Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth.

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.     

siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo

BACKGROUND/AIMS: We have investigated whether siRNA targeted against VEGF inhibits functional properties of endothelial cells in vitro and HCC tumor growth and blood vessel formation in vivo. METHODS: The influence of siRNA-VEGF on endothelial cell proliferation, apoptosis and tube formation were analyzed in vitro. Antitumoral effects were examined in an orthotopic tumor model after ex vivo transfer or intraperitoneal treatment of siRNA, respectively. Intratumoral microvessel density was assessed by CD31 staining. RESULTS: VEGF expression was inhibited in Hepa129 by 70% and in SVEC4-10 by 48% within two days after transfection. In vitro, endothelial cell proliferation and tube formation was reduced by 23% and 38%, respectively. Interference with VEGF signaling was demonstrated by reduced pAKT in hepatoma cells. Tumor growth was inhibited by ex vivo transfer or intraperitoneal application of siRNA-VEGF by 83% or 63% in orthotopic tumors within 14 days. VEGF protein was reduced in both models by 29% and 44%. Microvessel density dropped to 34% for tumors from ex vivo transfected cells and 39% for systemic treated tumors. CONCLUSIONS: The results show that VEGF knockdown can be associated with reduced endothelial cell proliferation and tube formation in vitro and decreased tumor growth and microvessel density in vivo.     

Usnic acid inhibits breast tumor angiogenesis and growth by suppressing VEGFR2-mediated AKT and ERK1/2 signaling pathways

Tumor growth depends on angiogenesis and inducing angiogenesis is one of the most important hallmarks in the cancer development. Treatment with small molecules that inhibit angiogenesis has been an effective strategy for anti-cancer therapy. Some anti-angiogenic factors are derived from traditional Chinese herbs. Usnic acid (UA), an active compound mainly found in lichens, has shown some biological and physiological activities. However, the role and mechanism of UA in tumor angiogenesis are still unknown. The aim of this study was to assess the effects of UA on tumor angiogenesis. In this study, we demonstrated that UA strongly inhibited in vivo angiogenesis in a chick embryo chorioallantoic membrane assay and vascular endothelial growth factor-induced mouse corneal angiogenesis model. In a mouse xenograft tumor model, UA suppressed Bcap-37 breast tumor growth and angiogenesis without affecting mice body weight. In an in vitro assay, UA not only significantly inhibited endothelial cell proliferation, migration and tube formation, but also induced morphological changes and apoptosis in endothelial cells. In addition, UA inhibited Bcap-37 tumor cell proliferation. Moreover, western blot analysis of cell signaling molecules indicated that UA blocked vascular endothelial growth factor receptor (VEGFR) 2 mediated Extracellular signal-regulated protein kinases 1 and 2(ERK1/2) and AKT/P70S6K signaling pathways in endothelial cells. These results provided the first evidence of the biological function and molecular mechanism of UA in tumor angiogenesis.     

IFN-gamma-mediated inhibition of tumor angiogenesis by natural killer T-cell ligand,alpha-galactosylceramide

Alpha-galactosylceramide (alpha-GalCer), which is a specific ligand for CD1d-restricted variable-alpha14 chain (V(alpha)14) natural killer T (NKT) cells, exerts a potent antitumor effect. We recently demonstrated that interferon-gamma (IFN-gamma) secreted by both NKT cells and NK cells plays a critical role in mediating the antimetastatic effect of alpha-GalCer; however, the IFN-gamma-dependent antitumor mechanisms remain poorly defined. In the present study, we demonstrate IFN-gamma-dependent inhibition of tumor angiogenesis by alpha-GalCer. In alpha-GalCer-treated mice, subcutaneous tumor growth and tumor-induced angiogenesis were inhibited in an IFN-gamma-dependent manner. The alpha-GalCer-activated splenic or hepatic mononuclear cells inhibited murine endothelial cell proliferation in vitro, and this inhibitory effect was mediated mostly by IFN-gamma produced by NKT cells and NK cells. NK cell depletion resulted in significant but partial inhibition of tumor growth and angiogenesis in vivo. These results suggest that the IFN-gamma-mediated inhibition of tumor angiogenesis is critically involved in the effector mechanisms of antitumor effects evoked by alpha-GalCer.     

Overexpression of ornithine decarboxylase enhances endothelial proliferation by suppressing endostatin expression

Angiogenesis, an essential process for tumor growth, is regulated by endothelial proliferation factors and their inhibitors such as endostatin. Endostatin, a carboxyl-terminal fragment of type XVIII collagen, inhibits endothelial proliferation, angiogenesis, and tumor growth. Ornithine decarboxylase (ODC), a molecule that is overexpressed in various cancers, is associated with promoting tumor growth and angiogenesis. We found that ODC-overexpressing human cancer cells and breast cancer specimens showed suppressed expression of type XVIII collagen and endostatin. We hypothesized that ODC overexpression may facilitate angiogenesis in tumors by suppressing endostatin expression. ODC-overexpressing COS cells, which showed suppressed type XVIII collagen and endostatin expression, were established. Conditioned media derived from these cells, containing decreased levels of endostatin, induced significant endothelial proliferation. ODC-overexpressing cells, when transplanted into nude mice, suppressed type XVIII collagen expression and promoted neovascularization in vivo. Thus, overexpression of ODC facilitates endothelial proliferation by suppressing endostatin expression.     

Notch signaling is involved in ovarian follicle development by regulating granulosa cell proliferation

Notch signaling is an evolutionarily conserved pathway, which regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Notch signaling are expressed in mammalian ovaries, but the exact functions of this pathway in follicle development is still unclear. In this study, primary follicles were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). We found that the cultured follicles completely stopped developing after L-658,458 and DAPT treatment, most of the granulosa cells were detached, and the oocytes were also degenerated with condensed cytoplasma. Further studies demonstrated that the proliferation of granulosa cells was dependent on the Notch signaling. L-658,458 and DAPT treatment inhibited proliferation of in vitro cultured primary granulosa cells and decreased the expression of c-Myc. Lentivirus mediated overexpression of Notch intracellular domain 2, and c-Myc could promote the proliferation of granulosa cells and rescue the growth inhibition induced by L-658,458 and DAPT. In conclusion, Notch signaling is involved in follicular development by regulating granulosa cell proliferation.     

Endothelial monocyte activating polypeptide II induces endothelial cell apoptosis and may inhibit tumor angiogenesis

Endothelial monocyte activating polypeptide II (EMAP-II) is a tumor-derived cytokine with potent effects on endothelial cells in vitro and in vivo including upregulation of tissue factor and the sensitization of human melanoma to systemic TNF treatment via its effects on the tumor vasculature. We investigated the effects of EMAP-II on tumor growth, angiogenesis, vasculogenesis, and apoptosis. EMAP-II inhibited endothelial cell proliferation, vasculogenesis, and neovessel formation. In vivo growth of human melanoma lines expressing high amounts of EMAP-II demonstrated slower growth, smaller tumors, and increased amounts of tumor necrosis than those expressing lower amounts of EMAP-II. EMAP-II induced endothelial-cell-specific apoptosis via a pathway that includes upregulation of the Fas-associated death domain and downregulation of Bcl-2. EMAP-II appears to have important effects on angiogenesis and may play a role in regulating tumor vascular growth.     

miR-153 inhibits the migration and the tube formation of endothelial cells by blocking the paracrine of angiopoietin 1 in breast cancer cells

The sprouting of endothelial cells is the first step of tumor angiogenesis. Our previous study suggests that miR-153 suppresses breast tumor angiogenesis partially through targeting hypoxia-induced factor (HIF1α). In this study, we demonstrated that miR-153 also suppresses the migration and the tube formation of endothelial cells through directly targeting angiopoietin 1 (ANG1) in breast cancer cells. There was a negative correlation between miR-153 and ANG1 levels in breast cancer. miR-153 blocked the expression and secretion of ANG1 in breast cancer cells through binding to ANG1 mRNA. Conditioned medium from the breast cancer cell, MCF7, treated with miR-153 had no effect on the proliferation of HUVECs, but significantly inhibited the migration and tube formation of HUVECs, which could be rescued by overexpression of ANG1. In addition, miR-153 also directly inhibited the proliferation and migration of MCF7 through downregulation of ANG1. These findings suggest that miR-153 suppresses the activity of tumor cells and the migration and tube formation of endothelial cells by silencing ANG1.     

Effect of Src kinase inhibition on metastasis and tumor angiogenesis in human pancreatic cancer

Tumor angiogenesis is a process that requires migration, proliferation, and differentiation of endothelial cells. We hypothesized that decrease in pancreatic tumor growth due to inhibition of Src activity is associated with the inability of Src kinase to trigger a network of such signaling processes, which finally leads to endothelial cell death and angiogenesis-restricted tumor dormancy. The therapeutic efficacy of Src kinase inhibitor AZM475271 was tested in nude mice orthotopically xenografted with L3.6pl pancreatic carcinoma cells. No liver metastases and peritoneal carcinosis were detected and a significant effect on the average pancreatic tumor burden was observed following treatment with AZM475271, which in turn correlated with a decrease in cell proliferation and an increase in apoptotic endothelial cells. AZM475271 was shown to significantly inhibit migration of human umbilical vein endothelial cells in an in vitro Boyden Chamber cell migration assay. In a rat aortic ring assay we could demonstrate as well inhibition of endothelial cell migration and sprouting following therapy with Src kinase inhibitor at similar doses. The most conclusive anti-angiogenic activity of AZM475271 was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor-induced neovascularization in response to systemic administration of AZM475271. Furthermore, we could show reduced proliferation of HUVECs determined with the TACS MTT Cell Viability Assay Kit. The blockade of Src kinase significantly reduced the level of VEGF in L3.6pl medium, the effect which was found also in the cell culture supernate from HUVECs. Inhibition of Src kinase by AZM475271 also showed prevention of survival signaling from VEGF and EGF receptors. Treatment with AZM475271 resulted in VEGF - dependent inhibition of tyrosine phosphorylation of FAK. HUVECs were also examined using propidium iodide staining for cell cycle analysis by FACS. Inhibition of Src kinase promoted HUVEC apoptosis in a dose-dependent manner. Taken together, our results suggest that the Src kinase inhibitor AZM475271, in addition to its effects on tumor cells, suppresses tumor growth and metastasis in vitro and in vivo potentially also by anti-angiogenic mechanisms.     

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.     

Notch1 siRNA 对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用研究

目的：探讨靶向Notch1基因小干扰RNA（ Notch1 siRNA,以下简称siNotch）对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用。方法将siNotch 转染小鼠恶性黑色素瘤细胞株B16F1,诱导RNA干扰,采用RT-PCR、Western blot、MTT法及细胞克隆试验体外检测细胞Notch1基因和蛋白表达及细胞增殖、克隆形成变化。随后分别将转染及对照细胞皮下接种于同基因C57BL/6小鼠（分别为转染组及对照组）,在成瘤过程中瘤内注射相应si-Notch1,记录肿瘤生长情况并绘制生长曲线,处死动物后取肿瘤比较各组肿瘤体积,并行HE及免疫组化染色观察肿瘤组织病理学变化和Notch1蛋白表达情况。结果 siNotch1转染B16F1细胞后,细胞增殖能力、克隆形成率及小鼠体内成瘤等均受到抑制（P＜0．01）,免疫组化显示转染组Notch1蛋白表达阳性细胞百分比明显低于对照组（P均＜0．01）。结论 siNotch1可降低细胞Notch1的表达,从而抑制小鼠MM细胞的增殖与生长,可作为治疗恶性黑色素瘤的新方法。     

Vascular endothelial growth factor C is increased in endometrium and promotes endothelial functions, vascular permeability and angiogenesis and growth of endometriosis

Endometriosis is an angiogenesis-dependent disease. Many studies demonstrated inhibition of angiogenesis leads to inhibition of endometriotic growth, however underlying mechanism is still not fully understood. Our previous study suggested vascular endothelial growth factor C (VEGF-C) as a target of anti-angiogenesis therapy for endometriosis. In this study, VEGF-C in endometrium and its role in angiogenesis of endometriosis were studied. Human endometrium were obtained from women with and without endometriosis for molecular studies. VEGF-A, VEGF-B, VEGF-C and VEGF-D mRNA and proteins in eutopic and ectopic endometrium were measured. Human endothelial cells were transfected with VEGF-C siRNA in vitro, effects of VEGF-C on endothelial cell migration, invasion and tube formation were investigated in vitro. Angiogenesis was inhibited in wild type mice, vascular permeability in dermal skin was determined in vivo. Transplanted endometrium were inhibited by VEGF-C siRNA in immunocompromised mice, development, growth and angiogenesis of the experimental endometriosis were compared in vivo. The results showed that VEGF-C mRNA and protein were increased in eutopic and ectopic endometrium of endometriosis patients. VEGF-C siRNA significantly inhibited endothelial cell migration and tube formation. VEGF-C siRNA significantly inhibited development and angiogenesis of the experimental endometriotic lesions in mice. Supplementation and over-expression of VEGF-C significantly reversed the inhibitory effects on the endothelial functions, vascular permeability and endometriotic growth. In conclusion, VEGF-C is increased in endometrium and it promotes endothelial functions, vascular permeability and development of experimental endometriosis. VEGF-C is important for angiogenesis in endometriosis.     

In vitro and in vivo angiogenesis in PC12 pheochromocytoma cells is mediated by vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels from existing vascular endothelium, is essential for tumor growth. Vascular endothelial growth factor (VEGF) is an endotheliumspecific mitogen and regulator of angiogenesis. Angiogenesis has been associated to the malignant phenotype of pheochromocytomas and is readily observed in experimental pheochromocytomas. Although VEGF gene expression has already been demonstrated in the rat PC12 cell line, the detailed mechanisms of action are not known. We have, therefore, studied angiogenesis in the rat PC12 pheochromocytoma cell line in vitro and in vivo. VEGF gene expression and accumulation of VEGF protein in cytoplasm and conditioned medium of PC12 cells was found. Conditioned medium from PC12 cells significantly increased proliferation of VEGF-dependent endothelial cells from human umbilical veins, and this effect reversed upon addition of a neutralizing anti-VEGF antibody. Dexamethasone and nerve growth factor (NGF) increased VEGF mRNA expression and accumulation of VEGF protein of PC12 subclones with established metastatic activity in vivo. PC12 cells xenotransplanted to nude mice had marked VEGF expression and induced host angiogenesis, confirmed by the presence of CD34-positive endothelial cells in the experimental PC12 tumors. When NGF-primed PC12 cells were immobilized in Matrigel supplemented with rising concentrations of the growth factor and xenotransplanted, increasing NGF resulted in tumors with smaller areas of necrosis and increased vital tumor volume. These results suggest that VEGF is a mediator of angiogenesis in the PC12 pheochromocytoma cell line, and that dexamethasone and NGF affect VEGF expression. Our data further suggest that NGF may contribute to angiogenesis in experimental pheochromocytoma.     

Impact of apolipoprotein(a) on in vitro angiogenesis

Angiostatin, which consists of the kringle I-IV domains of plasminogen and which is secreted into urine, is an efficient inhibitor of angiogenesis and tumor growth. Because N-terminal apolipoprotein(a) [apo(a)] fragments, which also contain several types of kringle IV domains, are found in urine as well, we evaluated the potential angiostatic properties of these urinary apo(a) fragments and of a recombinant form of apo(a) [r-apo(a)]. We used human microvascular endothelial cell (hMVEC)-based in vitro assays of tube formation in 3-dimensional fibrin matrixes. Purified urinary apo(a) fragments or r-apo(a) inhibited the basic fibroblast growth factor/tumor necrosis factor-alpha-induced formation of capillary-like structures. At concentrations varying from 0.2 to 10 microgram/mL, urinary apo(a) fragments inhibited tube formation by as much as 70%, whereas there was complete inhibition by r-apo(a). The highest concentrations of both inhibitors also reduced urokinase plasminogen activator production of basic fibroblast growth factor-induced hMVEC proliferation. The inhibitors had no effect on plasminogen activator inhibitor-1 expression. If our in vitro model for angiogenesis is valid for the in vivo situation as well, our data point toward the possibility that apo(a) may also be physiologically operative in modulating angiogenesis, as the concentration of free apo(a) found in humans exceeds that tested herein.     

Autocrine Role of Interleukin-8 in Induction of Endothelial Cell Proliferation,Survival, Migration and MMP-2 Production and Angiogenesis

Interleukin-8 (IL-8/CXCL8), a paracrine angiogenic factor, modulates multiple biologic functions in CXCR1 and CXCR2 expressing endothelial cells. Several reports suggest that inflammation, infection, cellular stress and tumor presence regulate IL-8 production in endothelial cells. In the present study, we test the hypothesis that IL-8 regulates multiple biological effects in endothelial cells in an autocrine manner. We examined the autocrine role of IL-8 in regulating angiogenesis by using a neutralizing antibody to IL-8, CXCR1 or CXCR2 in human vein umbilical endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMEC). Neutralizing antibody to IL-8, CXCR1 or CXCR2 inhibited endothelial cell proliferation, and MMP-2 production as compared to cells cultured with medium alone or control antibody. In addition, we observed that the number of apoptotic cells was significantly higher in anti-IL-8, anti-CXCR1 and anti-CXCR2 treated endothelial cells, which coincided with decreased survival-associated gene expression. We observed reduced migration of endothelial cells treated with anti-IL-8 and anti-CXCR2 antibody, but not anti-CXCR1 antibody as compared to controls. Further, we observed an inhibition of capillary tube formation and neovascularization following treatment with anti-IL-8, anti-CXCR1 and anti-CXCR2 antibodies. Together these data suggest that IL-8 functions as an important autocrine growth and angiogenic factor in regulating multiple biological activities in endothelial cells.     

Inhibition of PC cell-derived growth factor (PCDGF, epithelin/granulin precursor) expression by antisense PCDGF cDNA transfection inhibits tumorigenicity of the human breast carcinoma cell line MDA-MB-468

PC-cell derived growth factor (PCDGF) is an 88-kDa growth factor originally purified from the highly tumorigenic teratoma PC cell line and corresponds to the epithelin/granulin precursor. In teratoma cells, PCDGF expression was shown to be essential for tumorigenicity. We have reported that PCDGF was expressed in estrogen receptor-positive (ER(+)) human mammary epithelial cells in an estrogen-dependent fashion. In this study, we have investigated PCDGF expression in human mammary epithelial cell lines ranging from immortalized nontumorigenic cells to ER(+) and ER(-) breast carcinoma cells. Northern and Western blot analyses indicated that PCDGF mRNA and protein expression was low in nontumorigenic cells and increased in human breast carcinomas cell lines in a positive correlation with their tumorigenicity. Treatment of the ER(-) MDA-MB-468 cells with anti-PCDGF neutralizing antibody resulted in a dose-dependent inhibition of their proliferation, suggesting that secreted PCDGF acted as an autocrine growth factor for breast carcinoma cells. We then examined the in vitro and in vivo growth properties of MDA-MB-468 cells, where PCDGF expression had been inhibited by antisense PCDGF cDNA transfection. Inhibition of PCDGF expression resulted in a reduced proliferation rate in vitro and a 60-80% reduction in colony formation. Tumor formation in vivo was dramatically inhibited in antisense cells with a 90% inhibition of tumor incidence and tumor weight. These results demonstrate the importance of PCDGF overexpression for the proliferation and tumorigenicity of ER(-) breast carcinomas and suggest that PCDGF overexpression may play an important role in human breast cancer.     

Autocrine human growth hormone promotes tumor angiogenesis in mammary carcinoma

Accumulating literature implicates pathological angiogenesis and lymphangiogenesis as playing key roles in tumor progression. Autocrine human growth hormone (hGH) is a wild-type orthotopically expressed oncogene for the human mammary epithelial cell. Herein we demonstrate that autocrine hGH expression in the human mammary carcinoma cell line MCF-7 stimulated the survival, proliferation, migration, and invasion of a human microvascular endothelial cell line (HMEC-1). Autocrine/paracrine hGH secreted from mammary carcinoma cells also promoted HMEC-1 in vitro tube formation as a consequence of increased vascular endothelial growth factor-A (VEGF-A) expression. Semiquantitative RT-PCR analysis demonstrated that HMEC-1 cells express both hGH and the hGH receptor (hGHR). Functional antagonism of HMEC-1-derived hGH reduced HMEC-1 survival, proliferation, migration/invasion, and tube formation in vitro. Autocrine/paracrine hGH secreted by mammary carcinoma cells increased tumor blood and lymphatic microvessel density in a xenograft model of human mammary carcinoma. Autocrine hGH is therefore a potential master regulator of tumor neovascularization, coordinating two critical processes in mammary neoplastic progression, angiogenesis and lymphangiogenesis. Consideration of hGH antagonism to inhibit angiogenic processes in mammary carcinoma is therefore warranted.