Flavonoids inhibit VEGF/bFGF-induced angiogenesis in vitro by inhibiting the matrix-degrading proteases

Flavonoids have been proposed to act as chemopreventive agents in numerous epidemiological studies and have been shown to inhibit angiogenesis and proliferation of tumor cells and endothelial cells in vitro. Angiogenesis requires tightly controlled extracellular matrix degradation mediated by extracellular proteolytic enzymes including matrix metalloproteinases (MMPs) and serine proteases, in particular, the urokinase-type plasminogen activator (uPA)-plasmin system. In this study, we have investigated the antiangiogenic mechanism of the flavonoids, genistein, apigenin, and 3-hydroxyflavone in a human umbilical vein endothelial cell (HUVEC) model. The stimulation of serum-starved HUVECs with vascular endothelial growth factor/basic fibroblast growth factor (VEGF/bFGF) caused marked increase in MMP-1 production and induced the pro-MMP-2 activation accompanied by the increase in MT1-MMP expression. However, pretreatment with flavonoids before VEGF/bFGF stimulation completely abolished the VEGF/bFGF-stimulated increase in MMP-1 and MT1-MMP expression and pro-MMP-2 activation. Genistein blocked VEGF/bFGF-stimulated increase in TIMP-1 expression and decrease in TIMP-2 expression. Apigenin and 3-hydroxyflavone further decreased TIMP-1 expression below basal level and completely abolished TIMP-2 expression. VEGF and bFGF stimulation also significantly induced uPA expression, most strikingly the level of 33 kDa uPA, and increased the expression of PA inhibitor (PAI)-1. Genistein, apigenin, and 3-hydroxyflavone effectively blocked the generation of 33 kDa uPA, and further decreased the activity of the 55 kDa uPA and the expression of PAI-1 below the basal level. In conclusion, these data suggest that genistein, apigenin, and 3-hydroxyflavone inhibit in vitro angiogenesis, in part via preventing VEGF/bFGF-induced MMP-1 and uPA expression and the activation of pro-MMP-2, and via modulating their inhibitors, TIMP-1 and -2, and PAI-1.     

Membrane Fluidity, Invasiveness and Dynamic Phenotype of Metastatic Prostate Cancer Cells after Treatment with Soy Isoflavones

Soy isoflavones represent hopeful unconventional remedies in the therapy of prostate cancer. The aim of our study was to determine the effects of genistein and daidzein on the parameters that reflect metastatic potential, membrane fluidity, invasiveness and dynamic phenotype in Matrigel of LNCaP and PC-3 prostate cancer cells. Cell viability tests, using a wide range of concentrations of soy isoflavones (6–75 μg/ml for 72 h), were conducted to determine their IC₅₀ concentrations. Electron paramagnetic resonance investigations of prostate cancer cell membrane fluidity were performed at IC₅₀ concentrations of genistein and daidzein (12.5 and 25 μg/ml, respectively, for 10 min). Genistein provoked significant increases in the membrane order parameter (which is reciprocally proportional to membrane fluidity) of 0.722 ± 0.006 (LNCaP), 0.753 ± 0.010 (LNCaP + genistein), 0.723 ± 0.007 (PC-3) and 0.741 ± 0.004 (PC-3 + genistein); however, no such effects were observed for daidzein. While both genistein and daidzein reduced the proliferation of prostate cancer cells at their respective IC₅₀ concentrations, during the 72 h of incubation only genistein provoked effects on the dynamic phenotype and decreased invasiveness. The effect was more evident in PC-3 cells compared to LNCaP cells. Our results imply that (1) invasive activity is at least partially dependent on membrane fluidity, (2) genistein may exert its antimetastatic effects by changing the mechanical properties of prostate cancer cells and (3) daidzein should be applied at higher concentrations than genistein in order to achieve pharmacological effects.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

Genistein modulates prostate epithelial cell proliferation via estrogen- and extracellular signal-regulated kinase-dependent pathways

Epidemiological evidence suggests that consumption of soy is associated with a decreased risk for prostate cancer. Genistein, the most abundant isoflavone present in soy, is thought to be responsible, in part, for these anticancer effects. The present study examined the effects of genistein on cellular proliferation, extracellular signal-regulated kinase (ERK1/2) activity and apoptosis in a nontumorigenic human prostate epithelial cell line (RWPE-1). Low concentrations of genistein (0-12.5 micromol/L) significantly increased cell proliferation and ERK1/2 activity (P<.01) in RWPE-1 cells, while higher concentrations (50 and 100 micromol/L) of genistein significantly inhibited cell proliferation and ERK1/2 activity (P<.001). A similar biphasic effect of genistein on MEK1 activity, an ERK1/2 kinase, was also observed. Pretreatment of cells with a MEK1 inhibitor (PD 098059) significantly blocked genistein-induced proliferation and ERK1/2 activity (P<.01). In addition, treatment of cells with ICI 182,780, a pure antiestrogen, inhibited genistein-induced RWPE-1 proliferation and ERK1/2 signaling. Taken together, these results suggest that genistein modulates RWPE-1 cell proliferation and signal transduction via an estrogen-dependent pathway involving ERK1/2 activation.     

Caspase-3 status is a determinant of the differential responses to genistein between MDA-MB-231 and MCF-7 breast cancer cells

Genistein, a soy isoflavone with anti-tumor properties, has both estrogenic and non-estrogenic activities. Genistein sensitive/estrogen receptor negative (ER-) MDA-MB-231 cells and genistein resistant/ER+MCF-7 cells are frequently cited as examples of differential responses to genistein due to different ER status. Other factors that may affect genistein response, however, are largely unknown. Based on our finding that MCF-7 is caspase-3 deficient, we examined whether caspase-3 status plays a role in the differential responses between the two cell lines. We demonstrate that reconstitution of caspase-3 significantly sensitizes MCF-7 cells to genistein. Specific knockdown of caspase-3 in MDA-MB-231 cells renders the cells resistant to genistein. We also found that caspases-4 and -10 were downregulated in MCF-7 cells. Reconstitution of caspase-10 in MCF-7 cells, however, resulted in little sensitization. Moreover, we show that caspase-3 downregulation is very common in breast cancer cell lines and tumor tissues. Taken together, our data indicate that caspase-3 is a critical determinant of cellular response to genistein, which may have important implications in studying soy/genistein-mediated anti-tumor activities.     

三羟异黄酮对HER-2／neu高表达乳腺癌细胞血管生成相关因子表达的影响

三羟异黄酮(genistein)是大豆中的一种非营养成分,其结构与黄酮化合物类似,能竞争性地与雌激素受体结合,故称之为植物雌激素(phytoestmgen)。它具有广泛的生物学作用,如抗肿瘤、抗病毒、抗真菌、抗氧化、抗突变、抗高血压、抗增生等,其中genistein抑制肿瘤的血管生成是当前研究的热点之一。     

Human Tumor Cells Induce Angiogenesis through Positive Feedback between CD147 and Insulin-Like Growth Factor-I

Tumor angiogenesis is a complex process based upon a sequence of interactions between tumor cells and endothelial cells. Previous studies have shown that CD147 was correlated with tumor angiogenesis through increasing tumor cell secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In this study, we made a three-dimensional (3D) tumor angiogenesis model using a co-culture system of human hepatocellular carcinoma cells SMMC-7721 and humanumbilical vein endothelial cells (HUVECs) in vitro. We found that CD147-expressing cancer cells could promote HUVECs to form net-like structures resembling the neo-vasculature, whereas the ability of proliferation, migration and tube formation of HUVECs was significantly decreased in tumor conditioned medium (TCM) of SMMC-7721 cells transfected with specific CD147-siRNA. Furthermore, by assaying the change of pro-angiogenic factors in TCM, we found that the inhibition of CD147 expression led to significant decrease of VEGF and insulin-like growth factor-I (IGF-I) secretion. Interestingly, we also found that IGF-I up-regulated the expression of CD147 in both tumor cells and HUVECs. These findings suggest that there is a positive feedback between CD147 and IGF-I at the tumor-endothelial interface and CD147 initiates the formation of an angiogenesis niche.     

紫杉醇抑制前列腺癌增殖的体内实验研究

目的探讨紫杉醇对人前列腺癌细胞PC-3增殖的体内抑制作用。方法建立体内绿色荧光蛋白（GFP）标记的人雄激素非依赖性前列腺癌细胞PC-3裸鼠原位移植瘤模型,观察紫杉醇对裸鼠前列腺癌原位移植瘤的体积、重量的影响。结果裸鼠模型体内实验显示,与对照组（100μL生理盐水）相比,紫杉醇处理组（0.5 mg/kg）在给药第18天后能显著抑制前列腺肿瘤的体积（P〈0.05）;紫杉醇处理组在抑制前列腺肿瘤重量方面与对照组相比亦有明显抑制作用（P〈0.05）。与对照组相比G31P处理组VEGF（P〈0.05）的表达差异具有统计学意义（免疫组化法）。结论紫杉醇在体内实验中能明显抑制人雄激素非依赖性前列腺癌细胞系PC-3的增殖。     

Luteolin Inhibits Human Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2-Mediated Angiogenesis

Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.     

Genistein blocks homocysteine-induced alterations in the proteome of human endothelial cells

Dietary isoflavones from soy are suggested to protect endothelial cells from damaging effects of endothelial stressors and thereby to prevent atherosclerosis. In search of the molecular targets of isoflavone action, we analyzed the effects of the major soy isoflavone, genistein, on changes in protein expression levels induced by the endothelial stressor homocysteine (Hcy) in EA.hy 926 endothelial cells. Proteins from cells exposed for 24 h to 25 microM Hcy alone or in combination with 2.5 microM genistein were separated by two-dimensional gel electrophoresis and those with altered spot intensities were identified by peptide mass fingerprinting. Genistein reversed Hcy-induced changes of proteins involved in metabolism, detoxification, and gene regulation; and some of those effects can be linked functionally to the antiatherosclerotic properties of the soy isoflavone. Alterations of steady-state levels of cytoskeletal proteins by genistein suggested an effect on apoptosis. As a matter of fact genistein caused inhibition of Hcy-mediated apoptotic cell death as indicated by inhibition of DNA fragmentation and chromatin condensation. In conclusion, proteome analysis allows the rapid identification of cellular target proteins of genistein action in endothelial cells exposed to the endothelial stressor Hcy and therefore enables the identification of molecular pathways of its antiatherosclerotic action.     

Antiangiogenic activity of 11,11'-dideoxyverticillin, a natural product isolated from the fungus Shiraia bambusicola

The fungus Shiraia bambusicola yields the phytochemical 11,11'-dideoxyverticillin, which has been shown to possess potent anticancer activity both in vitro and in vivo. In this study, we reveal that 11,11'-dideoxyverticillin has anti-angiogenic activities and explore the potential mechanisms for this effect. Treatment with 11,11'-dideoxyverticillin inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) with IC(50) values of 0.17+/-0.05muM for VEGF-stimulated cells and 0.39+/-0.08muM for serum-stimulated cells. 11,11'-Dideoxyverticillin also antagonized the antiapoptotic effects of VEGF on serum-deprived HUVECs, inhibited VEGF-induced HUVEC migration in vitro, and blocked serum-induced HUVEC tube formation. Moreover, 11,11'-dideoxyverticillin completely blocked VEGF-induced microvessel sprouting from Matrigel-embedded rat aortic rings and vessel growth in Matrigel plugs in mice. In addition, 11,11'-dideoxyverticillin decreased VEGF secretion by MDA-MB-468 breast cancer cells, and significantly suppressed VEGF-induced tyrosine phosphorylation of Flt-1 and KDR/Flk-1. This inhibition of receptor phosphorylation was correlated with a marked decrease in VEGF-triggered pERK activation and a dramatic increase in pP38 MAPK, but no apparent change in pAkt. Together, these findings strongly suggest that 11,11'-dideoxyverticillin is a structurally novel angiogenesis inhibitor.     

Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology.

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.     

Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells

Ganoderma lucidum (G. lucidum) is a popular medicinal mushroom that has been used as a home remedy for the general promotion of health and longevity in East Asia. The dried powder of G. lucidum, which was recommended as a cancer chemotherapy agent in traditional Chinese medicine, is currently popularly used worldwide in the form of dietary supplements. We have previously demonstrated that G. lucidum induces apoptosis, inhibits cell proliferation, and suppresses cell migration of highly invasive human prostate cancer cells PC-3. However, the molecular mechanism(s) responsible for the inhibitory effects of G. lucidum on the prostate cancer cells has not been fully elucidated. In the present study, we examined the effect of G. lucidum on angiogenesis related to prostate cancer. We found that G. lucidum inhibits the early event in angiogenesis, capillary morphogenesis of the human aortic endothelial cells. These effects are caused by the inhibition of constitutively active AP-1 in prostate cancer cells, resulting in the down-regulation of secretion of VEGF and TGF-beta1 from PC-3 cells. Thus, G. lucidum modulates the phosphorylation of Erk1/2 and Akt kinases in PC-3 cells, which in turn inhibits the activity of AP-1. In summary, our results suggest that G. lucidum inhibits prostate cancer-dependent angiogenesis by modulating MAPK and Akt signaling and could have potential therapeutic use for the treatment of prostate cancer.     

Doxazosin inhibits human vascular endothelial cell adhesion, migration, and invasion

The quinazoline-derived alpha1-adrenoceptor antagonists, doxazosin and terazosin have been recently shown to induce an anoikis effect in human prostate cancer cells and to suppress prostate tumor vascularity in clinical specimens [Keledjian and Kyprianou, 2003]. This study sought to examine the ability of doxazosin to affect the growth of human vascular endothelial cells and to modulate vascular endothelial growth factor (VEGF)-mediated angiogenesis. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro model to determine the effect of doxazosin on cell growth, apoptosis, adhesion, migration, and angiogenic response of endothelial cells. The effect of doxazosin on cell viability and apoptosis induction of human endothelial cells, was evaluated on the basis of trypan blue and Hoechst 33342 staining, respectively. Doxazosin antagonized the VEGF-mediated angiogenic response of HUVEC cells, by abrogating cell adhesion to fibronectin and collagen-coated surfaces and inhibiting cell migration, via a potential downregulation of VEGF expression. Furthermore there was a significant suppression of in vitro angiogenesis by doxazosin on the basis of VEGF-mediated endothelial tube formation (P < 0.01). Fibroblast growth factor-2 (FGF-2) significantly enhanced HUVEC cell tube formation (P < 0.01) and this effect was suppressed by doxazosin. These findings provide new insight into the ability of doxazosin to suppress the growth and angiogenic response of human endothelial cells by interfering with VEGF and FGF-2 action. This evidence may have potential therapeutic significance in using this quinazoline-based compound as an antiangiogenic agent for the treatment of advanced prostate cancer.     

Inhibition effects of gold nanoparticles on proliferation and migration in hepatic carcinoma-conditioned HUVECs

Tumor angiogenesis is a complicated process based upon a sequence of interactions between tumor and vessel endothelial cells. Tumor conditioned medium has been widely used to stimulate endothelial cells in vitro angiogenesis. This work was aimed to investigate the effects of gold nanoparticles (GNPs) on angiogenesis in hepatic carcinoma-conditioned endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured with conditioned medium (CM) from the human hepatocarcinoma cell line HepG2 (HepG2-CM), and then treated with different concentrations of GNPs. The effects of GNPs on the viability, migration and active VEGF level of HUVECs were investigated by MTT assay, wound healing assay and transwell chamber assay, and ELISA assay, respectively. The data showed that GNPs significantly inhibited HUVECs proliferation and migration induced by HepG2-CM, and also reduced the levels of active VEGF in the co-culture system. Then, the alterations in morphology and ultrastructure of HUVECs detected by atomic force microscopy (AFM) showed that there appeared obvious pseudopodia, larger membrane particle sizes and much rougher surface in HUVECs after HepG2-CM treatment, which were all reversed after GNPs treatment. Changes in cytoskeleton of HUVECs determined by immunocytochemistry demonstrated that GNPs treatment remarkably inhibited the activation effect of HepG2-CM on HUVECs, which was associated with the disruption of actin filaments induced by GNPs. This study indicates that GNPs can significantly inhibit HepG2-CM activated endothelial cell proliferation and migration through down-regulation of VEGF activity and disruption of cell morphology, revealing the potential applications of GNPs as antiangiogenic agent for the treatment of hepatic carcinoma.     

Single chain Fv antibody against angiopoietin-2 inhibits VEGF-induced endothelial cell proliferation and migration in vitro

Angiopoietin-2 (Ang2) promotes tumor growth and metastasis by specifically priming endothelial cells for angiogenesis. Multiple angiogenic factors up-regulate expression of Ang2, suggesting that Ang2 may be the common pathway in growth factor initiated-angiogenesis. Using phage display technology, we generated single chain Fv molecule against human Ang2 (scFv-Ang2) with high affinity (K(d)=0.01 microM) from a mouse phage antibody library. Compared with control scFv, the mouse scFv-Ang2 completely inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) treated with vascular endothelial growth factor (VEGF, 10 ng/ml), but not that of the cells treated with either basic fibroblast growth factor, or angiotensin II, or Ang2. Chemotaxis assay showed that scFv-Ang2 could block completely Ang2-induced (100%) and partially VEGF-induced (49%) migration of HUVECs. The results indicate that Ang2 takes part in the VEGF-induced angiogenesis and scFv-Ang2 might be a promising compound in blocking both VEGF and Ang2 induced angiogenesis.     

Glycitein activates extracellular signal-regulated kinase via vascular endothelial growth factor receptor signaling in nontumorigenic (RWPE-1) prostate epithelial cells

Increased consumption of soy is associated with a decreased risk for prostate cancer; however, the specific cellular mechanisms responsible for this anticancer activity are unknown. Dietary modulation of signaling cascades controlling cellular growth, proliferation and differentiation has emerged as a potential chemopreventive mechanism. The present study examined the effects of four soy isoflavones (genistein, daidzein, glycitein and equol) on extracellularsignal-regulated kinase (ERK1/2) activity in a nontumorigenic prostate epithelial cell line (RWPE-1). All four isoflavones (10 micromol/L) significantly increased ERK1/2 activity in RWPE-1 cells, as determined by immunoblotting. Isoflavone-induced ERK1/2 activation was rapid and sustained for approximately 2 h posttreatment. Glycitein, the most potent activator of ERK1/2, decreased RWPE-1 cell proliferation by 40% (P<.01). Glycitein-induced ERK1/2 activation was dependent, in part, on tyrosine kinase activity associated with vascular endothelial growth factor receptor (VEGFR). The presence of both VEGFR1 and VEGFR2 in the RWPE-1 cell line was confirmed by immunocytochemistry. Treatment of RWPE-1 cells with VEGF(165) resulted in transient ERK1/2 activation and increased cellular proliferation. The ability of isoflavones to modulate ERK1/2 signaling cascade via VEGFR signaling in the prostate may be responsible, in part, for the anticancer activity of soy.