Inhibition of angiogenesis by suramin.

In this study, we have determined the ability of suramin to inhibit angiogenesis in the chick chorioallantoic membrane. Suramin alone showed significant angiostatic activity in a dose-related manner. Suramin also potentiated the activity of the angiostatic steroids, cortisol-21-phosphate, 17 alpha-hydroxyprogesterone, tetrahydrocortisol, and tetrahydrocortexolone. The presence of heparin decreased the angiostatic activity of suramin. These results suggest that suramin may decrease tumor growth by inhibiting angiogenesis. These novel findings indicate that suramin, perhaps in combination with angiostatic steroids, may be the basis for important new therapeutic approaches for diseases of neovascularization.     

Inhibition of neuroblastoma-induced angiogenesis by fenretinide.

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor- (VEGF-) and fibroblast growth factor-2- (FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF- and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48-72 hr of treatment with HPR, followed by a progressive accumulation of cells in G(1) at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.     

Inhibition of tumor-induced angiogenesis by sulfated chitin derivatives.

The effect of antimetastatic sulfated chitin derivatives (SCM-chitin III) on angiogenesis induced by B16-BL6 cells was examined in syngeneic mice. SCM-chitin III caused a marked decrease of the number of vessels toward tumor mass (angiogenic response) without affecting the tumor cell growth when coinjected with tumor cells (on day 0), or injected into tumor site on day 1 or 3 after tumor inoculation. In contrast, carboxymethyl chitin as well as heparin had no effect. Invasion of endothelial cells through reconstituted basement membrane (Matrigel) toward tumor-conditioned media was significantly inhibited by SCM-chitin III in a Transwell chamber assay. SCM-chitin III also inhibited the haptotactic migration of endothelial cells to fibronectin-substrate, but did not inhibit the chemotactic activity in tumor conditioned media in vitro. SCM-chitin III did not directly affect the viability and the growth of tumor cells and endothelial cells in vitro. These results suggest that inhibition of lung tumor metastasis by SCM-chitin III may in part be due to the inhibition of tumor-associated angiogenesis.     

Inhibition of angiogenesis and murine tumour growth by laminarin sulphate.

LAM S5 is a polysulphated derivative of the glucan laminarian that inhibits basic fibroblast growth factor (bFGF) binding and the bFGF-stimulated proliferation of fetal bovine heart endothelial (FBHE) cells. This report demonstrates that LAM S5 has anti-angiogenic activity, as shown by inhibition of tubule formation by endothelial cells cultured on Matrigel and inhibition of vascularisation of the chick chorioallantoic membrane. In addition, LAM S5 caused a tumour growth delay of the murine RIF-1 tumour of 2.6 days (P = 0.01).     

VEGF反义核酸抑制肿瘤血管形成的研究

目的探讨ＶＥＧＦ反义核酸作为抗新生血管形成药物和抗肿瘤药物的可能性。方法在小鼠角膜肿瘤模型上,测定两种ＶＥＧＦ反义核酸序列的５种不同修饰之反义核酸,对肿瘤血管形成和肿瘤生长的抑制作用。结果线性片段疗效较差,部分硫代和发夹结构两种修饰的反义核酸疗效满意。两种序列反义核酸混合使用最好。结论ＶＥＧＦ反义核酸有望成为抗肿瘤和抗新生血管形成的药物。     

Inhibition of angiogenesis and invasion in malignant gliomas

Malignant gliomas confer a dismal prognosis. As the molecular events that underlie tumor angiogenesis are elucidated, angiogenesis inhibition is emerging as a promising therapy for recurrent and newly diagnosed tumors. Data from animal studies suggest that angiogenesis inhibition may promote an invasive phenotype in tumor cells. This may represent an important mechanism of resistance to antiangiogenic therapies. Recent studies have begun to clarify the mechanisms by which glioma cells detach from the tumor mass, remodel the extracellular matrix and infiltrate normal brain. An array of potential therapeutic targets exists. Combination therapy with antiangiogenic and novel anti-invasion agents is a promising approach that may produce a synergistic antitumor effect and a survival benefit for patients with these devastating tumors.     

Inhibition of angiogenesis and invasion in malignant gliomas

Malignant gliomas confer a dismal prognosis. As the molecular events that underlie tumor angiogenesis are elucidated, angiogenesis inhibition is emerging as a promising therapy for recurrent and newly diagnosed tumors. Data from animal studies suggest that angiogenesis inhibition may promote an invasive phenotype in tumor cells. This may represent an important mechanism of resistance to antiangiogenic therapies. Recent studies have begun to clarify the mechanisms by which glioma cells detach from the tumor mass, remodel the extracellular matrix and infiltrate normal brain. An array of potential therapeutic targets exists. Combination therapy with antiangiogenic and novel anti-invasion agents is a promising approach that may produce a synergistic antitumor effect and a survival benefit for patients with these devastating tumors.     

Inhibition of angiogenesis by salmosin expressed in vitro

Recently, salmosin, a novel snake venom-derived disintegrin containing the Arg-Gly-Asp (RGD) sequence, was reported to be both antiangiogenic and antitumorigenic. The antitumor activity was substantiated by in vivo administration of recombinant salmosin into mice bearing tumors. However, it was difficult to prepare functionally active recombinant salmosin and to maintain a therapeutically effective concentration of the protein in the circulatory system by daily injections. Hence, we have suggested that salmosin gene transfer mediated by cationic liposomes may be a practical alternative for cancer treatment. Plasmids encoding the salmosin gene were constructed and then transferred by means of cationic liposomes into transformed human embryonic kidney (HEK) 293 cells. The transfected genes were able to produce functionally active salmosin proteins in vitro. In fact, the expressed salmosin remarkably inhibited proliferation of bovine capillary endothelial (BCE) cells and effectively inhibited the migration of highly metastatic B16BL6 mouse melanoma cells. Neovascularization in chick chorio-allantoic membranes (CAM) and in Matrigel implanted subcutaneously into mice was greatly inhibited in the presence of the expressed salmosin. Based on these experimental results, we suggest that the antitumor effect induced by salmosin gene transfection may be due to the antiangiogenic activity of the expressed salmosin proteins.     

Inhibition of tumor angiogenesis by hexuronyl hexosaminoglycan sulfate

The efficacy of heparin (HEP), the heparin analogue hexuronyl hexosaminoglycan sulfate (HHS), and hydrocortisone (HC) was studied in inhibiting the growth of four morphologically distinct pancreatic adenocarcinoma lines (CBP, LHP2, LSP3, and Pour-LVG) in hamsters. Animals were inoculated with LD100 doses of one of the four tumor lines and were randomly allocated to groups of five animals, which received in their drinking water either: HEP (1000 U/ml) alone, HHS (10 mg/ml) alone, HC (0.5 mg/ml) alone, HEP plus HC, HHS plus HC, or no additives (control). Tumors were measured, growth rates calculated, and nonparametric statistical comparisons made among the median growth rates of all of the treatment groups. All four tumors were tested in the rabbit cornea assay for their ability to induce angiogenesis. Extracts of tumors from control animals as well as from animals treated with HHS plus HC were prepared for quantitative testing in vitro by endothelial cell migration assay. All four tumor lines caused angiogenesis as measured in the rabbit cornea assay. A reduction in median tumor growth rates was observed in animals treated with HHS plus HC bearing the CBP, Pour-LVG, and LSP3 tumors. Similarly, in vitro capillary endothelial cell migration was decreased by HHS plus HC treatment in animals bearing CBP, Pour-LVG, and LSP3 tumors. Animals bearing the LHP2 tumor showed no effect of HHS plus HC treatment on tumor growth rate and no effect on endothelial cell migration. HEP alone, HHS alone, HC alone, and HEP plus HC showed no effect on tumor growth rate in any of the four tumors tested.     

Inhibition of angiogenesis by extracellular protein kinase A

The cyclic-AMP dependent protein kinase (PKA) signaling pathway regulates cell growth, development, metabolism, and gene expression. Peripheral blood of cancer patients but not normal individuals, shows increased catalytic subunit levels of PKA (PKAc). We showed here that this extracellular form of PKAc (ECPKA) from conditioned media of cultured cancer cells as well as purified PKAc inhibit angiogenesis, using the in utero chicken embryo chorioallantoic membrane assay. Inhibition of angiogenesis is partially reversed by PKI, a peptide inhibitor of PKA, thus suggesting an anti-angiogenic role for ECPKA. The significance of ECPKA in cancer is discussed.     

Inhibition of infiltration and angiogenesis by thrombospondin-1 in papillary thyroid carcinoma.

PURPOSE: Angiogenesis is essential for tumor growth and is controlled by the balance between angiogenic and antiangiogenic factors. We studied the expression of angiogenic factors and antiangiogenic factors in papillary thyroid carcinoma. EXPERIMENTAL DESIGN: We investigated immunohistochemically the expression patterns and levels of antiangiogenic factor and its receptor, thrombospondin-1 (TSP-1) and CD36, and four angiogenic factors, vascular endothelial growth factor (VEGF), VEGF-C, angiopoietin-2 (Ang-2), and Tie-2, in the primary tumors of 75 papillary thyroid carcinoma patients. We also examined the microvessel count (MVC), using CD31 staining. RESULTS: VEGF expression strongly correlated with other angiogenic factors. The cytoplasm of cancer cells stained positive for all factors. Tie-2 and TSP-1 receptor also appeared in endothelia of microvessels. TSP-1 inversely correlated with the degree of invasion of the primary tumor to other adjacent organs and with MVC. A higher MVC correlated with poorer survival. To clarify the balance between angiogenic and antiangiogenic factors in the same tumor, we calculated the ratio of each angiogenic factor against TSP-1 as the antiangiogenic factor. The ratios VEGF/TSP-1, VEGF-C/TSP-1, and Ang-2/TSP-1 significantly correlated with a higher MVC. Furthermore, the ratios VEGF/TSP-1 and Ang-2/TSP-1 significantly correlated with the degree of infiltration. CONCLUSIONS: To the best of our knowledge, this is the first report demonstrating that the balance between angiogenic and antiangiogenic factors correlates with distinct invasion to other organs and neovascularization of papillary thyroid carcinoma.     

牛蒡子苷元对肿瘤血管生成抑制作用的观察

目的:观察牛蒡子苷元(ARG)对裸鼠体内、体外肿瘤血管生成的影响,并探讨其作用机制。方法:利用人肝癌SMMC-7721细胞进行体内、外实验。体外实验分别采用RT-PCR、细胞免疫组化检测ARG作用前后SMMC-7721细胞中血管内皮生长因子(VEGF)基因及蛋白的表达;体内实验建立裸鼠SMMC-7721细胞肺转移瘤模型,成瘤后ARG干预,第30天取出肿瘤组织,免疫组化检测裸鼠瘤体CD34标记的血管内皮细胞,计算微血管密度(MVD)。结果:VEGF mRNA表达量经ARG及5-FU作用后,阴性对照组、3个药物组及阳性对照组依次为1.01±0.03、0.92±0.02、0.81±0.15、0.72±0.03和0.82±0.26;实验组VEGF蛋白表达率为44.16%,明显低于对照组的82.13%。ARG作用后VEGF mRNA及蛋白表达水平下降,P<0.05。裸鼠SMMC-7721细胞肺转移瘤的MVD减少,空白对照组、药物组和5-FU组裸鼠MVD值分别为39.43±3.31、19.29±2.06和21.57±2.82,P<0.01。结论:ARG通过下调裸鼠VEGF mRNA及蛋白的表达,抑制裸鼠SMMC-7721细胞肺转移瘤的血管生成。     

Inhibition of tumour-induced angiogenesis by systemically administered protamine sulphate

Systemic administration of protamine sulphate significantly decreased the intensity of angiogenesis induced in X-ray immunosuppressed (BALB/c X DBA/2W) F1 mice by either HEp-2 (human larynx carcinoma) cells or semi-syngeneic splenocytes injected intradermally. In vitro experiments have shown that protamine sulphate markedly decreases the proliferation of human endothelial cells as assessed by 3H-TdR incorporation assay. In contrast, the proliferation of HEp-2 cells was not affected, and only slight inhibition of normal human fibroblasts could be demonstrated. Heparin abolished the inhibitory effect of protamine sulphate, both in vivo and in vitro. These results suggest that the observed inhibitory effect of protamine sulphate on angiogenesis in vivo may be at least partially due to the interaction of this compound with endothelial cells.     

iNOS反义寡核苷酸对肿瘤血管生成抑制的研究

目的:探讨诱导型一氧化氮合成酶(induciblenitricoxidesynthase,iNOS)的反义寡核苷酸对肿瘤血管生成的抑制作用。方法:将人工合成的互补于iNOS基因翻译起始部位的反义-寡核苷酸(AS-ODN)及其错义-寡核苷酸(Mis-ODN)经脂质体介导转染入A549细胞后,了解其产生一氧化氮(nitricoxide,NO)的能力;将AS-ODN及其错义序列、空白对照分别在第1、4、7、12和18天注入接种有A549肺腺癌细胞的裸鼠的腹腔中,于第21天检测小鼠体内肿瘤的大小、肿瘤内血管密度(microvesseldensity,MVD)和肿瘤组织血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)表达的情况。结果:体外实验显示,iNOS的AS-ODN组和对照组相比,对A549细胞产生NO的抑制能力较强,并呈时间和剂量依赖性。在动物实验中,iNOS的AS-ODN对肿瘤的抑制作用较Mis-ODN和空白序列为强,差异均有统计学意义,F值分别为3·24和3·89,P值分别为0·044和0·032;AS-ODN组、Mis-ODN组及空白对照组中MVD的平均值分别为6·9±3·2、11·8±6·8和12·9±5·4,AS-ODN组MVD较Mis-ODN组和空白组明显降低,差异均有统计学意义,F值分别为5·53和7·63,P值分别为0·006和0·001;同时其VEGF的表达阳性率较Mis-ODN组明显下降,u=-2·457,P=0·017。结论:iN-OS的AS-ODN可降低肿瘤细胞产生NO的能力,在动物体内可降低肿瘤VEGF的表达和新生血管的生成,抑制肿瘤的生长。     

Inhibition of tumor cell invasion and angiogenesis by motuporamines

Tissue invasion is an important determinant of angiogenesis and metastasis and constitutes an attractive target for cancer therapy. We have developed an assay to identify agents that inhibit invasion by mechanisms other than inhibition of cell attachment or cytotoxicity. A screen of marine sponge extracts identified motuporamines as micromolar inhibitors of invasion of basement membrane gels by MDA-231 breast carcinoma, PC-3 prostate carcinoma, and U-87 and U-251 glioma cells. Motuporamine C inhibits cell migration in monolayer cultures and impairs actin-mediated membrane ruffling at the leading edge of lamellae. Motuporamine C also reduces beta1-integrin activation, raising the possibility that it interferes with "inside-out" signaling to integrins. In addition, motuporamine C inhibits angiogenesis in an in vitro sprouting assay with human endothelial cells and an in vivo chick chorioallantoic membrane assay. The motuporamines show little or no toxicity or inhibition of cell proliferation, and they are structurally simple and easy to synthesize, making them attractive drug candidates.     

Inhibition of Nodal suppresses angiogenesis and growth of human gliomas

Angiogenesis is the hallmark of malignant gliomas positively correlated with the vascular endothelial growth factor (VEGF) expression. We previously reported that expression levels of Nodal, a member of transforming growth factor-β super family, correlate with the malignant invasive behavior of human glioma cells. In this study, we show that knockdown of Nodal suppresses glioma angiogenesis by inhibition of VEGF. In human primary glioma specimens, expression of Nodal positively correlates with WHO glioma tumor grades and expression of VEGF in the corresponding glioma specimens. In human U87MG glioma cells, knockdown of endogenous Nodal by RNA interference (RNAi) significantly decreases colony formation and secretion of VEGF. In vivo, cellular depletion of Nodal in U87MG inhibited brain glioma growth and prolonged the survival of mice with U87MG/shNodal glioma compared with controls. Inhibition of Nodal suppressed tumor vessel growth in U87MG gliomas. Using Nodal inhibitor (SB431542), silencing Nodal, or overexpressing Nodal in the U87MG, GBM8401, and GBM glioma cells, our further experiments revealed that Nodal-induced VEGF expression might, at least in part, mediate through the ERK1/2-HIF-1α-mediated signaling pathway. Taken together, our data revealed that alteration of Nodal expression in glioma cells resulted in changes to VEGF secretion, and subsequent colony formation, in vivo tumor growth, and angiogenesis, all of which are consistent with the regulation of VEGF through the ERK1/2-HIF-1α-mediated signaling, suggesting that Nodal may serve as a potential therapeutic target for the treatment of human gliomas.     

Inhibition of gastric cancer-associated angiogenesis by antisense COX-2 transfectants

We characterized the effects of selective Cox-2 inhibition on the angiogenesis gastric cancer cell line SGC7901 by stable transfection of antisense Cox-2 cDNA (Cox-2-AS) or pharmacological inhibition by selective cox-2 inhibitor (NS398). The conditioned media obtained from SGC7901 treated with Cox-2-AS or NS398 suppressed the proliferation, migration and tube formation of human umbilical vein endothelial cells. Moreover, both treatments inhibited in vivo angiogenesis. These inhibitions could be partially reversed by the addition of PGE2. Our findings support that selective inhibition of Cox-2 alone plays an instrumental role on gastric cancer associated angiogenesis.     

Inhibition of heparanase activity and heparanase-induced angiogenesis by suramin analogues

Heparanase, a heparan sulfate-specific endo-beta-D-glucuronidase, plays an important role in tumor cell metastasis through the degradation of extracellular matrix heparan sulfate proteoglycans (ECM HSPG). Heparanase activity correlates with the metastatic propensity of tumor cells. Suramin, a polysulfonated naphthylurea, is an inhibitor of heparanase with suramin analogues shown to possess antiangiogenic and antiproliferative properties. We investigated the effects of selected suramin analogues (NF 127, NF 145 and NF 171) on heparanase activity and heparanase-driven angiogenesis. Studies of the ability of cellular extracts and purified heparanase from human, highly invasive and brain-metastatic melanoma (70W) cells revealed that heparanase expressed by these cells was effectively inhibited by suramin analogues in a dose-dependent manner. These analogues possessed more potent heparanase inhibitory activities than suramin: The concentrations required for 50% heparanase inhibition (IC(50)) were 20-30 microM, or at least 2 times lower than that for suramin. One hundred percent inhibition was observed at concentrations of 100 microM and higher. Of relevance, these compounds significantly decreased (i) the invasive capacity of human 70W cells by chemoinvasion assays performed with filters coated with purified HSPG or Matrigel trade mark, and (ii) blood vessel formation by in vivo angiogenic assays, thus linking their antiangiogenic properties with impedance of heparanase-induced angiogenesis. Specifically, inhibition of invasion by NF 127, NF 145 and NF 171 was found at 10 microM concentrations of compounds with a significant decrease of invasive values at concentrations as low as 1.5 microM. In addition, NF 127, NF 145 and NF 171 promoted nearly complete inhibition of heparanase-induced angiogenesis at values ranging from 236 microM (for NF 145) to 362 microM (for NF 127). These results further emphasize the importance of heparanase in invasive and angiogenic mechanisms and the potential clinical application of heparanase inhibitors such as suramin analogues in cancers and angiogenesis-dependent diseases.