Anti-Angiogenic Effects of Conjugated Docosahexaenoic Acid in Vitro and in Vivo

The anti-angiogenic effects of conjugated docosahexaenoic acid (CDHA), which was prepared by an alkaline treatment of docosahexaenoic acid and contained conjugated double bonds, were investigated in vitro and in vivo. CDHA inhibited tube formation by the bovine aortic endothelial cell (BAEC), and also inhibited the proliferation of BAEC at a concentration of CDHA that suppressed tube formation, but did not influence cell migration. The inhibition of BAEC growth caused by CDHA was accompanied by a marked change in cellular morphology. Nuclear condensation and brightness were observed in Hoechst 33342-stained cells treated with CDHA, indicating that CDHA induced apoptosis in BAEC. We also evaluated the angiogenesis inhibition of CDHA in vivo. The vessel formation which was triggered by tumor cells was clearly suppressed in mice orally given CDHA. Our findings suggest that CDHA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.     

Homocysteine inhibits angiogenesis in vitro and in vivo

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

Do conjugated eicosapentaenoic acid and conjugated docosahexaenoic acid induce apoptosis via lipid peroxidation in cultured human tumor cells?

Conjugated eicosapentaenoic acid (CEPA) and conjugated docosahexaenoic acid (CDHA) with triene structure, isomerized by alkaline treatment, showed intensive cytotoxicity with LD(50) at 12 and 16 microM, respectively, in DLD-1 cells (colorectal adenocarcinoma), while they had no effect on normal human fibroblast cell lines such as MRC-5, TIG-103, and KMS-6 cells. Cytotoxic action of CEPA and CDHA was also demonstrated in other tumor cell lines including HepG2, A549, MCF-7, and MKN-7 cells. alpha-Tocopherol suppressed cytotoxicity of CEPA and CDHA in tumor cells, and the cytotoxicity involved membrane phospholipid peroxidation. CEPA and CDHA induced DNA condensation and fragmentation in DLD-1 cells, indicating the involvement of apoptosis in this cytotoxic mechanism. Furthermore, previous reports have shown that lipid peroxidation product induces cell death, including apoptotic cell death in different cell lines. CEPA and CDHA have been demonstrated in cultured cells to cause cell death via lipid peroxidation and apoptosis in the absence of alpha-tocopherol.     

Regulation of angiogenesis-expression of VEGF receptors]

Angiogenesis, the formation of new blood vessels from pre-existing endothelium, is a crucial process for tumor growth, metastasis and inflammation. Therefore, it is focused on the anti-tumor therapy to prohibit angiogenesis in animal model and clinical studies. Eicosapentaenoic acid (EPA 20: 5,n-3) can restrain tumor growth and inflammation. In this paper, we examined the effects of EPA on tube formation. In EPA-pretreated endothelial cells angiogenesis was attenuated and also proliferation induced by VEGF, but not by b-FGF, was suppressed. The reason why EPA suppressed endothelial cell proliferation induced by VEGF was that EPA selectively inhibited the expression of KDR. As we mentioned, the regulation of angiogenesis in vivo may be involved in the expression of VEGF receptors.     

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.     

Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid.

Angiogenesis, the formation of new blood vessels, is an essential feature of malignant tumour development. Gamma linolenic acid (GLA), a n-6 polyunsaturated fatty acid (PUFA), inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, pancreatic cancer and hepatoma cells and also has anti-metastatic effects on endothelial cells. In the current study, we tested whether GLA inhibited angiogenesis induced by tumour cells. A rat aortic ring assay and in vitro tube formation of human vascular endothelial cells were used to determine angiogenesis (spontaneous, angiogenic factor- and tumour cells-induced). Inclusion of GLA in this 3-D matrix culture system significantly inhibited angiogenesis from aortic rings in a concentration-dependent manner. The results from tube formation of vascular endothelial cell further confirmed that GLA suppressed angiogenesis. Furthermore, in the cell motility assay (phagokinetic assay and endothelial wounding assay), a significant reduction of the motility of vascular endothelial cells by GLA was seen. It is concluded that gamma linolenic acid inhibits angiogenic factor and tumour-induced angiogenesis in vitro at least in part via its inhibitory effect on the motility of vascular endothelial cells.     

Antiangiogenic potential of 10-hydroxycamptothecin

To investigate the antiangiogenic potential of 10-hydroxycamptothecin (HCPT), the proliferation of human microvascular endothelial cells (HMEC) and seven human tumor cell lines were detected by SRB assay, and the endothelial cell migration and tube formation were assessed using two in vitro model systems. Also, inhibition of angiogenesis was determined with a modification of the chick embryo chorioallantoic membrane (CAM) assay in vivo. Morphological assessment of apoptosis was performed by fluorescence microscope. HCPT 0.313-5 micromol x L(-1) treatment resulted in a dose-dependent inhibition of proliferation, migration and tube formation in HMEC cells, and HCPT 6.25-25 nmol x egg(-1) inhibited angiogenesis in CAM assay. HCPT 1.25-5 micromol x L(-1) elicited typical morphological changes of apoptosis including condensed chromatin, nuclear fragmentation, and reduction in volume in HMEC cells. HCPT significantly inhibited angiogenesis both in vitro and in vivo at relatively low concentrations, and this effect was related with induction of apoptosis in HMEC cells. These results taken collectively suggest that HCPT may be a potent antiangiogenetic and cytotoxic drug and further investigation is warranted.     

Vascular basement membrane-derived multifunctional peptide, a novel inhibitor of angiogenesis and tumor growth

Vascular basement membrane-derived multifunctional peptide(VBMDMP)gene(fusion geneof the human immunoglobulin G3 upper hinge region and two tumstatin-derived fragments)obtained bychemical synthesis was cloned into vector pUC19,and introduced into the expression vector pGEX-4T-1 toconstruct a prokaryotic expression vector pGEX-4T-1-VBMDMP.Recombinant VBMDMP produced inEscherichia coli has been shown to have significant activity of antitumor growth and antimetastasis inLewis lung carcinoma transplanted into mouse C57B1/6.In the present study,we have studied the ability ofrVBMDMP to inhibit endothelial cell tube formation and proliferation,to induce apoptosis in vitro,and tosuppress tumor growth in vivo.The experimental results showed that rVBMDMP potently inhibited prolif-eration of human endothelial(HUVEC-12)cells and human colon cancer(SW480)cells in vitro,with noinhibition of proliferation in Chinese hamster ovary(CHO-K1)cells.rVBMDMP also significantly inhibitedhuman endothelial cell tube formation and suppressed tumor growth of SW480 cells in a mouse xenograftmodel.These results suggest that rVBMDMP is a powerful therapeutic agent for suppressing angiogenesisand tumor growth.     

Conjugated docosahexaenoic acid inhibits lipid accumulation in rats

Conjugated linoleic acid (CLA), which contains a conjugated double-bond system, and n-3 highly unsaturated fatty acids such as docosahexaenoic acid (DHA) are widely known to improve lipid metabolism. To examine the possibility that a fatty acid with a combination of these structural features might have stronger physiological effects, we prepared conjugated DHA (CDHA) by alkaline isomerization of DHA and examined its effects on lipid and sugar metabolism in rats. Rats were force fed with 200 mg of test oils [linoleic acid (LA), DHA, CLA or CDHA] everyday for 4 weeks. Compared with the animals from the other groups, those in the CDHA group showed a significant weight loss in white adipose tissue (57% of adipose tissue weight in the LA group) and significant decreases in the levels of liver triacylglycerol (TG; 65% of TG level in the LA group) as well as total cholesterol (TC; 88% of TC level in the LA group), indicating suppression of lipid accumulation in the liver and adipose tissue. In addition, plasma TG and TC levels significantly decreased (69% of TG level and 82% of TC level in the LA group), indicating improved lipid metabolism. In the liver, the fatty acid synthesis system was inhibited and the fatty acid beta-oxidation system was activated, whereas the free fatty acid, glucose and tumor necrosis factor alpha levels in the plasma were lowered following CDHA administration. Hence, intake of CDHA appears to suppress the accumulation of fat in the liver and epididymal adipose tissue and improves lipid and sugar metabolism in rats.     

Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis

Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors.     

Epicatechin conjugated with fatty acid is a potent inhibitor of DNA polymerase and angiogenesis

Anti-cancer and anti-angiogenesis effects of green tea catechins have been demonstrated. It has been found that chemical modification of tea catechins improves their biological activities. We examined the chemical modification of epicatechin enhanced anti-cancer and anti-angiogenic effects. Epicatechin conjugated with fatty acid (acyl-catechin) strongly inhibited DNA polymerase activity, HL-60 cancer cell growth and angiogenesis. Epicatechin conjugated with palmitic acid ((2R,3R)-3',4',5,7-tetrahydroxyflavan-3-yl hexadecanoate, epicatechin-C16) was the strongest inhibitor in DNA polymerase alpha, beta, lambda and angiogenesis assays. Epicatechin-C16 also suppressed human endothelial cell (HUVEC) tube formation on reconstituted basement membrane, suggesting that it affected not only DNA polymerase activity but also the signal transduction pathways needed for the tube formation in HUVECs. These results suggest that acylation of epicatechin is an effective chemical modification to improve the anti-cancer activity of epicatechin.     

Catechin conjugated with fatty acid inhibits DNA polymerase and angiogenesis

Catechins in green tea have anticancer and antiangiogenesis activities, with epigallocatechin-3-gallate (EGCG) being the most potent antiangiogenic tea catechin. This study examined whether chemical modification of catechin enhanced anticancer and antiangiogenic effects. Catechin, conjugated with fatty acid (acyl-catechin), strongly inhibited DNA polymerase, HL-60 cancer cell growth, and angiogenesis. Catechin conjugated with stearic acid [(2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl octadecanoate; catechin-C18] was the strongest inhibitor in DNA polymerase alpha and beta and angiogenesis assays. Catechin-C18 also suppressed human endothelial cell (HUVEC) tube formation on the reconstituted basement membrane, suggesting that it affected not only DNA polymerases but also signal transduction pathways in HUVECs. These data indicate that acyl-catechins target both DNA polymerases and angiogenesis as anticancer agents. These results suggest that acylation of catechin is an effective chemical modification to improve the anticancer activity of catechin.     

Inhibition of angiogenic attributes by decursin in endothelial cells and ex vivo rat aortic ring angiogenesis model

The present study was undertaken to observe the inhibition of angiogenesis by decursin. It was the first time to show that decursin offered strong anti-angiogenic activities under the biologically relevant growth (with serum) conditions. Decursin significantly inhibited human umbilical vein endothelial cell (HUVEC) proliferation concomitant with G1 phase cell cycle arrest. Decursin also inhibited HUVEC-capillary tube formation and invasion/migration in a dose-dependant manner which was associated with the suppression of matrix metalloproteinase (MMP) -2 and -9 activities. Decursin suppressed angiogenesis in ex vivo rat aortic ring angiogenesis model where it significantly inhibited blood capillary-network sprouting from rat aortic sections. Taken together, these findings suggested anti-angiogenic activity of decursin in biologically relevant condition, and warrants further pre-clinical studies for its potential clinical usefulness.     

3-O-Acetyloleanolic acid exhibits anti-angiogenic effects and induces apoptosis in human umbilical vein endothelial cells

3-O-Acetyloleanolic acid, a pentacyclic triterpenoid isolated from cowpea seeds, inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. HUVECs. The induced apoptosis was characterized by detection of cell surface annexin V and sub-G1 populations. The number of cells immunostained with annexin V-fluorescein isothiocyanate increased after treatment with 3-O-acetyloleanolic acid. The sub-G1 cell populations were also increased in treated HUVECs. 3-O-Acetyloleanolic acid induced activation of caspase 3, a critical mediator of apoptosis signaling. It also significantly inhibited angiogenesis in an in vivo Matrigel plug assay. 3-O-Acetyloleanolic acid thus exhibits anti-angiogenic effects and induces apoptosis in HUVECs and the results suggest that it has a potential use for suppression of the tumor growth stimulated by angiogenesis.     

Downregulation of microRNA-205 inhibits cell invasion and angiogenesis of cervical cancer through TSLC1-mediated Akt signaling pathway

Cervical cancer (CC) is a common gynecological cancer and a leading cause of cancer-related deaths in women globally. Therefore, this study explores the action of microRNA-205 (miR-205) in the invasion, migration, and angiogenesis of CC through binding to tumor suppressor lung cancer 1 (TSLC1). Initially, the microarray analysis was used to select the candidate gene and the regulatory microRNA. Then, the target relationship between miR-205 and TSLC1 as well as the expression of miR-205, TSLC1, and p-Akt/total Akt in CC cells were determined. Afterwards, CC cell invasion and migration were detected after the treatment of miR-205 mimics/inhibitors and short hairpin RNA against TSLC1. After coculture of cancer cells and vascular endothelial cells, cell proliferation, tube formation, and microvessel density (MVD) were detected to determine the roles of miR-205 in angiogenesis. Finally, tumor growth in nude mice was measured in vivo. TSLC1 was determined as the candidate gene, which was found to be targeted and negatively regulated by miR-205. Then, downregulated miR-205 or forced TSLC1 expression inhibited invasion, migration, and angiogenesis in CC, corresponding to suppressed cell proliferation, tube formation, and expression of IL-8, VEGF, and bFGF, as well as the inhibited activation of the Akt signaling pathway. Furthermore, downregulation of miR-205 was found to exert an inhibitory role in tumor formation and MVD by elevating TSLC1 in CC in vivo. This study demonstrated that downregulated miR-205 inhibited cell invasion, migration, and angiogenesis in CC by inactivating the Akt signaling pathway via TSLC1 upregulation.     

Differential effects of interferon gamma and alpha on in vitro model of angiogenesis.

The formation of blood vessels in vivo (angiogenesis) is an important process and is usually initiated in response to injury, tumor growth, or normal tissue development. We have studied the effect of human interferon (IFN) alpha (alpha) and gamma (gamma) on the capillary-like network formation in an in vitro model of angiogenesis using human umbilical vein endothelial cells (HUVEC). When HUVEC cells are plated on Matrigel (reconstituted basement membrane matrix enriched in laminin), a network of capillary like structures (endotubes) rapidly forms. IFN-alpha enhanced the tube formation in a dose-dependent manner, whereas IFN-gamma significantly inhibited the tube formation. In addition, both the enhancement and inhibition of angiogenesis by IFN-alpha and gamma was found to be greater if the cells were pretreated with IFN for 12 hr before plating on the Matrigel. These results suggest that IFN may play an important role in several vascular processes including early stages of wound healing, recanalization of thrombi, tumor growth, metastasis, normal growth, and development.     

Wogonin suppresses tumor growth in vivo and VEGF-induced angiogenesis through inhibiting tyrosine phosphorylation of VEGFR2

Previous studies revealed that wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, possessed anticancer activity both in vitro and in vivo. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we found for the first time that wogonin inhibited the growth and tumor angiogenesis of human gastric carcinoma in nude mice. We explored the inhibitory effect of wogonin on angiogenesis stimulated by vascular endothelial growth factor (VEGF) in vitro. Wogonin suppressed the VEGF-stimulated migration and tube formation of human umbilical vein endothelial cells (HUVECs). It also restrained VEGF-induced tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This inhibition of receptor phosphorylation was correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that wogonin might be a promising antitumor drug.     

A model system for tumor angiogenesis: involvement of transforming growth factor-alpha in tube formation of human microvascular endothelial cells induced by esophageal cancer cells.

Tumor growth is dependent on angiogenesis, which is thought to be mediated through growth factors, such as transforming growth factor-alpha (TGF-alpha) and -beta (TGF-beta), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), produced by tumor cells. We have developed a model system for tumor angiogenesis in vitro: tube formation of human omentum microvascular endothelial (HOME) cells in type I collagen gels when these cells are co-cultured with tumor cells. Exogenously added TGF-alpha induced tube formation of HOME cells in collagen gel. In contrast, TGF-beta inhibited the TGF-alpha-induced tube formation of endothelial cells. We investigated whether tube formation could be induced in HOME cells in collagen gel when the HOME cells were co-cultured with three esophageal cancer cell lines, TE1, TE2, and TE5. TE1 and TE2 cells expressed both TGF-alpha and TGF-beta mRNA, but the level of TGF-alpha mRNA in TE2 was found to be much lower than in TE1 cells. TE5 did not express either TGF-alpha or TGF-beta. The tube formation of HOME cell was induced when they were co-cultured with TE1 cells, while both TE2 and TE5 cell lines induced tube formation at much lower rates than TE1. TE1-induced tube formation of HOME cells was specifically blocked by co-administration of anti-TGF-alpha-antibody, but not by anti-bFGF-antibody. The present study suggests that, in our model system, esophageal tumor angiogenesis is partly controlled by TGF-alpha, possibly through a paracrine pathway.