Shikonin, acetylshikonin, and isobutyroylshikonin inhibit VEGF-induced angiogenesis and suppress tumor growth in lewis lung carcinoma-bearing mice

Lithospermum erythrorhizon has been used for treatment of inflammatory diseases and cancer as a folk remedy. Based on the evidences that anti-inflammatory agents frequently exert antiangiogenic activity, thus we examined comparatively the antiangiogenic activities of three naphthoquinone derivatives (shikonin, acetylshikonin, and isobutyroylshikonin) isolated from the plant. Three derivatives exhibited weak cytotoxicity against human umbilical vein endothelial cells (HUVECs) with IC50 of over 20 microM. Shikonin had more specific inhibitory effects on proliferation and vascular endothelial growth factor (VEGF) production by VEGF compared with different derivatives. All of derivatives significantly suppressed the migration of VEGF treated HUVECs at different optimal concentrations. Also, shikonin and acetylshikonin significantly disrupted VEGF-induced tube formation. Furthermore, three derivatives effectively downregulated the expression of urokinase-type plasminogen activator (uPA), but not its receptor uPAR. Additionally, shikonin significantly inhibited tumor growth in LLC-bearing mice, whereas its derivatives had relatively mild effects. Taken together, our findings suggest that shikonin and its derivatives exhibit the antiangiogenic and antitumorigenic effects by suppressing proliferation and angiogenic factors.     

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.     

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.     

Uncaria rhynchophylla induces angiogenesis in vitro and in vivo

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, and angiogenic factors and matrix protein interactions modulate this process. The aim of this study was to determine the angiogenic properties of Uncaria rhynchophylla. Uncaria rhynchophylla significantly enhanced human umbilical vein endothelial cells (HUVECs) proliferation in a dose-dependent manner. Neutralization of vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) by monoclonal antibody suppressed the Uncaria rhynchophylla stimulatory effect on proliferation. In addition, Uncaria rhynchophylla significantly increased chemotactic-migration on gelatin and tubular structures on Matrigel of HUVECs in a dose-dependent manner. Interestingly, Uncaria rhynchophylla dose-dependently increased VEGF, and bFGF gene expression and protein secretion of HUVEC. The angiogenic activity of Uncaria rhynchophylla was confirmed using an in vivo Matrigel angiogenesis model, showing promotion of blood vessel formation. These results suggest that Uncaria rhynchophylla could potentially used to accelerate vascular wound healing or to promote the growth of collateral blood vessel in ischemic tissues.     

Quercetin activates an angiogenic pathway, hypoxia inducible factor (HIF)-1-vascular endothelial growth factor, by inhibiting HIF-prolyl hydroxylase: a structural analysis of quercetin for inhibiting HIF-prolyl hydroxylase

We investigated a molecular mechanism underlying quercetin-mediated amelioration of colonic mucosal injury and analyzed chemical structure contributing to the quercetin's effect. Quercetin up-regulated vascular endothelial growth factor (VEGF), an ulcer healing factor, not only in colon epithelial cell lines but also in the inflamed colonic tissue. VEGF derived from quercetin-treated colon epithelial cells promoted tube formation. The VEGF induction was dependent on quercetin-mediated hypoxia-inducible factor-1 (HIF-1) activation. Quercetin delayed HIF-1alpha protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1alpha hydroxylation and subsequent von Hippel Lindau-dependent HIF-1alpha degradation. HPH inhibition by quercetin was neutralized significantly by an elevated dose of iron. Consistent with this, cellular induction of HIF-1alpha by quercetin was abolished by pretreatment with iron. Two iron-chelating moieties in quercetin, -OH at position 3 of the C ring and/or -OH at positions 3' and 4' of the B ring, enabled the flavonoid to inhibit HPH and subsequently induce HIF-1alpha. Our data suggest that the clinical effect of quercetin may be partly attributed to the activation of an angiogenic pathway HIF-1-VEGF via inhibiting HPH and the chelating moieties of quercetin were required for inhibiting HPH.     

Inhibition of angiogenesis by a tenascin-c peptide which is capable of activating beta1-integrins

In addition to humoral angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), integrin-mediated adhesion of vascular endothelial cells to the extracellular matrix plays an important role in neovascularization. We recently found that TNIIIA2, a peptide derived from tenascin-C, induces functional activation of beta1 integrins. Here we investigated the effect of TNIIIA2 on vascular endothelial cell migration and proliferation, key processes for angiogenesis. TNIIIA2 was shown to activate beta1-integrins on human dermal microvascular endothelial cells (HDMEC). HDMEC adhered to fibronectin mainly via integrin alpha5beta1 and their haptotactic migration on that substrate was inhibited by TNIIIA2, in concomitant with a marked inhibition of Rac activation. TNIIIA2-treatment unaffected autophosphorylation of focal adhesion kinase (FAK), but induced its physical association with phospho-paxillin (Tyr118), suggesting the FAK/paxillin-dependent negative regulation of Rac activation. HDMEC proliferation on the fibronectin substrate was also inhibited by TNIIIA2-treatment, and this was accompanied either by an increase in the population of G 0/G1 cells and, conversely, a decrease in the population of S and G2/M cells or by dephosphorylation/inactivation of MAP-kinase (ERK1/2). Inhibited HDMEC migration and proliferation were both restored by pretreating the cells with a fibronectin peptide, FNIII14, which is capable of inactivating beta1-integrins. The chorioallantoic membrane assay demonstrated an antiangiogenic effect of TNIIIA2 in vivo. Thus, TNIIIA2 appears to negatively regulate angiogenesis by inhibiting migration and proliferation of endothelial cells. The ability to activate beta1-integrins may be responsible for the antiangiogenic effect of TNIIIA2, although it cannot be excluded the possibility that an additional mechanism(s) may play a role.     

Angiogenesis: new targets for the development of anticancer chemotherapies

Angiogenesis is the process by which new blood vessels are formed from preexisting microvasculature. To ensure an adequate blood supply, tumor cells release angiogenic factors that are capable of promoting nearby blood vessels to extend vascular branches to the tumor. In addition, larger tumors have been shown to release angiogeneic inhibitory factors that prevent blood vessels from sending branches to smaller, more distant tumors that compete for oxygen and nutrients. Angiogenesis is a complex multistep biochemical process, and offers several potential molecular targets for non-cytotoxic anticancer therapies. Strategies for exploiting tumor angiogenesis for novel cancer drug discovery include: (i) inhibition of proteolytic enzymes that breakdown the extracellular matrix surrounding existing capillaries; (ii) inhibition of endothelial cell migration; (iii) inhibition of endothelial cell proliferation; (iv) enhancement of tumor endothelial cell apoptosis. There is also a host of miscellaneous agents that inhibit angiogenesis for which the specific mechanisms are not clear. Several methods have been developed for measuring antiangiogenic activity both in vitro and in vivo. Although there has been intensive research efforts focused at the phenomena of angiogenesis, as well as the search for antiangiogenic agents for more than two decades, many questions remain unanswered with regard to the overall biochemical mechanisms of the angiogenesis process and the potential therapeutic utility of angiogenic inhibitors. Nevertheless potent angiogenic inhibitors capable of blocking tumor growth have been discovered, and appear to have potential for development into novel anticancer therapeutics. However there are still hurdles to be overcome before these inhibitors become mainstream therapies.     

Hyperforin acts as an angiogenesis inhibitor

Hyperforin is a plant compound from Hypericum perforatum that inhibits tumor cell proliferation in vitro by induction of apoptosis. Here, we report that hyperforin also acts as an angiogenesis inhibitor in vitro and in vivo. In vitro, hyperforin blocked microvessel formation of human dermal microvascular endothelial cells (HDMEC) on a complex extracellular matrix. Furthermore, hyperforin reduced proliferation of HDMEC in a dose-dependent manner, without displaying toxic effects or inducing apoptosis of the cells. To evaluate the antiangiogenic activity of hyperforin in vivo, Wistar rats were subcutaneously injected with MT-450 mammary carcinoma cells and were treated with peritumoral injections of hyperforin or solvent. Hyperforin significantly inhibited tumor growth, induced apoptosis of tumor cells and reduced tumor vascularization, as shown by in situ staining of CD31-positive microvessels in the tumor stroma. These data suggest that, in addition to the induction of tumor cell apoptosis, hyperforin can also suppress angiogenesis by a direct, non-toxic effect on endothelial cells.     

复尔康注射液抗血管生成的体外实验研究

目的研究复尔康注射液在体外对血管生成的抑制作用及对肿瘤细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法采用细胞计数试剂盒(cell counting kit-8,CCK8)、细胞刮痕法(cell wounding healing assay)和小管形成试验(tube formation assay)等检测复尔康注射液对人微血管内皮细胞株(human microvascular endothelial cells,HMECs)增殖、迁移和小管形成的影响;采用CCK8、免疫组化SABC法检测复尔康注射液对HepG2细胞增殖和VEGF表达的影响。结果复尔康注射液明显抑制HMEC细胞的增殖,IC50为(11.44±3.97)mg.L-1;复尔康注射液质量浓度在3.125 mg.L-1时,表现出抑制HMEC细胞迁移和小管形成的作用,且随着质量浓度的增加,抑制作用愈明显,呈现出较好的剂量依赖关系;复尔康注射液还能明显抑制HepG2细胞的增殖,减弱VEGF的分泌表达。结论抗血管生成作用可能是复尔康注射液抗肿瘤作用的机制之一。     

Antiangiogenic activity of Diallyl Sulfide (DAS)

Antiangiogenic activity of Diallyl sulfide (DAS) was studied using in vivo as well as in vitro models. In vivo antiangiogenic activity was studied using B16F-10 melanoma cell induced capillary formation in C57BL/6 mice. DAS significantly inhibited tumour directed capillary formation. Studies of serum cytokine profile of angiogenesis induced animals clearly showed that DAS significantly reduced the production of proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha and GM-CSF which are known proangiogenic factors. The serum level of VEGF, an important proangiogenic factor, in angiogenesis induced animals was found to be significantly reduced upon treatment with DAS which may be due to its efficacy in the down regulation of VEGF mRNA expression. Administration of DAS significantly enhanced the production of antiangiogenic factors such as IL-2 and TIMP. In vitro studies using rat aortic ring assay showed that administration of DAS at no n-toxic concentrations significantly inhibited microvessel sprouting. Studies using Human umbilical vein endothelial cells (HUVECs) clearly demonstrated that administration of DAS significantly retarded endothelial cell proliferation, migration, invasion and tube formation. These data clearly suggests that antiangiogenic activity of DAS can be related to its negative regulation of proangiogenic factors such as VEGF and proinflammatory cytokines and positive regulation of antiangiogenic factors such as IL-2 and TIMP.     

CD151 gene delivery increases eNOS activity and induces ECV304 migration, proliferation and tube formation

AIM: To investigate the effects of CD151 on the activity of endothelial NO synthase (eNOS), and ECV304 migration, proliferation and tube formation. METHODS: pAAV-CD151 and pAAV-anti-CD151 were constructed and used to transiently transfect ECV304 mediated with Lipofectamine 2000. After transfection, the expression of CD151 was measured by Western blotting. Cell migration assay was performed using Boyden transwell; proliferation assay was evaluated using the 3- [4,5-dimethylthiazol-2-yl]-2,5, diphenyltetrazolium bromide (MTT) method, and tube formation test was examined on matrigel. eNOS activity was assayed by L- [3H]citrulline production from L-[3H]arginine. The involvement of eNOS was explored using an eNOS inhibitor (L-NAME) and the effects in the process were observed. RESULTS: CD151 promotes cell migration, proliferation and tube formation. In addition, CD151 increases eNOS activity. Moreover, cell migration, proliferation and tube formation induced by CD151 are inhibited when L-NAME is used, which indicates that there is an involvement of eNOS in CD151-induced cell migration, cell proliferation and tube formation. CONCLUSION: CD151 promotes ECV304 migration, proliferation and tube formation. The mechanism is that CD151 increases eNOS activity. This result also suggests that eNOS is involved in the angiogenic effects of CD151.     

Targeting of the COX-2/PGE2 axis enhances the antitumor activity of T7 peptide in vitro and in vivo

T7 peptide is considered as an antiangiogenic polypeptide. The presents study aimed to further detect the antiangiogenic mechanisms of T7 peptide and determine whether combining T7 peptide and meloxicam (COX-2/PGE2 specific inhibitor) could offer a better therapy to combat hepatocellular carcinoma (HCC). T7 peptide suppressed the proliferation, migration, tube formation, and promoted the apoptosis of endothelial cells under both normoxic and hypoxic conditions via integrin α3β1 and αvβ3 pathways. Cell proliferation, migration, apoptosis, or tube formation ability were detected, and the expression of integrin-associated regulatory proteins was detected. The anti-tumor activity of T7 peptide, meloxicam, and their combination were evaluated in HCC tumor models established in mice. T7 peptide suppressed the proliferation, migration, tube formation, and promoted the apoptosis of endothelial cells under both normoxic and hypoxic conditions via integrin α3β1 and αvβ3 pathways. Meloxicam enhanced the activity of T7 peptide under hypoxic condition. T7 peptide partly inhibited COX-2 expression via integrin α3β1 not αvβ3-dependent pathways under hypoxic condition. T7 peptide regulated apoptosis associated protein through MAPK-dependent and -independent pathways under hypoxic condition. The MAPK pathway was activated by the COX-2/PGE2 axis under hypoxic condition. The combination of T7 and meloxicam showed a stronger anti-tumor effect against HCC tumors in mice. The data highlight that meloxicam enhanced the antiangiogenic activity of T7 peptide in vitro and in vivo.     

Effects of rhizoxin,a microbial angiogenesis inhibitor,on angiogenic endothelial cell functions

Our previous study revealed that rhizoxin ([1S-[1R*,3R*,5S*,8R*(1R*,2S*,3E,5E,7E),10R*,11S*,13S*,14E,16S*,17S*]]-10-hydroxy-8-[2-methoxy-1,3,7-trimethyl-8-(2-methyl-4-oxazolyl)-3,5,7-octatrienyl]-11,16-dimethyl-4,7,12,18-tetraoxatetracyclo[15.3.1.03,5.011,13]heneicos-14-ene-6,19-dione) has a potent inhibitory effect on in vivo angiogenesis. However, little is known regarding the mechanism by which rhizoxin exhibits antiangiogenic activity. In this study, we examined its effects on the functions of endothelial cells associated with neovascular formation in vivo, using cultured vascular endothelial cells. Rhizoxin concentration-dependently inhibited the proliferation of bovine carotid artery endothelial cells, human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the IC(50) values being 7, 5 and 0.4 nM, respectively. In addition, it reduced the extracellular plasminogen activator level in bovine vascular endothelial cells in the low nM range, and suppressed the migration of human dermal microvascular endothelial cells in the pM range. Furthermore, it blocked the tubular morphogenesis of human umbilical vein endothelial cells and human dermal microvascular endothelial cells on Matrigel in a concentration-dependent manner; the IC(50) values being 40 and 130 pM, respectively. These results suggest that rhizoxin exhibits antiangiogenic activity through the combined inhibition of some functions of endothelial cells responsible for induction of in vivo angiogenesis.     

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。     

Nomilin inhibits tumor-specific angiogenesis by downregulating VEGF, NO and proinflammatory cytokine profile and also by inhibiting the activation of MMP-2 and MMP-9

Angiogenesis is a crucial step in the growth and metastasis of cancers. Antiangiogenic activity of nomilin was studied using in vivo as well as in vitro models. Nomilin significantly inhibited tumor directed capillary formation. Serum proinflammatory cytokines such as IL-1β, IL-6, TNF-α and GM-CSF and also serum NO levels were significantly reduced by the treatment of nomilin. Administration of nomilin significantly reduced the serum level of VEGF, a proangiogenic factor and increased the antiangiogenic factors IL-2 and TIMP-1. In vitro studies using rat aortic ring assay showed that administration of nomilin at non-toxic concentrations significantly inhibited microvessel sprouting. Studies using human umbilical vein endothelial cells clearly demonstrated that administration of nomilin significantly retarded endothelial cell proliferation, migration, invasion and tube formation. These data clearly demonstrate the antiangiogenic potential of nomilin by downregulating the activation of MMPs, production of VEGF, NO and proinflammatory cytokines as well as upregulating IL-2 and TIMP.