Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component in the root and rhizome of Rheum palmatum, is a tyrosine kinase inhibitor with a number of biological activities, including antitumor effects. Here, we examine the effects of emodin on vascular endothelial growth factor (VEGF)-A-induced angiogenesis, both in vitro and in vivo. In vitro, emodin dose-dependently inhibits proliferation, migration into the denuded area, invasion through a layer of Matrigel and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated with VEGF-A. Emodin also inhibits basic fibroblast growth factor-induced proliferation and migration of HUVECs and VEGF-A-induced tube formation of human dermal microvascular endothelial cells. Specifically, emodin induces the cell cycle arrest of HUVECs in the G0/G1 phase by suppressing cyclin D1 and E expression and retinoblastoma protein phosphorylation, and suppresses Matrigel invasion by inhibiting the basal secretion of matrix metalloproteinase-2 and VEGF-A-stimulated urokinase plasminogen activator receptor expression. Additionally, emodin effectively inhibits phosphorylation of VEGF-A receptor-2 (KDR/Flk-1) and downstream effector molecules, including focal adhesion kinase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, Akt and endothelial nitric oxide synthase. In vivo, emodin strongly suppresses neovessel formation in the chorioallantoic membrane of chick and VEGF-A-induced angiogenesis of the Matrigel plug in mice. Our data collectively demonstrate that emodin effectively inhibits VEGF-A-induced angiogenesis in vitro and in vivo. Moreover, inhibition of phosphorylation of KDR/Flk-1 and downstream effector molecules is a possible underlying mechanism of the anti-angiogenic activity of emodin. Based on these data, we propose that an interaction of emodin with KDR/Flk-1 may be involved in the inhibitory function of emodin toward VEGF-A-induced angiogenesis in vitro and responsible for its potent anti-angiogenic in vivo.     

Penta-O-galloyl-beta-D-glucose suppresses tumor growth via inhibition of angiogenesis and stimulation of apoptosis: roles of cyclooxygenase-2 and mitogen-activated protein kinase pathways

Recent studies have revealed that 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG) has anti-tumorigenic activity in vitro. In the present work, we evaluated the in vitro and in vivo antiangiogenic and antitumor activities of PGG and examined its molecular mechanisms. PGG significantly inhibited the proliferation and tube formation in basic fibroblast growth factor (bFGF)-treated human umbilical vein endothelial cells (HUVECs) at non-cytotoxic concentrations. PGG effectively disrupted the bFGF-induced neo-vascularization in chick chorioallantoic membrane (CAM) and in Matrigel plugs in the mice. When mice were intraperitoneally injected, PGG also significantly inhibited tumor angiogenesis induced by Lewis lung carcinoma (LLC) and the growth of LLC by 57 and 91% of control tumor weight at 4 and 20 mg/kg, respectively. Immunohistochemical analysis revealed decreased microvessel density, decreased expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), reduced tumor cell proliferation and increased tumor cell apoptosis. Similarly, PGG significantly attenuated the expression of COX-2 and VEGF and reduced the secretion of VEGF and prostaglandin E2 in bFGF-treated HUVECs. Furthermore, the COX-2 inhibitor NS398 significantly inhibited tube formation and neo-vascularization in CAM, supporting the role of COX-2 in PGG inhibition of angiogenesis. PGG diminished the phosphorylation of extracellular signal regulated kinase 1/2, Jun NH2-terminal kinase and activated phospho-p38 mitogen-activated protein kinase (MAPK) in a dose-dependent manner in bFGF-treated HUVECs. In addition, p38 inhibitor SB203580 abolished the downregulation of COX-2, VEGF and the antiproliferative activity by PGG. Taken together, our data demonstrate that PGG exerts antitumor activity primarily via inhibition of angiogenesis through COX-2 and MAPK- dependent pathways.     

Anti-angiogenic genistein inhibits VEGF-induced endothelial cell activation by decreasing PTK activity and MAPK activation

Genistein (Gen), a soy isoflavone, is considered to exert potent antitumor effect partially through its anti-angiogenesis property. However, the precise molecular mechanism is still unknown. Our previous investigations have demonstrated that genistein down-regulates expression of pro-angiogenic factors via inhibiting protein tyrosine kinase (PTK) activity both in breast cancer cells and in xenograft tumors. In the present experiment, we chose cultured human umbilical vein endothelial cells (HUVECs), which have a considerable role in tumor angiogenesis formation, to explore the influence of genistein on VEGF-stimulated endothelial cell activation and the underlying mechanism. Stimulation of human primary HUVECs by VEGF not only increased endothelial cell protein tyrosine kinase (PTK) activity but also augmented matrix metalloproteinase-2 (MMP-2), -9 secretions and increased MMP-2, -9 activities. Treatment of ECs with genistein induced VEGF-loaded endothelial apoptosis by inhibiting production and activity of matrix metalloproteinases (MMPs). In addition, exposure to genistein decreased activation of JNK and p38, not ERK-1/2, induced by VEGF. Collectively, our findings suggested that the inhibition of PTK activity and MAPK activation and the decrease in MMPs production and activity by genistein interrupt VEGF-stimulated endothelial cell activation, which thereby may represent a mechanism that would explain the anti-angiogenesis effect of genistein and its cancer-protective function.     

Regulation of tumor angiogenesis by fastatin, the fourth FAS1 domain of betaig-h3, via alphavbeta3 integrin

We previously reported that the FAS1 domains of betaig-h3 bear motifs that mediate endothelial cell adhesion and migration via interactions with alphavbeta3 integrin and regulate angiogenesis. In the present study, we show that the fourth FAS1 domain, designated fastatin, inhibits endothelial adhesion and migration, not only to betaig-h3, but also fibronectin and vitronectin, in a RGD-dependent manner. Fastatin and other FAS1 domains suppress endothelial cell tube formation and in vivo neovascularization in a Matrigel plug assay. The antiangiogenic activity of fastatin is associated with antitumor activity in mouse tumor models. Fastatin additionally induces apoptosis in several cells expressing alphavbeta3 integrin, including endothelial cells. Binding of fastatin to alphavbeta3 integrin inhibits phosphorylation of focal adhesion kinase, Raf, extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin. Fastatin is thus the first endogenous angiogenesis regulator identified that inhibits both endothelial cell migration and growth by binding to alphavbeta3 integrin. Our data suggest that FAS1 domains from all possible forms of the four human FAS1 family proteins are potential endogenous regulators for pathologic angiogenesis. Moreover, FAS1 domains such as fastatin may be developed into drugs for blocking tumor angiogenesis.     

Anti-angiogenic effects of a nutrient mixture on human umbilical vein endothelial cells

Matrix metalloproteinases (MMPs) have been recognized as key players in the degradation of the extracellular matrix (ECM) by migration and proliferation of endothelial cells and their subsequent invasion of the underlying stroma. The prevention of ECM degradation through the inhibition of MMP activity has been shown to be a promising therapeutic approach to block the invasion that occurs during angiogenesis. In previous studies, we demonstrated the anti-tumor effect of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, green tea extract, arginine, N-acetyl cysteine, selenium, copper and manganese on various tumor cell lines in vivo and in vitro. The aim of the present study was to determine whether this mixture has anti-angiogenic effects on human umbilical vein endothelial cells (HUVECs). At near confluence, the HUVEC cell cultures were tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose for proliferation, migration, MMP expression, and invasion. Cell proliferation was evaluated by MTT assay, invasion potential by Matrigel invasion, MMP expression by gelatinase zymography, and cell migration by a 2 mm-wide scratch in plates. For tube formation, HUVECs were cultured in previously polymerized Matrigel. NM inhibited HUVEC migration, MMP expression and invasion through Matrigel in a dose-dependent manner. Zymography showed a dose-dependent inhibition of MMP-2 expression with virtual total inhibition at a 500 microg/ml concentration. Invasion through Matrigel was totally inhibited at 500 microg/ml NM. NM reduced cell migration by scratch test in a dose-dependent fashion with total inhibition at a 500 microg/ml concentration. NM also inhibited the tube formation of HUVECs, but did not significantly inhibit cell proliferation. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation with anti-angiogenic effects, such as inhibiting vascular tube formation and endothelial cell invasion and migration.     

Vascular endothelial growth factor-mediated activation of p38 is dependent upon Src and RAFTK/Pyk2

Vascular endothelial growth factor (VEGF) induces activation of p38 mitogen-activated protein kinase (MAPK) in primary endothelial cells and may be critical for VEGF-induced angiogenesis. We investigated the molecular basis for p38 activation in response to VEGF. The expression of a C-terminal splice variant of FAK, FRNK, had no affect on VEGF-induced activation of p38; however, expression of a dominant-negative RAFTK/Pyk2 mutant led to a decrease in the activation of p38, but had no affect on extracellular signal-regulated kinase (ERK). Since calcium regulates RAFTK/Pyk2, we investigated its role in p38 activity. Preincubation with EGTA suppressed p38 activation, while calcium ionophore induced p38 activity. Inhibition of phospholipase C (PLC) resulted in complete inhibition of ERK, while having no affect on p38 activity. These data suggested a bifurcation in the regulation of MAPKs that occurs at the level of PLC and RAFTK/Pyk2 activation. Src family kinases interact with RAFTK/Pyk2. Inhibition of Src by either pharmacological or genetic means decreased p38 activity. Finally, we found that both Src and RAFTK/Pyk2 were essential for endothelial cell migration. These data identified a novel regulatory network involving extracellular calcium, RAFTK/Pyk2, Src and p38. This signaling network appears to be critical for VEGF-induced endothelial cell migration.     

Characterization of gastrin-induced proangiogenic effects in vivo in orthotopic U373 experimental human glioblastomas and in vitro in human umbilical vein endothelial cells.

PURPOSE: This study aims to investigate the role of gastrin-17 (G17) on angiogenesis features in gliomas both in vitro and in vivo. EXPERIMENTAL DESIGN: The influences of G17 and G17 receptor antagonists were characterized in vitro in terms of angiogenesis on human umbilical vein endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with respect to U373 orthotopic glioma xenografts. The influence of phosphatidylinositol 3'-kinase, protein kinase C, and nuclear factor-kappaB inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis. G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced modifications in nuclear factor-kappaB DNA binding activity were characterized by means of specific enzyme-linked immunosorbent assays. The influence of G17 on E- and P-selectin expression was determined by means of computer-assisted microscopy, whereas the influence of E- and P-selectin on HUVEC migration was approached by means of antisense oligonucleotides. The chemotactic influence of G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted videomicroscopy with Dunn chambers. RESULTS: Messenger RNAs for cholecystokinin (CCK)A, CCKB, and CCKC receptors were present in HUVECs and microvessels dissected from a human glioblastoma. Whereas G17 significantly increased the levels of angiogenesis in vivo in the U373 experimental glioma model and in vitro in the HUVECs, the CCKB receptor antagonist L365,260 significantly counteracted the G17-mediated proangiogenic effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor alpha (CXCR1) and IL-8 receptor beta (CXCR2) mRNAs were not detected in these endothelial cells. Gastrin significantly (but only transiently) decreased the level of expression of E-selectin, but not P-selectin, whereas IL-8 increased the expression of E-selectin. Specific antisense oligonucleotides against E- and P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on Matrigel. CONCLUSIONS: The present study shows that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on HUVECs. This effect depends in part on the level of E-selectin activation, but not on IL-8 expression/release by HUVECs.     

Inhibition of angiogenesis and invasion by 3,3'-diindolylmethane is mediated by the nuclear factor-kappaB downstream target genes MMP-9 and uPA that regulated bioavailability of vascular endothelial growth factor in prostate cancer

Progression of prostate cancer is believed to be dependent on angiogenesis induced by tumor cells. 3,3'-Diindolylmethane (DIM) has been shown to repress neovascularization in a Matrigel plug assay and inhibit cell proliferation, migration, invasion, and capillary tube formation of cultured human umbilical vein endothelial cells. However, the molecular mechanism, by which DIM inhibits angiogenesis and invasion, has not been fully elucidated. Therefore, we sought to explore the molecular mechanism by which DIM inhibits angiogenesis and invasion, specifically by investigating the role of angiogenic factors secreted by prostate cancer cells which control all steps of angiogenesis. We found that BioResponse DIM (B-DIM), a formulated DIM with higher bioavailability, inhibited angiogenesis and invasion by reducing the bioavailability of vascular endothelial growth factor (VEGF) via repressing extracellular matrix-degrading proteases, such as matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (uPA), in human prostate cancer cells and reduced vascularity (angiogenesis) in vivo using Matrigel plug assay. We also found that B-DIM treatment inhibited DNA binding activity of nuclear factor-kappaB (NF-kappaB), which is known to mediate the expression of many NF-kappaB downstream target genes, including VEGF, IL-8, uPA, and MMP-9, all of which are involved in angiogenesis, invasion, and metastasis. Our data suggest that inhibition of NF-kappaB DNA binding activity by B-DIM contributes to the regulated bioavailability of VEGF by MMP-9 and uPA and, in turn, inhibits invasion and angiogenesis, which could be mechanistically linked with the antitumor activity of B-DIM as observed previously by our laboratory in a prostate cancer animal model.     

Inhibition of growth factor-induced Ras signaling in vascular endothelial cells and angiogenesis by 3,3'-diindolylmethane

3,3'-Diindolylmethane (DIM), an indole derivative produced on consumption of broccoli and other cruciferous vegetables, has been shown to have multiple anticancer effects in both in vivo and in vitro models. The present study was carried out to clarify the mechanism of DIM's antiangiogenic activity. We found that DIM can inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation and DNA synthesis in human umbilical vascular endothelial cells (HUVECs). Consistent with this inhibition, VEGF-induced extracellular signal-regulated kinase (ERK1/2) phosphorylation was greatly reduced. However, VEGF receptor phosphorylation induced by VEGF was not affected by DIM, indicating that DIM does not exert a direct and specific effect on the tyrosine kinase activity of this receptor. Further studies showed that DIM had a similar inhibitory effect on ERK1/2 phosphorylation induced by a variety of growth factors. Furthermore, Ras-GTP content, which dramatically increased after HUVECs were challenged by either individual growth factors or serum, was reduced by approximately 80% with 25 muM DIM treatment, which in turn resulted in the reduced activities of Raf and MEK, culminating in the drop of ERK1/2 activation. Overexpression of constitutively active GTPase mutant, Ras G12V, in HUVECs reversed the inhibitory effect of DIM on ERK1/2 activation. In a rodent Matrigel plug model, the presence of DIM strongly reduced VEGF-induced neovascularization, indicating that DIM is active in vivo. These data provide evidence that DIM inhibits Ras signaling induced by VEGF and other growth factors, which interferes with its downstream biological effects necessary for angiogenesis.     

Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling

Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.     

Synergistic induction of in vivo angiogenesis by the combination of insulin-like growth factor-II and epidermal growth factor

Tumor growth is accelerated by induction of angiogenesis regulated by many cytokines and growth factors. In a tumor mass, various angiogenic factors and their receptors are simultaneously expressed and the overlapped expressions of various factors may contribute to the aggressive growth of tumor. However, the possible combined effect and mechanism of growth factors involved in angiogenesis are still under investigated. Insulin-like growth factor-II (IGF-II) has been identified as an angiogenic factor and highly expressed in solid tumors. Here we demonstrated that another angiogenic factor, epidermal growth factor (EGF), synergistically induced the angiogenic activity when co-treated with IGF-II in vivo. We performed mouse Matrigel plug assay. Cotreatment of IGF-II and EGF resulted in a significant induction of functional new vessels in mouse Matrigel plug more than additive amount of vessels induced by each growth factor. However, synergism of these two factors was not found in in vitro angiogenic assays, i.e., in migration and proliferation assays. The metalloproteinase-2 (MMP-2) protein level was enhanced by co-treatment of IGF-II and EGF, similar to that of individual treatment of them or PMA or bFGF. EGF down-regulated hypoxia-induced IGF-II binding protein-3 (IGFBP-3), which may contribute to the enhancement of free IGF-II accessibility to its receptors in vivo. Moreover, the combination of IGF-II with EGF significantly induced bFGF mRNA level, a potent angiogenic molecule, which may also contribute to synergistic effect in vivo. These results suggest that IGF-II and EGF may synergistically cooperate to induce angiogenesis in vivo by indirect mechanisms, i.e., synergistic induction of another angiogenic factor and modulation of IGF-II's bioavailability.     

二氢杨梅素通过ERK/VEGFA/VEGFR2通路对体内外胃癌血管生成的影响

背景与目的：二氢杨梅素（dihydromyricetin,DHM）通过抑制细胞周期、促进细胞凋亡和抑制新生血管生成等机制发挥抗肿瘤作用。探讨DHM对胃癌的抗癌作用,并研究其可能作用机制。方法：不同剂量DHM作用体外人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）和胃癌细胞系MKN28后,采用细胞计数试剂盒（cell counting kit-8,CCK-8）法筛选无毒剂量的DHM,Transwell小室实验测定细胞侵袭;小管形成实验测定HUVEC体外血管生成能力;采用蛋白质印迹法（Western blot）检测血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）、phospho-血管内皮生长因子受体（vascular endothelial growth factor receptor, VEGFR）2、细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）、phospho-c-Jun氨基端激酶（c-Jun NH2-terminal kinase,JNK）和phospho-p38表达水平;基质胶塞实验测定DHM对体内血管形成的影响;通过皮下肿瘤细胞注射法建立MKN28裸鼠异种移植瘤模型,观察DHM给药对体内胃癌生长的影响,Ki-67增殖指数及CD34、VEGFA在移植瘤组织中的表达通过免疫组织化学法测定。结果：（0.5~2.5 μmol/L）的DHM对HUVEC和MKN28细胞生长无明显抑制作用,该浓度范围的DHM呈浓度依赖性地抑制HUVEC侵袭和小管形成,VEGFA（20 μg/L）可明显逆转DHM对HUVEC侵袭和小管形成的抑制效果;DHM处理可导致MKN28细胞中VEGFA和phospho-ERK表达呈剂量依赖性下降,而对p-p38和p-JNK的表达无明显影响;DHM处理可导致HUVEC中phospho-VEGFR2表达呈剂量依赖性下降;低剂量（30 mg/kg）DHM对裸鼠无明显的不良反应,但可抑制体内血管形成,同时有效延缓体内胃癌生长,体内肿瘤组织中的CD34和VEGFA的表达受到DHM的显著下调作用,但低剂量DHM对Ki-67无明显作用。结论：低剂量的DHM可有效抑制体内外血管生成和体内胃癌生长,该作用至少部分是通过ERK/VEGFA/VEGFR2信号通路来实现的。     

Inhibition of telomerase in the endothelial cells disrupts tumor angiogenesis in glioblastoma xenografts

Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs). In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis.     

Novel approach to analysis of in vitro tumor angiogenesis with a variable-pressure scanning electron microscope: suppression by matrix metalloproteinase inhibitor SI-27

Degradation of basement membrane by metallo-proteinases (MMP) is a critical step in tumor angiogenesis. To evaluate in vitro angiogenesis, several models have been employed, including bovine cornea, fenestrated rat brain, Matrigel, and others. These models did not provide quantitative analysis of capillary formation. The current study aimed for a novel approach to in vitro assay of angiogenesis with a “wet scanning electron microscope (SEM)” to investigate suppression of tumor angiogenesis by the MMP inhibitor, SI-27. The effects of noncytotoxic concentrations of SI-27 (1–100μM) were determined on nonmitogenic vascular endothelial growth factor (VEGF) (10ng/ml)-mediated cell motility and in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). Activities of MMP and tissue inhibitor of metalloproteinase (TIMP) were determined by enzyme-linked immunosorbent assay (ELISA). Subsequently, the inhibitory effect of SI-27 was examined on in vitro angiogenesis stimulated by supernatants of human glioma cell lines (U87MG, U251MG, or U373MG). In vitro angiogenesis was quantitatively analyzed with a variable-pressure SEM. Cell motility and in vitro angiogenesis by HUVECs were significantly increased by VEGF along with elevated MMP-1 and-2 activity, whereas SI-27 significantly suppressed VEGF-mediated in vitro angiogenesis and inactivated both MMP-1 and MMP-2, but not inhibited cell motility. The angiogenesis promoted by glioma supernatants showed a significant reduction in the presence of SI-27. SI-27, a novel MMP inhibitor, inhibited tumor angiogenesis in vitro. It can be anticipated to prevent tumor growth through its angiosuppressive effect. Quantitative analysis with a variable-pressure SEM is a novel approach to in vitro angiogenesis assay.     

雷公藤红素抑制血管生成的实验研究

目的：探讨雷公藤红素对血管生成的抑制作用。方法：采用MTT法测定雷公藤红素对血管内皮细胞株(ECV)增殖的影响,观察雷公藤红素对ECV迁移实验和小管形成实验的作用,以及对鸡胚尿囊膜血管生成的影响。体内实验采用Matrigel plug方法。结果：雷公藤红素可明显抑制ECV的体外增殖,IC50为1．33μg／ml;可抑制ECV的迁移和小管形成,并且呈明显的剂量依赖性;同时具有抑制鸡胚尿囊膜血管生成和Matrigel plug中的血管新生的作用。结论：雷公藤红素具有明显抑制血管生成的作用,有可能成为有效的血管生成抑制剂。     

ING4对缺氧状态下血管内皮细胞增殖、迁移及HIF-1α表达的抑制作用

目的:在缺氧状态下,观察ING4对血管内皮细胞增殖、迁移及血管形成相关因子VEGF、MMP-2、MMP-9活性的影响,并探讨ING4对HIF-1α的调控作用。方法:人脐静脉内皮细胞HUVECs体外培养,构建ING4质粒及干扰RNA转染至HUVECs,应用CoCl2处理细胞模拟缺氧环境,MTT法检测细胞增殖,Transwell实验检测细胞迁移,Real time PCR及Western blot实验检测血管形成相关因子VEGF、MMP-2、MMP-9、HIF-1α及其靶基因AK3的mRNA及蛋白表达水平。结果:MTT及Transwell实验结果显示在缺氧状态下,转染ING4质粒能够抑制人脐静脉内皮细胞株HUVECs的增殖及迁移,而转染ING4 siRNA则能够促进HUVECs增殖及迁移,Real time PCR及Western blot实验结果显示转染ING4质粒能够抑制血管形成相关因子VEGF、MMP-2、MMP-9 mRNA及蛋白的表达水平,并且能够抑制HIF-1α及其靶基因AK3的表达水平。结论:在缺氧状态下,ING4能够通过抑制血管内皮细胞的增殖及迁移能力,下调血管形成相关因子VEGF、MMP-2、MMP-9的表达水平,进而抑制新血管形成,其机制可能与ING4能够下调缺氧状态下HIF-1α及其靶基因AK3的表达水平有关。     

Bee venom inhibits tumor angiogenesis and metastasis by inhibiting tyrosine phosphorylation of VEGFR-2 in LLC-tumor-bearing mice

Bee venom (BV) treatment is the therapeutic application of honeybee venom (HBV) for treating various diseases in Oriental medicine. In the present work, the authors investigated the functional specificity of BV as an angiogenesis inhibitor using in vitro models and in vivo mouse angiogenesis and lung metastasis models. BV significantly inhibited the viability of Lewis lung carcinoma (LLC) cells but did not affect peripheral blood mononuclear lymphocytes (PBML) cells. BV also inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and capillary-like tube formation of human umbilical vein endothelial cells (HUVECs). Western blotting analysis showed that BV inhibited AKT and MAPK phosphorylation in LLC cells and HUVECs and down regulated expression of VEGF and VEGFR-2 of LLC cells and HUVECs. Also, BV effectively disrupted VEGF-induced neovascularization in Matrigel plugs in our in vivo angiogenesis assay. When given subcutaneously, BV also significantly suppressed tumor angiogenesis through inhibition of VEGF and VEGFR-2 in LLC model. Mice bearing subcutaneous LLC tumors were treated with 1 μg/ml or 10 μg/ml of BV. They showed reductions ranging between 49% and 62% in primary tumor volume and reduction of spontaneous pulmonary metastasis occurrences. Furthermore, BV treatment in the spontaneous lung metastases model after primary tumor excision prolonged their median survival time from 27 to 58 days. These results suggest that the tumor-specific anti-angiogenic activity of BV takes effect during different stages of tumor progression by blocking the tyrosine phosphorylation of VEGFR-2, and validate the application of BV in lung cancer treatment.     

Angiogenic activity of human soluble intercellular adhesion molecule-1.

Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in a number of pathological conditions associated with angiogenesis, including tumor growth. Because the increased levels of sICAM-1 suggested that it may be angiogenic, we tested the ability of sICAM-1 to promote angiogenesis. Human recombinant sICAM-1 stimulates chemokinetic endothelial cell migration, endothelial cell tube formation on Matrigel, and sprouting of aortic rings. sICAM-1 also mediates angiogenesis in the chick chorioallantoic membrane assay. Additionally, we found a Mr 49,000 molecule that binds to sICAM-1 that may be the surface ligand on endothelial cells. The evidence that sICAM-1 has angiogenic activity suggests a possible role linking inflammation and neovascularization. Furthermore, sICAM-1 may enhance tumor growth by promoting angiogenesis and escape from immunosurveillance.     

Barbigerone, an isoflavone, inhibits tumor angiogenesis and human non-small-cell lung cancer xenografts growth through VEGFR2 signaling pathways

Purpose

We previously reported that barbigerone (BA), an isoflavone isolated from Suberect Spatholobus, exhibited inhibitory effects on proliferation of many cancer cell lines in vitro. The objective of this study was to explore whether BA could effectively suppress tumor angiogenesis and tumor growth.

Methods

Zebrafish model and Matrigel assay were performed to access the anti-angiogenesis effects of BA. A549 and SPC-A1 tumor xenografts in mice models were used to examine the antitumor activity of BA. The anti-angiogenic effects and underlying mechanisms were also investigated using human umbilical vein endothelial cells (HUVECs) and A549 cells.

Results

In zebrafish model, 2.5???mol/L of BA significantly inhibited angiogenesis. Intravenous administration of BA effectively inhibited the tumor growth of A549 and SPC-A1 xenograft models in mice. The anti-angiogenic effect was indicated by CD31 immunohistochemical staining, Matrigel plug assay, and mouse aortic ring assay. BA could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, and capillary-like tuber formation of HUVECs in a dose-dependent manner, suggesting that BA inhibited tumorigenesis by targeting angiogenesis. Western blots revealed that BA directly inhibited the phosphorylation of VEGFR2, followed by inhibiting the activations of its downstream protein kinases, including ERK, p38, FAK, AKT, and expression of iNOS, but had no effect on COX2. Additionally, BA could also down-regulate VEGF secretion in A549 cancer cells, which may correlate with the suppression of ERK, AKT activation, indicating that BA inhibits tumor angiogenesis and tumor growth through VEGFR2 signaling pathways.

Conclusions

These findings suggest that BA may be a novel candidate in inhibiting tumor angiogenesis and NSCLC tumor growth.