the course of a blind screening for new potential inhibitors of angiogenesis. In our study, we compare the potential anti-angiogenic activities of puupehenone and another 11 related compounds that were either natural products from marine origin or their synthetic derivatives. The effects of these compounds were determined with cell growth and differentiation assays on bovine aorta endothelial cells. Our results show that these compounds are weak inhibitors to cell growth and are not selective for endothelial cells. However, contrary to cell growth, the differentiation of endothelial cells into tubular structures was completely inhibited by 7 of these compounds at concentrations equal or lower than 3 microM. Three of these compounds, isozonarol, 8-epipuupehedione and 8 epi-9,11-dihydropuupehedione, completely inhibited the in vivo angiogenesis in the CAM assay at doses equal or lower than 30 nmol/egg. Further characterisation showed that these 3 terpenes also inhibited endothelial cell production of urokinase and invasion. One compound (8-epipuupehedione) inhibited endothelial cell migration in a dose-dependent manner. The anti-angiogenic properties of the selected compounds, the simplicity of their structures and the feasibility of their synthesis make them attractive drugs for further evaluation in the treatment of angiogenesis-related pathologies.     

EGCG对胃癌血管新生中VEGF信号通路的多靶点作用

目的:探讨EGCG对胃癌血管生成抑制作用及其信号通路。方法:建立裸鼠异位胃癌模型,经腹腔注射EGCG,检测肿瘤生长及肿瘤组织微血管密度;不同浓度EGCG处理胃癌细胞24 h,检测胃癌VEGF蛋白和mRNA表达及VEGF分泌;同时不同浓度EGCG处理脐静脉内皮细胞,检测内皮细胞生长、迁移和体外小管形成。结果:EGCG显著抑制胃癌生长和肿瘤血管生成,平均肿瘤抑制率60.4%;EGCG显著抑制胃癌VEGF蛋白、mRNA表达和VEGF分泌;EGCG时间和剂量依赖性地抑制VEGF诱导的内皮细胞增殖,同时也剂量依赖性地抑制VEGF诱导的内皮细胞的迁移和小管生成。结论:EGCG多靶点作用于VEGF信号通路,抑制胃癌生长和血管生成。     

A selective retinoid X receptor agonist bexarotene (LGD1069, targretin) inhibits angiogenesis and metastasis in solid tumours

The present study determined the influence of a retinoid X receptor agonist bexarotene on angiogenesis and metastasis in solid tumours. In the experimental lung metastasis xenograft models, treatment with bexarotene inhibited the development of the lung tumour nodule formation compared to control. In vivo angiogenesis assay utilising gelfoam sponges, bexarotene reduced angiogenesis in sponges containing vascular endothelial growth factor, epidermal growth factor and basic fibroblast growth factor to various extent. To determine the basis of these observations, human breast and non-small-cell lung cancer cells were subjected to migration and invasion assays in the presence of bexarotene. Our data showed that bexarotene decrease migration and invasiveness of tumour cells in a dose-dependent manner. Furthermore, bexarotene inhibited angiogenesis by directly inhibiting human umbilical vein endothelial cell growth and indirectly inhibiting tumour cell-mediated migration of human umbilical vein endothelial cells through Matrigel matrix. Analysis of tumour-conditioned medium indicated that bexarotene decreased the secretion of angiogenic factors and matrix metalloproteinases and increased the tissue inhibitor of matrix metalloproteinases. The ability of bexarotene to inhibit angiogenesis and metastasis was dependent on activation of its heterodimerisation partner peroxisome proliferator-activated receptor gamma. Collectively, our results suggest a role of bexarotene in treatment of angiogenesis and metastasis in solid tumours.     

Microvesicles releasing by oral cancer cells enhance endothelial cell angiogenesis via Shh/RhoA signaling pathway

The present study aimed to investigate the significance of hedgehog signaling pathway in association with clinicopathology parameters and its effect on angiogenesis in oral squamous cell carcinoma (OSCC). The expression of Sonic Hh (Shh) and Gli1 were done on primary tumors and metastatic lymph nodes in OSCC samples from 80 patients by immunohistochemical analysis. The western blot was used to examine the expression of Shh in OSCC cell lines and OSCC-derived microvesicles (MVs). The role of Shh carried by MVs to induce endothelial cell angiogenesis was further investigated by matrigel assay. Our results indicated that the expression of Shh was positive associated with microvesseldentisty(MVD), TNM stage, tumor recurrence and lymph node metastasis. Moreover, Shh and Gli1 expression were higher in paired metastatic lymph nodes compared with expression of their primary tumors. The expression of Shh was abundant in Cal27, and present in SCC4, SCC9, and the amount of Shh protein in Cal27 targeting MVs was increased significantly than Cal27 cell group, up to ～ fifth-fold. The Cal27 derived MVs increased significantly angiogenesis of HUVECs in vitro, and this effect was blocked with exoenzyme C3 transferase (C3) and shRNA targeting RhoA by suppressing RhoA expression and activation. The data suggested that OSCC derived Shh carried by MVs may facilitate the tumor growth and modulate the preparation of a vascular network in primary tumor and/or premetastatic niche.